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Institute for MicrobiologySchool of Veterinary Medicine Hannover
The role of iron inActinobacillus pleuropneumoniae infection:Identification and in vivo characterization of
virulence-associated genes
THESIS
submitted in partial fulfilment of the requirements for the degree
PHILOSOPHICAL DOCTOR- Ph.D. -
in the field ofMicrobiology
at the School of Veterinary Medicine Hannover
byNina Baltes,
Düsseldorf, Germany
Hannover, Germany 2002
Supervisor: Prof. Dr. G.-F. Gerlach (Institute for Microbiology,
Veterinary School Hannover, Germany)
Advisory committee: Prof. Dr. G.-F. GerlachProf. Dr. B. Tümmler (Clinical research group OE6711,
Medical School Hannover, Germany)
Prof. Dr. L. Haas (Institute for Virology, Veterinary School
Hannover, Germany)
External evaluation: Paul Langford, Ph.D. (Molecular Infectious Diseases
Group, Department of Paediatrics, Imperial College of
Science, Technology and Medicine, St. Mary's Hospital
Campus, London, United Kingdom)
Oral examination: June 3rd, 2002
This work has feen funded by the Deutsche Forschungsgemeinschaft (DFG), grant
no. GE 522/ 3-1 and 3-2
The pathogen is nothing, the terrain is everything.
Louis Pasteur
This study has been published in part:
Publications:
TONPITAK, W., S. THIEDE, W. OSWALD, N. BALTES and G.-F. GERLACH (2000):
Actinobacillus pleuropneumoniae iron transport: a set of exbBD genes is transcriptionally linked to the
tbpB gene and required for utilization of transferrin-bound iron.
Infect. Immun. 68, 1164-1170
BALTES, N., W. TONPITAK, G.-F. GERLACH, I. HENNIG-PAUKA, A. HOFFMANN-MOUJAHID, M.
GANTER and H. J. ROTHKOTTER (2001):
Actinobacillus pleuropneumoniae iron transport and urease activity: effects on bacterial virulence and
host immune response.
Infect. Immun. 69,(1) 472-478
BALTES, N., HENNIG-PAUKA, I., GERLACH, G.-F.(2002)
Both transferrin binding proteins are virulence factors in Actinobacillus pleuropneumoniae serotype 7
infection.
FEMS Microbiology Letters, in press
Abstracts:
TONPITAK, W., S. THIEDE, W. OSWALD, N. BALTES and G.-F. GERLACH (2000):
"Eisentransport bei Actinobacillus pleuropneumoniae: Zusammenhang zwischen den Genen der
transferrinbindenden Proteine und einem Set von tonB-, exbB-, und exbD-Genen"
Tagung der deutschen veterinärmedizinische Gesellschaft – Fachgruppe Bakteriologie und Mykologie,
15.-17. Juni 2000, Leipzig, Germany
BALTES, N., TONPITAK, W., GERLACH, G.-F., HENNIG-PAUKA, I., HOFFMANN-MOUJAHID, A.,
GANTER, M., ROTHKOTTER, H.J. (2000)
Actinobacillus pleuropneumoniae iron transport and urease activity: effects on bacterial virulence and
host immune response
81st conference of Research Workers in Animal Diseases (CRWAD)
12.-14. November 2000, Chicago, U.S.A.
BALTES, N., HENNIG-PAUKA, I., GERLACH, G.-F.(2001)
Actinobacillus pleuropneumoniae transferrin binding proteins are essential virulence factors in
serotype 7 infection
82nd conference of Research Workers in Animal Diseases (CRWAD)
11.-13. November 2001, St. Louis, U.S.A.
Table of contents
A Introduction.............................................................................15
B Literature review .....................................................................16
B.1 Actinobacillus pleuropneumoniae.......................................................... 16B.1.1 Taxonomy .................................................................................................. 16
B.1.2 Significance and epidemiology................................................................... 16
B.1.3 Infection, disease, and immunity ................................................................ 17
B.1.4 Virulence factors ........................................................................................ 19
B.1.4.1 LPS and capsule ........................................................................................ 19
B.1.4.2 RTX toxins.................................................................................................. 20
B.1.4.3 Outer membrane proteins .......................................................................... 21
B.1.4.4 Urease ....................................................................................................... 21
B.1.4.5 Other factors .............................................................................................. 22
B.2 Regulation of virulence factors............................................................... 22B.2.1 Temperature .............................................................................................. 22
B.2.2 Osmolarity.................................................................................................. 22
B.2.3 pH .............................................................................................................. 23
B.2.4 Quorum sensing......................................................................................... 23
B.2.5 Oxidative stress.......................................................................................... 23
B.2.6 Host-specific inducers ................................................................................ 24
B.3 Molecular mechanisms of virulence factor regulation ......................... 24B.3.1 Two-component systems ........................................................................... 24
B.3.2 Helix-turn-helix proteins ............................................................................. 25
B.3.3 Alternative sigma factors............................................................................ 25
B.3.4 Antisense RNA........................................................................................... 25
B.3.5 Stabilization of transcripts .......................................................................... 26
B.4 Iron in bacterial infection ........................................................................ 26B.4.1 Role of iron................................................................................................. 26
B.4.2 Iron limitation in the host ............................................................................ 26
B.4.3 Iron uptake by bacteria............................................................................... 27
B.4.3.1 Siderophore dependent iron uptake ........................................................... 27
B.4.3.2 Siderophore independent iron uptake ........................................................ 29
B.4.3.3 Iron transport through bacterial membranes .............................................. 30
B.4.4 Regulation of bacterial iron uptake via the Fur repressor protein............... 33
B.5 Dimethylsulfoxide (DMSO) reductase .................................................... 34
B.6 Representational Difference Analysis of cDNA (cDNA RDA) ............... 35
B.7 Infection models for A. pleuropneumoniae ........................................... 36
B.8 Working hypothesis................................................................................. 38
C Materials and methods ...........................................................39
C.1 Chemicals, reagents and equipment...................................................... 39
C.2 Buffers and solutions .............................................................................. 39
C.3 Bacterial cultures ..................................................................................... 39C.3.1 Bacterial strains.......................................................................................... 39
C.3.2 Media and growth conditions ..................................................................... 39
C.3.3 Antibiotic solutions and supplements ......................................................... 40
C.4 Bacteriological methods ......................................................................... 40C.4.1 Urease assay ............................................................................................. 41
C.4.2 Plate bioassay............................................................................................ 41
C.5 Manipulation of nucleic acids ................................................................. 44C.5.1 Plasmids .................................................................................................... 44
C.5.2 Primers....................................................................................................... 44
C.5.3 Isolation of DNA ......................................................................................... 44
C.5.3.1 Plasmid DNA.............................................................................................. 44
C.5.3.2 Total chromosomal DNA of A. pleuropneumoniae ..................................... 44
C.5.4 Polymerase chain reaction......................................................................... 54
C.5.4.1 Preparation of DNA template by colony boiling. ......................................... 54
C.5.5 Representational difference analysis (RDA) of cDNA ................................ 56
C.5.5.1 Preparation of RNA.................................................................................... 56
C.5.5.2 Second strand synthesis ............................................................................ 57
C.5.5.3 Representation of 'tester' and 'driver' ......................................................... 57
C.5.5.4 Preparation of tester and driver.................................................................. 58
C.5.5.5 Hybridization and subsequent PCR ........................................................... 58
C.5.5.6 Isolation of RDA fragments ........................................................................ 58
C.5.6 Pulsed field gel electrophoresis (PFGE) .................................................... 58
C.5.6.1 Isolation of agarose-embedded chromosomal A. pleuropneumoniae
DNA ........................................................................................................... 58
C.5.6.2 Restriction endonuclease digestion of DNA embedded in agarose
plugs .......................................................................................................... 59
C.5.6.3 Pulsed field gel electrophoresis.................................................................. 60
C.5.7 Construction of A. pleuropneumoniae isogenic deletion mutants............... 60
C.5.7.1 Transconjugation from E. coli to A. pleuropneumoniae by filter mating
technique ................................................................................................... 60
C.5.7.2 Sucrose counterselection........................................................................... 61
C.5.8 Nucleic acid detection ................................................................................ 62
C.5.8.1 Southern blotting........................................................................................ 62
C.5.8.2 Northern blotting......................................................................................... 62
C.5.8.3 Labeling of probes with 32P-dCTP.............................................................. 62
C.5.8.4 Southern hybridization ............................................................................... 63
C.5.8.5 Northern hybridization ................................................................................ 63
C.5.8.6 DNA colony blotting.................................................................................... 63
C.5.9 Nucleotide sequencing and sequence analysis.......................................... 64
C.6 Manipulation of proteins ......................................................................... 64C.6.1 Preparation of protein aggregates.............................................................. 64
C.6.2 Determination of protein concentration ...................................................... 65
C.6.3 Preparation of proteins from A. pleuropneumoniae by whole cell lysis ...... 65
C.6.4 Preparation of antisera............................................................................... 66
C.6.4.1 Purification of antisera................................................................................ 66
C.6.5 Protein detection ........................................................................................ 67
C.6.5.1 Western blotting in a tank transfer system ................................................. 67
C.6.5.2 Immunoblotting........................................................................................... 67
C.7 Challenge experiment in the pig ............................................................. 68C.7.1 Challenge experiment timeline................................................................... 68
C.7.2 Origin and housing of the animals.............................................................. 68
C.7.3 Aerosol infection chamber.......................................................................... 68
C.7.4 Preparation of bacteria for aerosolization................................................... 69
C.7.5 Aerosol infection......................................................................................... 69
C.7.5.1 Surveillance of the animals during the experiment..................................... 69
C.7.6 Bronchoalveolar lavage fluid (BALF).......................................................... 71
C.7.6.1 Assessment of bacteriological status of BALF ........................................... 71
C.7.7 Post mortem examination .......................................................................... 72
C.7.7.1 Determination of lung lesion scores ........................................................... 72
C.7.7.2 Bacteriological examination of organ samples ........................................... 72
C.7.8 Enzyme Linked Immunosorbent Assay (ELISA)......................................... 72
C.7.9 Enzyme Linked Immunosorbent (ELI) spot analyses ................................. 73
C.7.10 Statistics..................................................................................................... 74
D Results.....................................................................................75
D.1 Role of urease and ExbB in A. pleuropneumoniae infection ............... 75D.1.1 Challenge experiment with A. pleuropneumoniae AP76 and isogenic
deletion mutants App∆ureC and App∆exbB ............................................... 75
D.1.1.1 Clinical symptoms in infected pigs ............................................................. 75
D.1.1.2 Bacterial reisolation kinetics and pathomorphological changes in
challenged pigs .......................................................................................... 76
D.1.1.3 Systemic and local immune response of challenged pigs. ......................... 76
D.2 Role of transferrin binding proteins in A. pleuropneumoniaeinfection .................................................................................................... 81
D.2.1 Construction of isogenic mutants lacking transferrin binding protein
expression.................................................................................................. 81
D.2.1.1 Transconjugation plasmids ........................................................................ 81
D.2.1.1.1 pTF205EM302 for the introduction of the tbpB deletion ...................... 82
D.2.1.1.2 pTF205mut404 for the introduction of the tbpA deletion...................... 82
D.2.1.2 Construction of isogenic mutants ............................................................... 85
D.2.2 Challenge experiment with A. pleuropneumoniae ∆ureC and isogenic
deletion mutants A. pleuropneumoniae ∆tbpB, A. pleuropneumoniae
∆tbpA and A. pleuropneumoniae ∆tbpBA................................................... 87
D.2.2.1 Clinical symptoms in infected pigs ............................................................. 87
D.2.2.2 Bacterial reisolation kinetics and pathomorphological changes in
challenged pigs .......................................................................................... 87
D.2.2.3 Systemic immune response of challenged pigs ......................................... 88
D.3 Identification of genes expressed in vivo .............................................. 88D.3.1 Representational Difference Analysis of cDNA .......................................... 88
D.3.2 Isolation of larger fragments containing RDA fragments ............................ 91
D.3.2.1 Construction of a sorted A. pleuropneumoniae genomic library................. 91
D.3.2.2 Colony blotting ........................................................................................... 92
D.3.3 Localization of RDA fragments on the A. pleuropneumoniae genomic
map ............................................................................................................ 92
D.3.4 Characterization of large fragments ........................................................... 94
D.3.5 A. pleuropneumoniae dimethylsulfoxide (DMSO) reductase subunit
DmsA ......................................................................................................... 95
D.3.5.1 Sequence analyses.................................................................................... 95
D.3.5.2 dmsA transcription ..................................................................................... 97
D.3.5.3 A. pleuropneumoniae DmsA expression .................................................... 97
D.3.5.4 Presence of dmsA in serotype reference strains........................................ 99
D.3.6 A. pleuropneumoniae ferric hydroxamate uptake receptor (FhuA)
homologue ............................................................................................... 100
D.3.6.1 Sequence analyses.................................................................................. 100
D.3.6.2 fhuA transcription ..................................................................................... 103
D.3.6.3 Presence of fhuA in serotype reference strains ....................................... 103
D.3.6.4 A. pleuropneumoniae FhuA expression ................................................... 105
D.3.7 Construction of deletion mutants.............................................................. 107
D.3.7.1 Construction of pBMKD1 for the introduction of a dmsA deletion into A.
pleuropneumoniae ................................................................................... 107
D.3.7.2 Construction of pECF2 for the introduction of an fhuA deletion into A.
pleuropneumoniae ................................................................................... 108
D.3.7.3 Construction and analysis of isogenic dmsA and fhuA deletion mutants . 111
D.3.8 Functional analysis of FhuA ..................................................................... 113
D.3.9 Role of DmsA and the FhuA homologue in A. pleuropneumoniae
infection.................................................................................................... 113
D.3.9.1 Aerosol infection experiment.................................................................... 113
D.3.9.2 Post mortem examination ........................................................................ 115
D.3.9.3 Systemic immune response ..................................................................... 115
E Discussion ............................................................................118
E.1 Role of urease and ExbB in A. pleuropneumoniae infection ............. 118
E.2 Role of transferrin binding proteins in A. pleuropneumoniaeinfection .................................................................................................. 121
E.3 Identification of genes expressed in vivo ............................................ 122
F Summary ...............................................................................130
G Zusammenfassung ...............................................................132
H References ............................................................................134
I Appendix ...............................................................................173
I.1 Chemicals ............................................................................................... 173
I.2 Buffers and solutions ............................................................................ 176
I.3 Sequence of A. pleuropneumoniae dmsA ........................................... 180
I.4 Sequence of A. pleuropneumoniae fhuA ............................................. 185
I.5 Animal experiments ............................................................................... 190
I.6 Index of figures ...................................................................................... 196
I.7 Index of tables ........................................................................................ 197
Acknowledgements...........................................................................198
Danksagung.......................................................................................199
List of abbreviations
® registered trademarkA. bidest. Aqua bidestillataA. dest. Aqua destillataA. pleuropneumoniae Actinobacillus pleuropneumoniaebp base pairsBq BecquereldATP deoxyadenosine triphosphatedCTP deoxycytosine triphosphatedGTP deoxyguanosine triphosphatedTTP deoxythymidine triphosphateDa DaltonDNA deoxyribonucleic acidDNAse deoxyribonucleasedNTP deoxynucleotide triphosphateEDTA ethylenediamine tetraacetic acidFig. Figureh hourk kilokb kilo base pairsLB Luria BertaniM Molarm milliµ micromin minuten nanoNAD nicotinamide adenine dinucleotideODxxx optical density at xxx nanometersORF open reading framePCR polymerase chain reactionPFGE pulsed field gel electrophoresisPPLO pleuropneumonia like organismRNA ribonucleic acidRNase ribonucleaserpm rounds per minutesec secondUV ultravioletV voltVol volumev/v volume by volumew/v weight by volume
INTRODUCTION
15
A Introduction
Porcine Actinobacillus Pleuropneumonia (PAP) is an economically important
respiratory disease of fattening pigs occurring worldwide. Disease symptoms vary
from acute pleuropneumonia characterized by necrotic and hemorrhagic lesions in
young animals to chronic disease with reduced weight gains in older animals;
subclinical infections without disease symptoms can also occur. Infected animals can
carry and shed the pathogen for up to several months and thus be the potential
source of new outbreaks of the disease.
Actinobacillus (A.) pleuropneumoniae, the cause of PAP, is a facultatively anaerobic
gramnegative rod and belongs to the family of Pasteurellaceae. It is considered to be
an obligatory and non-invasive, strictly extracellular parasite of the porcine respiratory
tract that resides primarily on the epithelium of bronchioli and alveoli. Several poten-
tial virulence factors such as the Apx toxins (A. pleuropneumoniae-specific RTX-
toxins), transferrin-binding proteins, and a capsule have been identified. Different
isoforms of these factors are closely associated with the different A. pleuropneumo-
niae serotypes. The resulting clonal population structure of the species is highly
relevant clinically, as the resulting antigenic heterogeneity is responsible for the
limited crossprotection induced by vaccination with common bacterin vaccines.
To date, the factors enabling A. pleuropneumoniae to initially establish infection and
to persist in the host are not fully understood. Although it has been shown that
contact with the host induces the expression of novel proteins, the nature of these
antigens has not been elucidated. Novel approaches like "Signature-Tagged
Mutagenesis" (STM) an "In Vivo Expression Technique" (IVET) have served to
identify several factors relevant in A. pleuropneumoniae virulence in acute infection.
The mechanisms involved in persistence, however, have not been elucidated. This
therefore was the primary goal of this study. Here, one focus was on the
determination of the in vivo relevance of A. pleuropneumoniae iron uptake via the
transferrin binding proteins; the second focus was the identification of alternative iron-
uptake mechanisms and other potential virulence factors supporting A. pleuropneu-
moniae persistence in the host.
LITERATURE REVIEW
16
B Literature review
B.1 Actinobacillus pleuropneumoniae
B.1.1 Taxonomy
Actinobacillus (A.) pleuropneumoniae is a gramnegative non-motile rod that belongs
to the family of Pasteurellaceae. A large majority of A. pleuropneumoniae are nicotine
amide dinucleotide (NAD) dependent and grow in minute and hemolytic colonies on
sheep blood agar. A CAMP-like phenomenon can be observed in the presence of a
Staphylococcus aureus strain (MANNHEIM 2002). A. pleuropneumoniae was
originally classified as Haemophilus (H.) pleuropneumoniae (SHOPE et al. 1964). H.
parahaemolyticus was another synonym, based on the similarity between A.
pleuropneumoniae and the human pathogen H. haemolyticus (NICOLET 1968).
Finally, the species was transferred to the genus Actinobacillus based on DNA
studies showing a high homology to A. lignieresii (POHL et al. 1983).
Not all A. pleuropneumoniae strains require NAD for growth. Based on NAD-depen-
dance, A. pleuropneumoniae strains are grouped into biotypes 1 and 2, where bio-
type 1 is NAD dependent, biotype 2 is NAD-independent. (POHL et al. 1983; NICO-
LET 1992; NIELSEN et al. 1997b). To date, twelve serotypes for biotype 1 and six for
biotype 2 are recognized (BOSSE et al. 2002). The A. pleuropneumoniae biotype 1
serotypes 1 and 5 are subdivided into subtypes a and b, respectively. Recently, a
biotype 1 strain has been proposed as serotype 15 (BLACKALL et al. 2002). Cross-
reactions among serotypes are common since O antigens of serotypes 1, 9 and 11, 4
and 7 as well as 3, 6 and 8 are almost identical (RYCROFT and GARSIDE 2000).
Actinobacillus pleuropneumoniae biotype 1 strains are more virulent than biotype 2
strains, and from biotype 1 strains, serotypes 1, 5, 9 and 10 have been observed to
be the most virulent, although these findings have not been confirmed experimentally
(HAESEBROUCK et al. 1997).
B.1.2 Significance and epidemiology
Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia,
was first identified in Great Britain in 1957 by Pattison and coworkers (NICOLET
LITERATURE REVIEW
17
1992). It is highly host specific for pigs, although it has occasionally been isolated
from lambs (NIELSEN 1986; HERVAS et al. 1996). Actinobacillus pleuropneumoniae
is distributed worldwide; however, serotypes and biotypes are not evenly distributed.
For example, while serotypes 1, 5, and 7 prevail in the United States, serotypes 1, 3,
and 5 are most frequently isolated in Canada, and serotypes 1, 2, 3, 5, and 9 are the
serotypes of importance in Europe (BLAHA 1992; CHIERS et al. 2002).
The primary route of infection is via droplets at close range (NICOLET et al. 1969), by
shared air space or by direct contact with infected pigs (TAYLOR 1995;
TORREMORELL et al. 1997; JOBERT et al. 2000). Tenacity of the organism is low
and results in limited survival times of the bacterium in the environment unless hu-
midity is high, temperatures are low or the organism is protected by organic matter.
Thus, disinfection is highly efficacious, and transmission via personnel or fomites is of
limited importance (FENWICK and HENRY 1994; TAYLOR 1995). Outbreak of the
disease is facilitated by crowding, poor hygiene, poor ventilation and sudden
changes in environmental conditions (NICOLET 1992; FENWICK and HENRY 1994).
A major risk is posed by clinically healthy carrier animals which, upon introduction
into an A. pleuropneumoniae-free herd, may be the cause of a severe outbreak
(RYCROFT and GARSIDE 2000). The economic importance of the disease is due to
deaths in acute disease as well as reduced growth rates of convalescent and chroni-
cally infected pigs (STRAW et al. 1989).
B.1.3 Infection, disease, and immunity
Porcine pleuropneumonia may affect pigs of all ages, although pigs aged 10-16
weeks are most susceptible (FENWICK and HENRY 1994). The disease is
characterized by fibrinous and necrotizing pleuropneumonia, sometimes associated
with pulmonary hemorrhage and affection of pericardium and joints. In the chronic
state, lung lesions present themselves as sequestered abscesses with persistent
pleural adhesions (MATSCHULLAT 1983; BERTRAM 1985; LIGGETT et al. 1987;
DIDIER et al. 2002).
Outbreaks with high mortality often occur shortly after weaning (NICOLET 1992).
Deaths occur mostly within the first four days after infection (SEBUNYA and
SAUNDERS 1983), and surviving animals often do not fully recover, surviving with
LITERATURE REVIEW
18
residual lung lesions and pleural adhesions contributing to secondary bacterial infec-
tions and retarded growth due to interference with respiratory functions. In older ani-
mals, disease occurs more sporadically, with an increased frequency of chronic or
subclinical infections. Such animals may serve as reservoirs, as the disease is fre-
quently not correctly diagnosed (FENWICK and HENRY 1994).
The mechanisms of protective immunity against A. pleuropneumoniae are not fully
understood (RYCROFT and GARSIDE 2000). Newborn piglets are protected by
maternal antibodies (NIELSEN 1975; CRUIJSEN et al. 1992) directed primarily
against Apx toxins. After weaning, the level of maternal antibodies declines, thereby
facilitating infection in animals aged 10-16 weeks. After infection, antibody titers are
first detectable after ten days, reach a maximum after 3 to 4 weeks, and persist for
several months (HAESEBROUCK et al. 1997). This humoral immune response is
thought to be crucial in the defense against A. pleuropneumoniae infection, with
immunoglobulin (Ig) G being of major importance (DEVENISH et al. 1990; BOSSE et
al. 1992); this hypothesis is supported by passive transfer experiments with serum
from convalescent pigs, resulting in specific serum IgG titers similar to those in
immunized pigs (BOSSE et al. 1992). In experimental infections involving aerosol
delivery of live A. pleuropneumoniae, it was shown that levels of IgA, IgM and IgG
levels as well as lymphocytes and plasma cells increased significantly in bronchoal-
veolar lavage fluid (BALF, DELVENTHAL et al. 1992; HENSEL et al. 1994b).
Natural infection results in protective immunity towards the homologous serotype and
variable immunity towards heterologous serotypes (NIELSEN 1985; JOLIE et al.
1995; CRUIJSEN et al. 1995; HENSEL et al. 1996), with the cross-serotype protec-
tion likely being the result of an immune response to cross-reacting antigens such as
lipopolysaccharide (LPS) components, outer membrane proteins, and cytolysins
(JOLIE et al. 1994).
Current commercial vaccines are either whole cell bacterins or subunit vaccines
containing various combinations of serotype 1, 2, 5, 7 and 9 preparations. These
vaccines can reduce mortality or prevent the occurrence of clinical signs, but will not
prevent colonization or, in some cases, lung lesions (NICOLET 1992; FENWICK and
HENRY 1994; HAESEBROUCK et al. 1997; CHIERS et al. 1998).
LITERATURE REVIEW
19
More recently, a novel approach to vaccine construction has been developed, using
bacterial envelopes, so-called ghosts. A. pleuropneumoniae ghost formation is
achieved by expression of the phage PhiX174-derived protein E, resulting in trans-
membrane pore formation and subsequent loss of cytoplasmic contents and DNA
(SZOSTAK et al. 1996). The intramuscular application of A. pleuropneumoniae sero-
type 9 ghosts conferred immunity against homologous serotype challenge and pre-
vented colonization (HENSEL et al. 2000). A subunit vaccine constructed from A.
pleuropneumoniae serotype 2 and 9 cultures grown under iron-deficient conditions
abolished or strongly reduced clinical symptoms, but did not prevent colonization
(GOETHE et al. 2000). However, to date there is no vaccine commercially available
for A. pleuropneumoniae that protects against all serotypes, confers a protective
immune response after a single application, and allows the differentiation between
immune responses to infection and vaccination.
B.1.4 Virulence factors
Virulence factors are defined as bacterial products that aid in growth or survival of a
bacterium in the host, contributing to infection and disease (MEKALANOS 1992;
MAHAN et al. 1996).
B.1.4.1 LPS and capsule
A. pleuropneumoniae possesses lipopolysaccharide (LPS), fimbrial, and capsular
adhesins (INZANA et al. 1988; BELANGER et al. 1990; PARADIS et al. 1994;
ZHANG et al. 2000) responsible for the initial attachment of the organism to
respiratory epithelium. LPS contains the hydrophobic lipid A which is responsible for
LPS toxicity through stimulation of cytokine release (BAARSCH et al. 1995), and a
hydrophilic part consisting of a core oligosaccharide and a heteropolysaccharide side
chain (O-antigen). LPS has been suggested to be the main adhesin of A.
pleuropneumoniae (PARADIS et al. 1994) and is responsible for the binding of
hemoglobin (BELANGER et al. 1995). It is also partly responsible for colony
morphology. Long O-antigen side chains result in "smooth", short chains in "rough"
colony morphology, with the "smooth" type showing more efficient adherence to
tracheal rings in vitro (BELANGER et al. 1990). The capsule of A. pleuropneumoniae
consists of non-branching polysaccharide chains built from repeating disaccharides,
LITERATURE REVIEW
20
and contains mainly hexosamine (85%) as well as phosphate, proteins, nucleic acids
(0.2%) and endotoxin 0.01% (INZANA 1987). The capsule in itself is non-toxic
(FENWICK and OSBURN 1986), but the thickness of the capsule has an influence on
virulence in that strains with thicker capsules are more virulent (JENSEN and
BERTRAM 1986; ROSENDAL and MACINNES 1990). This may be explained by the
higher extent of steric hindrance of antibodies directed against subcapsular somatic
antigens (INZANA et al. 1988).
B.1.4.2 RTX toxins
RTX toxins are pore-forming cytolytic proteins present in many gram-negative patho-
gens (WELCH 1991) and characterized by repetitive glycin-rich sequences, nine
amino acids in length (RTX - repeats in toxin), near the carboxy terminus. They are
capable of lysing erythrocytes and/or nucleated cells (THOMPSON et al. 1993). A
hydrophobic region is responsible for pore formation in the targeted membrane, and
a carboxyterminal signal sequence mediates transport through the bacterial cell
membranes by type I secretion systems (GENTSCHEV et al. 2002). To date, four
RTX toxins have been characterized in A. pleuropneumoniae and designated as
ApxI-ApxIV. The distribution of Apx toxins varies between serotypes. The genes en-
coding ApxIV are present in all serotypes, but expression has not been observed in
culture (SCHALLER et al. 1999). The ApxII toxin is produced by all serotypes except
serotype 10, ApxI by serotypes 1, 5, 9, 10, and 11, and ApxIII by serotypes 2, 3, 4, 6,
and 8 (KAMP et al. 1991; FREY et al. 1993; JANSEN et al. 1994). Thus, nearly all
serotypes express three different Apx toxins, which is unusual for bacteria expressing
RTX toxins (WELCH 1991). The ApxI toxin (105 kDa) is strongly hemolytic and cyto-
toxic (FREY and NICOLET 1988; KAMP et al. 1991), the ApxII toxin (120 kDa) is
weakly cytotoxic and weakly hemolytic (FREY et al. 1994), and the ApxIII toxin (103-
105 kDa) is non-hemolytic but strongly cytotoxic to alveolar macrophages and poly-
morphonuclear granulocytes (PMNs, KAMP et al. 1991; RYCROFT et al. 1991). Re-
combinant Apx IV toxin is weakly hemolytic (SCHALLER et al. 1999). The ApxI, ApxII
and recombinant ApxIV toxins are able to induce a CAMP-like phenomenon (FREY
et al. 1994; SCHALLER et al. 1999). In A. pleuropneumoniae infection, Apx toxins
play a major role; inoculation of pigs with culture supernatant or recombinant Apx
LITERATURE REVIEW
21
toxins is able to cause pleuropneumonic symptoms with only minor differences to an
infection induced by viable A. pleuropneumoniae (VAN LEENGOED and KAMP
1989; KAMP et al. 1997). Immunization with ApxI toxin from A. pleuropneumoniae
serotype 1 prevents acute death, but still allows colonization and development of
chronic pleuropneumonia (DEVENISH et al. 1990).
B.1.4.3 Outer membrane proteins
Some outer membrane proteins (OMPs) of A. pleuropneumoniae have been
associated with virulence. A maltose-inducible protein that is recognized by serum
from convalescent pigs has been identified and shows structural similarity to a porin
protein of Pasteurella multocida (DENEER and POTTER 1989). A constitutively ex-
pressed 40 kDa lipoprotein, OmlA, was first identified in serotype 1 (GERLACH et al.
1993) and serotype 5 (BUNKA et al. 1995); subsequently, its sequence has been
determined for all A. pleuropneumoniae reference strains via PCR (GRAM and
AHRENS 1998).
Under iron restricted conditions, A. pleuropneumoniae expresses two outer mem-
brane proteins (NIVEN et al. 1989) that bind porcine transferrin. These proteins will
be presented in B.4.3.2.
B.1.4.4 Urease
Urease catalyzes hydrolysis of urea to ammonia, a preferred nitrogen source for
bacteria, and carbamic acid. The role of urease in A. pleuropneumoniae infection has
not been fully elucidated. It has been demonstrated that urease is not required for A.
pleuropneumoniae infection (TASCON CABRERO et al. 1997), and a spontaneously
urease-negative A. pleuropneumoniae wild type strain has been isolated is a case of
acute pleuropneumonia (BLANCHARD et al. 1993). There is no experimental evi-
dence available for the role of urease in chronic A. pleuropneumoniae infection, how-
ever, there is experimental evidence that urease may contribute to the ability of A.
pleuropneumoniae to establish infection. A clinical challenge trial showed that, at low
challenge doses, urease negative mutants were unable to establish infection
(BOSSE and MACINNES 2000). By increasing the pH value, urease might contribute
to the creation of more favorable conditions for survival and growth in the mucus
LITERATURE REVIEW
22
layer covering the airway epithelium, thereby counteracting the decrease of pH in
acute inflammatory processes.
B.1.4.5 Other factors
Other virulence factors of A. pleuropneumoniae include an IgA protease that has
been suggested to facilitate colonization of the lower respiratory tract (KILIAN et al.
1979; NEGRETE-ABASCAL et al. 1994) and a Cu,Zn superoxide dismutase that pro-
tects A. pleuropneumoniae against oxygen radicals in vitro (LANGFORD et al. 1996),
but is not required in A. pleuropneumoniae virulence (SHEEHAN et al. 2000). The
ohr gene, encoding an organic hydroperoxide reductase, was recently identified
(SHEA and MULKS 2002) and could play a role in detoxification of organic peroxides
generated during infection.
B.2 Regulation of virulence factors
The expression of bacterial virulence determinants is often regulated in response to
environmental signals (MEKALANOS 1992), enabling the bacterium to flexibly adapt
to the drastically different conditions in the host organism. The signals inducing ex-
pression of virulence factors are not completely understood, but some have been
characterized and will be presented here.
B.2.1 Temperature
The transition from environment to host organism is associated with a drastical
change in temperature, which is responded to with changes in virulence factor
expression by many bacteria (MEKALANOS 1992). For example, Shigella spp.
respond to a temperature shift from 30°C to 37°C by expressing a range of plasmid-
encoded virulence factors that are essential for invasion of colon epithelium as well
as for replication inside cells and spreading to adjacent cells. These factors are
transcriptionally regulated, with regulatory genes being situated both on the plasmid
and on the chromosome (MAURELLI and SANSONETTI 1988; HALE 1991).
B.2.2 Osmolarity
The osmolarity of the culture medium influences the supercoiling of bacterial DNA.
High osmolarity reinforces supercoiling, thereby influencing expression of various
genes (GALAN and CURTISS III 1990). In Vibrio cholerae, expression of cholera
LITERATURE REVIEW
23
toxin, Tcp pili, and other virulence factors reaches optimum levels at an osmolarity
that is physiological in host tissue (MILLER and MEKALANOS 1988). In Pseudomo-
nas aeruginosa, the expression of the alginate capsule is influenced by osmolarity in
some strains (DERETIC et al. 1989).
B.2.3 pH
In Salmonella (S.) Typhimurium the pH value may serve as a signal for expression of
virulence factors, aiding in survival under the low pH conditions in the stomach and in
phagolysosomes (GORDEN and SMALL 1993; FOSTER 1999).
B.2.4 Quorum sensing
Quorum sensing describes communication between bacteria by means of small
autoinducer signal molecules, homoserine lactones, that are secreted by some
gramnegative bacteria. As bacterial population density increases, concentration of
signal molecules is raised until a threshold is reached and gene regulation
mechanisms are triggered (WHITEHEAD et al. 2001). First discovered in
bioluminescent marine Vibrio fischeri (FUQUA et al. 1994; FUQUA et al. 1996),
quorum sensing systems have since been identified in a number of gramnegative
and grampositive pathogens. In Pseudomonas aeruginosa, quorum sensing is
required for normal biofilm formation and virulence (PARSEK and GREENBERG
2000). Not all quorum sensing systems utilize homoserine lactones for interbacterial
communication. In Staphylococcus spp., the agr quorum sensing system uses short
signal peptide molecules and is responsible for the regulation of virulence-associated
exoproteins in Staphylococcus aureus (KLEEREBEZEM et al. 1997; OTTO et al.
1998; DUFOUR et al. 2002). Recently, quorum sensing autoinducer activity was
reported in Mannheimia haemolytica and other Pasteurella spp., including A.
pleuropneumoniae (MALOTT and LO 2002).
B.2.5 Oxidative stress
The oxidative stress caused by the release of cytotoxic oxygen radicals by macro-
phages induces proteins that protect the bacterium from these toxic compounds. The
OxyR protein of E. coli is regulated in this manner and, in turn, activates the tran-
LITERATURE REVIEW
24
scription of at least nine genes in response to hydrogen peroxide stress, among them
a catalase and a hydroperoxidase (MAHAN et al. 1996).
B.2.6 Host-specific inducers
Recognition of host-specific signals is an effective mechanism for the induction of
virulence factor expression. Phenolic compounds that accumulate in infected roots of
plants induce virulence factor expression in Agrobacterium tumefaciens in conjunc-
tion with various monosaccharides (ANKENBAUER and NESTER 1990).
The enhancement of growth by interleukin-1 has been reported in E. coli (PORAT et
al. 1991), and tumor necrosis factor alpha (TNF-α) has been shown to enhance inva-
sion of HeLa cells by Shigella flexneri through interaction between TNF-α-bacterium-
complexes and the TNF-α receptors of the cells (LUO et al. 1993). However, cytokine
receptors have not been identified in bacteria, and the underlying mechanisms re-
main unclear.
Host-induced expression of virulence factors has been demonstrated in A.
pleuropneumoniae as the result of the addition of bronchoalveolar lavage fluid
(BALF) to culture medium. The addition of BALF from animals infected with A.
pleuropneumoniae induced expression of the small transferrin binding protein TbpB
and a number of yet unidentified proteins. TbpB is also expressed under iron-
restricted conditions (TEUTENBERG-RIEDEL 1998). The ApxIV toxin that was
described recently is not expressed in vitro; however, experimentally infected pigs
produce antibodies against it, indicating that ApxIV is expressed in vivo (SCHALLER
et al. 1999).
B.3 Molecular mechanisms of virulence factor regulation
B.3.1 Two-component systems
Many functions in gramnegative bacteria are regulated by two-component regulatory
systems that confer the ability to sense, and respond to, environmental stimuli. These
systems consist of a transmembrane sensory protein capable of sensing an external
signal, and a cytoplasmic regulatory protein that acts as a transcriptional activator or
repressor. The former consists of an extracellular sensory domain and a histidine
kinase that is autophosphorylated in response to a stimulus, resulting in transfer of a
LITERATURE REVIEW
25
phosphate group to the response regulator protein, thereby altering its DNA binding
properties (MILLER et al. 1989). Examples of two-component regulatory systems are
the BvgA/BvgS system in Bordetella pertussis, regulating the expression of fimbriae
and toxins (UHL and MILLER 1996), and the ToxR/ToxS system in Vibrio cholerae
(DIRITA 1992). A few examples for the stimuli which activate these systems are
given in the paragraphs above.
B.3.2 Helix-turn-helix proteins
Transcriptional activators from the AraC and LysR families are proteins containing a
helix-turn-helix motif that binds to regulatory regions upstream of regulated genes
(CALDWELL and GULIG 1991; FINLAY and FALKOW 1997). An example for an
AraC-like protein system is VirF (LcrF) of Yersinia (Y.) spp. (HOE et al. 1992;
SKURNIK and TOIVANEN 1992). VirF is a global regulator of Yops (Yersinia outer
proteins), virulence factors that are membrane-bound or secreted. A LysR-like sys-
tem, SpvR, is found in Salmonella spp. (CALDWELL and GULIG 1991); it is plasmid-
encoded and regulates plasmid-encoded virulence factors responsible for long term
survival of Salmonella in mice (GULIG et al. 1993).
B.3.3 Alternative sigma factors
Alternative sigma factors bound to RNA polymerase recognize specific promoters,
enabling RNA polymerase to bind to the template DNA. An important group of alter-
native sigma factors are heat shock sigma factors like σ32. The E. coli rpoH gene en-
codes σ32; the protein is unstable at 30°C, with its stability being enhanced and its
translation rate being considerably higher at 42-45°C. σ32 recognizes promoters of
heat shock genes, resulting in upregulated expression of heat shock proteins like
Hsp70 and Hsp60, which are chaperones involved in correct folding and transloca-
tion of proteins (ANG et al. 1991; KNIPPERS et al. 2002).
B.3.4 Antisense RNA
Regulation through antisense RNA occurs in control of transcription, translation,
plasmid copy number, and in the shifting between a lytic and a lysogenic life cycle in
bacteriophages. Antisense RNA binds to complementary mRNA, thus inhibiting the
binding of RNA polymerase, regulatory proteins or ribosomes (INOUYE and
LITERATURE REVIEW
26
DELIHAS 1988; SIMONS and KLECKNER 1988). In Vibrio anguillarum, a plasmid-
encoded antisense RNA (RNAα) is involved in negative regulation of iron-regulated
genes (WALDBESER et al. 1995).
B.3.5 Stabilization of transcripts
Stabilization of transcripts is an effective way of influencing translation rates. The
termination factor Rho of E. coli has been found to stabilize mRNA, possibly by bind-
ing to it. Rho-negative mutants of E. coli show shorter halflife of total mRNA
(SOZHAMANNAN and STITT 1997). A similar effect has been identified for an A.
pleuropneumoniae analogue of Rho. Recombinant RhoAP stabilizes the mRNA of
tbpBA, the transferrin binding protein operon (THIEDE 1998).
B.4 Iron in bacterial infection
B.4.1 Role of iron
Iron is the fourth most abundant element on earth (CROSA 1997), and it is essential
for growth of virtually all cells with the exception of lactobacilli that utilize manganese
and cobalt as biocatalysts in place of iron (GUERINOT 1994). Iron is required for im-
portant cell functions, such as the transport and storage of oxygen, as a catalyst in
electron transport processes, and as cofactor for enzymes in DNA synthesis (LITWIN
and CALDERWOOD 1993; EARHART 1996). An excess of free iron is toxic as a re-
sult of the Fenton reaction which involves the iron-catalyzed production of toxic
hydroxyl radicals. Since extrusion mechanisms are unknown in bacteria, iron uptake
is strictly regulated (MIETZNER and MORSE 1994). In the presence of oxygen, fer-
rous iron is oxidized to the ferric state and may form ferric hydroxide which is quite
stable and also insoluble in aqueous solutions at neutral or alkaline pH, thus ren-
dering it inaccessible to bacteria (LITWIN and CALDERWOOD 1993; GUERINOT
1994). Bacteria have developed a multitude of systems for iron uptake that, due to
iron being essential for bacterial growth, are closely related to virulence (MARTINEZ
et al. 1990).
B.4.2 Iron limitation in the host
In the mammalian host, the majority of iron is located intracellularly, bound to proteins
such as hemoglobin, myoglobin or cytochrome c, or stored in ferritin or heme. Ex-
LITERATURE REVIEW
27
tracellular iron is usually tightly attached to the high-affinity iron-binding proteins,
transferrin and lactoferrin. The concentration of freely available iron is approximately
10-12 µM, which is much lower than the concentration of 0.05-0.5 µM required for
bacterial growth (MARTINEZ et al. 1990; GRIFFITHS 1991). Additionally, infection
triggers hypoferremia, reducing plasma iron levels by up to 50%. The mechanisms
are thought to be mediated by interleukin-1 which is released by macrophages or
monocytes after stimulation by microorganisms or their products. Suggested mecha-
nisms are the reduction of plasma transferrin levels and the release of apo-lactoferrin
by polymorphonuclear granulocytes. Apo-lactoferrin shows a higher affinity to iron
than transferrin, and therefore, iron can be removed from the extracellular space by
cells of the reticuloendothelial system (WEINBERG 1984). Also, increased ferritin
synthesis serves to decrease the release of tissue iron to transferrin. On the intracel-
lular level, where iron levels are higher than outside of the cells, expression of trans-
ferrin receptors can be downregulated to lower iron uptake and thus make less iron
available to intracellular pathogens (WOOLDRIDGE and WILLIAMS 1993).
B.4.3 Iron uptake by bacteria
To overcome iron limitation in the host, bacteria possess a variety of iron-acquisition
systems to compete with host iron-binding systems, either by directly chelating iron
from host sources or by utilizing iron-binding compounds from the host (LITWIN and
CALDERWOOD 1993; PAYNE 1993).
B.4.3.1 Siderophore dependent iron uptake
Siderophores are small (400-1000 Da), high-affinity iron chelators synthesized by
microorganisms including bacteria under iron-deficient conditions and released into
the environment. They are capable of removing ferric iron from insoluble complexes
or from high affinity host iron-binding compounds like transferrin and lactoferrin. The
siderophore-iron complexes are then bound by specific receptors on the bacterial cell
surface and internalized (BAGG and NEILANDS 1987; BRIAT 1992). The majority of
siderophores belong to two chemical classes, the catechols and the hydroxamates
(LITWIN and CALDERWOOD 1993; MIETZNER and MORSE 1994; EARHART
1996). The prototype catecholate siderophore, enterobactin, is found in many
members of the family of Enterobacteriaceae. The synthesis of enterobactin involves
LITERATURE REVIEW
28
the entABC and entDEFG genes located on the chromosomal ent gene cluster
(BULLEN et al. 1978; CROSA 1989).
The prototype of the hydroxamate class of siderophores, Aerobactin, was originally
isolated from Aerobacter aerogenes. It is a conjugate of 6 (N-acetyl-N-
hydroxyamino)-2aminohexanoic acid and citric acid. The genes required for aerobac-
tin synthesis are either located on the ColV-K30 plasmid or on the chromosome.
Aerobactin synthesis is associated with invasiveness in E. coli and in human patho-
gens such as Shigella flexneri (MIETZNER and MORSE 1994). A well-characterized
siderophore from Pseudomonas aeruginosa is pyochelin, which is expressed upon
induction by respiratory mucus from cystic fibrosis patients (WANG et al. 1996).
Some bacteria that do not produce siderophores express receptors for siderophores
synthesized by other pro- and eukaryotic microorganisms. For example, Neisseria
(N.) gonorrhoeae, N. meningitidis and Listeria (L.) monocytogenes are capable of
using siderophores produced by other organisms as their sole iron source
(ANDERSON et al. 1994; GENCO and DESAI 1996; COULANGES et al. 1997).
Probably the best characterized siderophore receptor for non-bacterial siderophores
is the ferrichrome receptor FhuA in E. coli (BRAUN et al. 1973; WAYNE and
NEILANDS 1975). Ferrichrome is a hydroxamate siderophore synthesized by fungi
from the genera Aspergillus, Penicillium and Ustilago (NEILANDS 1984). Structurally,
it is a cyclic hexapeptide composed of three glycine and three modified ornithine
residues that bind ferric iron via hydroxamate groups (VAN DER HELM et al. 1980).
The ferrichrome receptor of E. coli, FhuA, is TonB/ExbBD1-dependent (SCHOFFLER
and BRAUN 1989; GUNTER and BRAUN 1990), and also serves as the receptor for
colicin M and phagesT1, T5 and φ80 (LAZDUNSKI et al. 1998). Similar receptors are
found in Vibrio cholerae (ROGERS et al. 2000), Campylobacter jejuni (GALINDO et
al. 2001) Rhizobium leguminosarum (YEOMAN et al. 2000) and Bradyrhizobium
japonicum (LEVIER and GUERINOT 1996). A common feature among these species
is the transcriptional upregulation of the fhuA gene or the respective fhuA homo-
1 B.4.3.3
LITERATURE REVIEW
29
logues under iron-deficient conditions (HANTKE and BRAUN 1975). In E.coli, four
fhu genes are involved in hydroxamate uptake. These genes are organized in one
operon in the order fhuA-fhuC-fhuD-fhuB (FECKER and BRAUN 1983). The periplas-
mic FhuD protein binds the ferrisiderophore upon its TonB mediated transport
through FhuA and transfers it to FhuB, which is responsible for the transport through
the cytoplasmic membrane. FhuC, situated on the cytoplasmic side of the membrane,
shows characteristics of an ATP binding protein, and it is proposed that ATP hydroly-
sis provides the energy for transport through the cytoplasmic membrane (BRAUN et
al. 1991, Fig. 1).
Siderophore utilization hast been demonstrated in A. pleuropneumoniae, and appar-
ently, some strains are capable of producing siderophores that are neither catecho-
lates nor hydroxamates (DIARRA et al. 1996). To date, a siderophore receptor has
not been identified in A. pleuropneumoniae. Western blot analysis using two mono-
clonal antibodies directed against the C- and N- termini of E. coli FhuA failed to de-
tect cross-reacting A. pleuropneumoniae outer membrane proteins (DIARRA et al.
1996).
B.4.3.2 Siderophore independent iron uptake
Citrate may serve as a weak chelator for iron, and although high concentrations of
citrate are required for effective binding, many bacteria have developed uptake sys-
tems for ferric dicitrate, for example E. coli, L. monocytogenes and pathogenic
Neisseria spp. Derepression of the receptor requires the presence of citrate in the
extracellular space (WOOLDRIDGE and WILLIAMS 1993). In E. coli, the responsible
fecABCDE genes are TonB-dependent (B.4.3.3), while in N. gonorrhoeae, iron
dicitrate uptake is independent from TonB (MARTINEZ et al. 1990; GENCO and
DESAI 1996; COULANGES et al. 1997).
A number of bacteria can extract iron from heme or hemoglobin present in serum due
to hemolysis. Escherichia coli and Yersinia spp. are merely able to use iron from
heme while N. meningitidis, N. gonorrhoeae, H. influenzae, Vibrio cholerae and
Campylobacter jejuni as well as some A. pleuropneumoniae strains can utilize both
heme and hemoglobin (WOOLDRIDGE and WILLIAMS 1993; GUERINOT 1994;
BELANGER et al. 1995). Free hemoglobin is rapidly bound by haptoglobin, free
LITERATURE REVIEW
30
heme is bound by hemopexin and albumin which are inaccessible for most
microorganisms; however, N. meningitidis, N. gonorrhoeae, and H. influenzae are
capable of utilizing some of these compounds as well (GENCO and DESAI 1996;
MACIVER et al. 1996).
The ability to bind transferrin and lactoferrin and to obtain iron from them is wide-
spread among members of the families of Pasteurellaceae and Neisseriaceae. Up-
take of transferrin-bound iron is mediated by a two-component receptor protein com-
plex situated in the outer membrane. The larger transferrin binding protein, TbpA
(also TfbB or Tbp1) has a molecular mass of approximately 100 kDa, the smaller
one, TbpB (TfbA, Tbp2) has a molecular mass of 60-85 kDa. Lactoferrin receptor
proteins are designated as LbpB and LbpA. These bacterial transferrin and lactoferrin
receptors are strictly specific for the transport proteins of the respective natural host
(SCHRYVERS and LEE 1989; GERLACH et al. 1992a; GONZALEZ et al. 1995;
GRAY-OWEN and SCHRYVERS 1996). Uptake of transferrin-bound iron is TonB-
dependent in N. gonorrhoeae (BISWAS et al. 1997), N. meningitidis (STOJILJKOVIC
and SRINIVASAN 1997), and it is ExbB-dependent in A. pleuropneumoniae
(TONPITAK et al. 2000). Transferrin binding proteins have long been known to be
important in bacterial virulence (SCHRYVERS and GONZALEZ 1990), but their role
in infection is still not fully understood. For Neisseria spp. it is known that, in vitro,
only the TbpA protein is required for utilization of transferrin (ANDERSON et al. 1994)
whereas the TbpB protein shows different affinities to iron-saturated and apo-
transferrin (CORNELISSEN and SPARLING 1996). N. gonorrhoeae mutants lacking
transferrin receptor expression are avirulent in a human infection model
(CORNELISSEN et al. 1998). The A. pleuropneumoniae TbpB protein also shows
selective binding for iron-saturated transferrin (GERLACH et al. 1992a) and, in
addition, confers a protective immune response (ROSSI-CAMPOS et al. 1992).
Interestingly, its expression is downregulated over the course of infection (HENNIG et
al. 1999).
B.4.3.3 Iron transport through bacterial membranes
Passage of ferric iron complexes like ferrisiderophores through the outer membrane
depends on the TonB protein (26 kDa), which provides energy to the outer mem-
LITERATURE REVIEW
31
brane receptor. TonB is part of an energy coupling and transport system, the TonB-
ExbBD system (EARHART 1996). It is anchored in the cytoplasmic membrane and
spans the periplasmic space, which is evidenced by the ability of the ferrichrome re-
ceptor FhuA to stabilize TonB (BRAUN et al. 1991). The TonB protein interacts with
outer membrane receptors via the TonB box, a consensus sequence near the N-ter-
minal end of the receptor protein. TonB itself has three functional domains: a hydro-
phobic N-terminal domain which anchors the protein in the cytoplasmic membrane
and interacts with ExbB, a hydrophilic elongated, rigid central domain spanning the
periplasmic space (BRAUN 1995), and a hydrophobic C-terminal domain which is
likely responsible for interaction with outer membrane proteins. TonB is proposed to
respond to the proton gradient and assume an "energized conformation" that opens
the receptor channels (BRAUN and KILLMANN 1999). ExbB (25 kDa), spanning the
cytoplasmic membrane, and ExbD, situated in the cytoplasmic membrane (15.5 kDa),
assist TonB (EARHART 1996). In E. coli, ExbBD function may be partially comple-
mented by the structurally homologous TolQR system which is involved in colicin
transport, and vice-versa (BRAUN and HERRMANN 1993). Iron-laden ferrichrome
binds to the receptor FhuA which, aided by the energy provided by the TonB-ExbBD
system, transports the ferrisiderophore into the periplasmic space where it is bound
by the periplasmic binding protein FhuD and transported to the cytoplasmic mem-
brane.
Transport through the cytoplasmic membrane is mediated by a protein complex
composed of FhuB, a permease, and FhuC, an ATP binding protein. Unlike FhuA
which is specific for ferrichrome, the FhuB-FhuC complex transports all ferric
hydroxamates (KOSTER 1991). Similar systems exist for other compounds and in
other organisms like N. gonorrhoeae (ANDERSON et al. 1994; GUERINOT 1994;
MIETZNER and MORSE 1994; GRAY-OWEN and SCHRYVERS 1996). Once
arrived in the cytoplasm, iron is mobilized from siderophores by ferrisiderophore re-
ductases in E. coli, Pseudomonas aeruginosa and Bacillus subtilis, releasing ferric
iron into the cytoplasm (GAYNES et al. 1981; FISCHER et al. 1990; HALLE and
MEYER 1992).
LITERATURE REVIEW
32
In A. pleuropneumoniae, transferrin is bound by the TbpB-TbpA heterodimer
(GONZALEZ et al. 1990; GERLACH et al. 1992b; WILKE et al. 1997), then iron is
released from the host protein and most likely transported through the outer mem-
brane. The TbpA protein shows homology to TonB-dependent outer membrane pro-
teins from E.coli (GONZALEZ et al. 1990; BELL et al. 1990), and a putative TonB box
with the sequence DVYVTGT was identified in the TbpA protein (GONZALEZ et al.
1995). In A. pleuropneumoniae, exbB and exbD homologous genes are located up-
stream of the tbpBA genes, with exb and tbp genes forming one operon (TONPITAK
et al. 2000). The ExbB and ExbD proteins are required for the uptake of transferrin-
bound iron in vitro, as demonstrated by the inability of an exbB-negative isogenic
deletion mutant to utilize porcine transferrin as the sole iron source (TONPITAK et al.
2000). The organisation of iron compound receptors, their energy coupling and cyto-
plasmic membrane transporter systems are illustrated for Fhu and Tpb proteins in
Fig. 1.
In addition, in A. pleuropneumoniae, a periplasmic ferric uptake system which might
have similar functions as E. coli FhuDBC, has been cloned, sequenced and termed
afuABC. This system is able to complement iron transport function in an E. coli aroB
mutant deficient in enterochelin synthesis. The complemented aroB mutant will grow
on medium containing iron chelator in a concentration that inhibits growth of the un-
complemented strain (CHIN et al. 1996). The AfuA protein is proposed to be a peri-
plasmic binding protein, the AfuB protein a membrane permease, and the AfuC pro-
tein shows the characteristics of an ATP binding protein (CHIN et al. 1996). However,
a possible involvement of AfuABC in uptake of transferrin-bound iron has not been
investigated. Structurally similar systems have been identified in other organisms; a
hitABC operon has been identified in Haemophilus influenzae (ADHIKARI et al.
1995), an FbpABC system is found in N. gonorrhoeae (ADHIKARI et al. 1996), and
an SfuABC system in Serratia marcescens (ANGERER et al. 1990).
LITERATURE REVIEW
33
Fig. 1: Energy coupling mechanisms of high-affinity iron uptake systems. Left: ferrichrome up-
take system of E. coli (BRAUN et al. 1991); right: Transferrin binding proteins of A. pleuropneumoniae.
FhuA, TbpA, TbpB, receptor proteins; TonB, spanning periplasmic space; ExbBD, in cytoplasmic
membrane; FhuD, periplasmic transporter protein; FhuB, permease, FhuC, ATP-binding protein; OM,
outer membrane; P, periplasm; CM, cytoplasmic membrane. Proteins corresponding to FhuDBC for
TbpBA-acquired iron are unknown.
B.4.4 Regulation of bacterial iron uptake via the Fur repressor protein
In most bacteria, high affinity iron uptake systems are regulated through an iron-de-
pendent regulatory mechanism. This mechanism is best characterized in E. coli
(LITWIN and CALDERWOOD 1993). A single regulatory protein is responsible for the
coordinated regulation of gene expression by iron. The fur (ferric uptake regulator)
gene (SCHAFFER et al. 1985) encodes a repressor protein that uses ferrous iron as
a co-repressor to inhibit transcription of iron-regulated genes under iron-replete con-
ditions. When iron is scarce, the repressor detaches from the operator region of the
respective genes to allow transcription (BAGG and NEILANDS 1987; EARHART
1996). Fur protein homologues have been identified in numerous species, among
them S. typhimurium (ERNST et al. 1978), Yersinia pestis (STAGGS and PERRY
1991), Vibrio cholerae (LITWIN et al. 1992), and others. Fur binds to the "Fur box"
containing the palindromic consensus sequence GAT AAT GAT AAT CAT TAT C in
LITERATURE REVIEW
34
the promoter region of the iron-regulated gene (CALDERWOOD and MEKALANOS
1988; OCHSNER et al. 1995). A Fur box showing over 50% identity with the consen-
sus sequence has been postulated for A. pleuropneumoniae (GONZALEZ et al.
1995) 100 bp upstream of the tbpB start codon; however, more recent research has
shown that the transferrin binding genes are co-transcribed with exbBD, situated di-
rectly upstream of tbpB (TONPITAK et al. 2000).
Besides the genes relevant in iron metabolism, the Fur regulon encompasses many
virulence factors, like the Shiga toxin of Shigella dysenteriae or the Shiga-like toxin of
E. coli (CALDERWOOD and MEKALANOS 1987). Also, in E. coli, the superoxide
dismutase gene sodB is positively regulated by Fur, but independent from iron
(NIEDERHOFFER et al. 1990). This regulation of virulence-associated genes through
Fur is an extremely efficient way of responding to an environmental stimulus that, to
the bacterium, signals entry into the host.
B.5 Dimethylsulfoxide (DMSO) reductase
E. coli possesses an enzyme that allows the use of DMSO and various other sub-
strates as a terminal electron acceptor in cell respiration under anaerobic conditions,
DMSO reductase (BILOUS and WEINER 1985). The enzyme consists of three sub-
units, DmsA, DmsB and DmsC. DmsA (87 kDa) is the catalytic subunit containing a
molybdopterin cofactor (WEINER et al. 1988), DmsB (23 kDa) is an electron carrier
containing four iron-sulfur clusters, with DmsC (30 kDa) serving as membrane anchor
for the other two subunits (BILOUS and WEINER 1988). The corresponding genes
dmsABC are organized in an operon (BILOUS et al. 1988). DMSO reductases have
been identified in several organisms besides E. coli, namely Rhodobacter (R.) cap-
sulatus (MCEWAN et al. 1991), R. sphaeroides (JOHNSON et al. 1990), and Haemo-
philus influenzae (LOOSMORE et al. 1996). The latter contains a dmsABC operon,
however, no data on translated proteins is available. In E.coli, the enzyme complex is
situated at the cytoplasmic face of the inner membrane, DmsA and DmsB are
extrinsic proteins facing the cytoplasm, the membrane anchor DmsC is an intrinsic
membrane protein. In R. capsulatus, DMSO reductase consists of a single
periplasmic polypeptide of 82 kDa containing a molybdenum cofactor.
LITERATURE REVIEW
35
Substrates of the enzyme include a variety of S- and N-oxides, e.g. DMSO, trimethyl-
amine-N-oxide (TMAO), and adenosine-N-oxide as well as sodium chlorate and
hydroxylamine (WEINER et al. 1988). In E. coli, expression of DMSO reductase re-
quires anaerobic growth conditions (BILOUS and WEINER 1985), is enhanced by the
addition of ferrous sulfate to culture medium (WEINER et al. 1988), and appears to
be regulated by the Fnr (fumarate and nitrate reduction) protein (COTTER and
GUNSALUS 1989). An Fnr homologue, HlyX, that complements E. coli fnr mutants
and also induces a latent E. coli hemolysin, has been identified in A.
pleuropneumoniae (MACINNES et al. 1990; GREEN and BALDWIN 1997).
With respect to its membrane targeting, the DMSO reductase of E. coli is exceptional.
The DmsA subunit containing the molybdenum cofactor is targeted to the membrane
via the twin arginine translocation pathway, TAT (BERKS 1996), also termed Mem-
brane Targeting and Translocation, MTT (SARGENT et al. 1998), that normally ex-
ports folded cofactor-containing proteins to the periplasm. Proteins transported
through this pathway contain a typical twin arginine motif in their signal sequence, the
consensus sequence is (S/T)-R-R-x-F-L-K, with the twin arginine residues being in-
variable (BERKS 1996).
B.6 Representational Difference Analysis of cDNA (cDNA RDA)
The technique of representational difference analysis was originally developed for the
analysis of differences between two closely related complex genomes (LISITSYN
1993). The technique is based on the principle of subtractive hybridization, with an
added PCR step devised to reduce complexity and size of the investigated gene
fragments, followed by several steps of alternating subtraction and amplification
leading to the selective amplification of target sequences. DNA is digested with re-
striction endonucleases, and only the DNA containing the putative differences
("tester") is ligated to double-stranded adaptor molecules consisting of a long (24mer)
and a short (12mer) oligonucleotide. The short oligonucleotide is used to increase the
efficiency of ligation and is not covalently bound; it disassociates in subsequent
steps. Tester DNA is then hybridized to an excess of the DNA used for comparison
("driver"). The subsequent PCR after fill-in of cohesive ends uses the 24mer primer to
selectively amplify tester-tester hybrids that have formed because there is no
LITERATURE REVIEW
36
adequate partner present in the driver population. Thus, differences between the two
genomes are selectively enriched. Driver-driver hybrids are unamplifiable due to the
lack of ligated primer molecules, tester-driver hybrids are multiplied in a linear fashion
only, because the primer is present on only one end of the molecule (LISITSYN
1993).
This technique was adapted for use with cDNA (cDNA RDA, HUBANK and SCHATZ
1994), because a reliable, technically simple technique for the detection of differen-
tially expressed genes was not available. For example, the differential display tech-
nique (LIANG and PARDEE 1992) relies on random-primed amplification of mRNAs
of two populations and comparison of banding patterns. However, this technique am-
plifies all mRNA species, while RDA eliminates sequences present in both popula-
tions. Another advantage of cDNA RDA is the possibility of isolating rare transcripts
due to the initial amplification step. However, the same step can also be responsible
for the loss of some sequences, as a result of the position of restriction sites, frag-
ment size or amplification kinetics. Furthermore, with an increasing number of ampli-
fication rounds, fragment size decreases as small fragments are amplified more ef-
fectively. This leads to the loss of potential difference products (HUBANK and
SCHATZ 1999).
The cDNA RDA technique has been used successfully to detect differentially ex-
pressed genes in Ewing's sarcoma cells (BRAUN 1995). It has been adapted for use
with bacterial RNA in N. meningitidis (BOWLER et al. 1999), where cDNA from a
lactoferrin binding protein gene (lbpA) deletion mutant and an intact parent strain
were used in an RDA experiment to identify lbpA. In intracellular M. bovis, increased
expression of mycoseroic acid synthase in was detected using this technique (LI et
al. 2001). In S. aureus, a protocol which optimized the effectivity of hybridization
("micro-RDA") has been developed and was used to identify genes expressed in
biofilm forming bacteria versus planctonic populations (BECKER et al. 2001).
B.7 Infection models for A. pleuropneumoniae
Experimental infection with a respiratory pathogen requires a model that produces
reliable and reproducible results in terms of clinical signs and pathology. Ideally, the
LITERATURE REVIEW
37
mode of administration itself influences the animal only minimally, as to avoid stress-
induced alteration of the course of infection. For a respiratory pathogen, administra-
tion of bacteria to the airways seems to be the best choice. Four routes of ad-
ministration are commonly used: intranasal, intratracheal, intrabronchial, and aerosol
administration. For A. pleuropneumoniae, all four techniques have been established.
Intranasal application involves the application of a defined number of bacteria to one
or both nostrils in fluid form (BRANDRETH and SMITH 1987), intratracheal applica-
tion is performed by injection (INZANA et al. 1993), intrabronchial administration re-
quires the use of a flexible endoscope as described for bronchoalveolar lavage
(HENSEL et al. 1994a; GOETHE et al. 2000), and aerosols are either administered
over a nasal mask (HENSEL et al. 1993) or in an aerosol chamber large enough to
accommodate one or several pigs (JACOBSEN et al. 1996).
All setups with the exception of aerosol administration require anesthesia and/ or
fixation of the pigs. Aerosol infection most adequately mimics the natural mode of
infection, and therefore it appears to be the preferable mode of delivery. An aerosol
chamber close to the pig stable allows leading of the animals to the chamber which
they will enter at free will, particularly if small amounts of food are offered inside.
Once locked in the chamber, most pigs will rapidly adapt to the situation and lie
down, remaining calm for the entire time of exposure (~45 min).
Aerosols suitable for a challenge experiments can be generated in various ways.
HENSEL et al. (1993) used a computer-controlled system in which the generation of
aerosols from freeze-dried powder over a rotating brush generator was adapted to
individual respiratory volumes, JACOBSEN et al. (1995) relied on an ultrasonic nebu-
lizer. As a damaging effect of ultrasound on viable bacteria cannot be excluded, a
nozzle operated by compressed air seems preferable.
LITERATURE REVIEW
38
B.8 Working hypothesis
As the work described in the literature review demonstrates, comparatively little is
known about the means of A. pleuropneumoniae to establish and maintain infection,
and a cross-serotype protective marker vaccine is not available. As a consequence,
novel strategies for the elucidation of pathogenesis and virulence need to be em-
ployed.
The study presented here had the following goals:
- to investigate the virulence of isogenic A. pleuropneumoniae mutant strains, in
which the uptake of transferrin-bound iron is abolished or impaired, in an animal in-
fection model, thereby determining the role of uptake of transferrin-bound iron in in-
fection, and
- to identify and characterize possible alternative iron-uptake mechanisms as well as
other host-induced virulence-associated factors via an ex vivo approach, using
bronchoalveolar lavage fluid to mimic in vivo conditions.
MATERIALS AND METHODS
39
C Materials and methods
C.1 Chemicals, reagents and equipment
Chemicals and reagents used in this study are summarized in the appendix (I.1),
equipment and specific biologicals are indicated in footnotes.
C.2 Buffers and solutions
Buffers and solutions used in this study are summarized in the appendix (I.2) or
noted in the text where appropriate.
C.3 Bacterial cultures
C.3.1 Bacterial strains
Bacterial strains used in this study are listed in Table 1.
C.3.2 Media and growth conditions
Escherichia (E.) coli strains were cultured in Luria-Bertani (LB) medium supple-
mented with the appropriate antibiotics (100µg/ml ampicillin, 50 µg/ml kanamycin, 25
µg/ml chloramphenicol); for cultivation of E. coli β2155, 1 mM diaminopimelic acid
was added. Bacteria were incubated at 37°C in an incubator1 or in a shaking incuba-
tor2.
Actinobacillus (A.) pleuropneumoniae strains were cultured in supplemented PPLO
medium with 0.1% Tween® 80 unless stated otherwise. For the selection of A.
pleuropneumoniae transconjugants, 25 µg/ml kanamycin or 5 µg/ml chloramphenicol
were added depending on the transconjugation vector. Iron restriction was induced
by addition of 100 µM 2,2 dipyridyl or 200 µM diethylenetriamine-pentaacetic acid
calcium trisodium salt hydrate (Na3CaDTPA).
1 Memmert GmbH & Co. KG, Schwalbach2 Incubator shaker Series 25, New Brunswick Scientific Co., Inc., Edison, NJ, U.S.A.
MATERIALS AND METHODS
40
The cultures were incubated at 37°C in a 5% CO2 incubator1 or in a shaking incuba-
tor. For anaerobic culture, media were pre-incubated overnight in anaerobic jars
using the AnaeroGenTM system2 before inoculation with liquid aerobic A.
pleuropneumoniae cultures (10 % of the total culture volume). Cultures were then
incubated with stirring at 37°C for five hours.
MediaLB broth: 10 g Bacto® tryptone, 5 g yeast extract, 5 g NaCl, add distilled
water to 1 liter, autoclave
LB agar: LB broth with 1.5% agar (w/v), autoclave
PPLO broth: 21 g/l PPLO® broth, add distilled water to 1 liter, sterile filter
PPLO agar: 35 g/l PPLO® agar, add distilled water to 1 liter, autoclave
C.3.3 Antibiotic solutions and supplements
Ampicillin: stock solution 100 mg/ml in 70% ethanol, with
addition of concentrated HCl until all substance is
completely dissolved.
Chloramphenicol: stock solution 25 mg/ml in 70% ethanol
Kanamycin: stock solution 50 mg/ml in A. bidest.
Diaminopimelic acid: stock solution 100 mM in A. bidest., a few drops of
concentrated HCl were added until the solution
cleared
PPLO supplement stock solution:1 g/l L-glutamine, 26 g/l L-cysteine dihydrochloride,
1 g/l L-cystine dihydrochloride, 1g/l nicotinamide
dinucleotide (NAD) in 10% D (+) glucose monohy-
drate
Antibiotic stock solutions and diaminopimelic acid stock solution were sterilized by
filtration1 and stored at –20°C.
C.4 Bacteriological methods
1 Heraeus CO2-Auto-Zero, Heraeus Instruments GmbH Labortechnik, Hanau, Germany2 Oxoid GmbH, Wesel, Germany
MATERIALS AND METHODS
41
C.4.1 Urease assay
To test whether a bacterial colony exhibited urease activity, bacterial colonies were
overlaid with urease test agarose (0.5% agarose, 20 mg/ml urea,100 µg/ml phenol
red), either on an agar plate or after transfer to a nitrocellulose disk2 or filter paper
disc3. A red colony colour and halo indicating urease activity was visible after 1-5
min, negative colonies turned yellow.
C.4.2 Plate bioassay
A plate bioassay was used to determine the capability of A. pleuropneumoniae to
utilize porcine transferrin and iron-saturated ferrichrome as the sole iron source. BHI
agar was prepared containing 10 µg/ ml NAD and 250 µM diethylene triamine-
pentaacetic acid calcium trisodium salt hydrate (Na3CaDTPA), an iron chelator.
A. pleuropneumoniae was grown in liquid culture until it reached OD660= 0.2. Iron re-
striction was induced by the addition of Na3CaDTPA to a final concentration of
200 µM. The culture was incubated for an additional three hours, with OD
measurement at 2 and 3 hours post induction to confirm that bacterial growth stag-
nated due to iron depletion. 100 µl of a 1:10 dilution (in PPLO broth) was then plated
on BHI agar plates supplemented with Na3CaDTPA, and filter disks4 (9 mm diameter)
were placed on the agar plates. These filter disks were then loaded with 75 µl of iron-
saturated porcine transferrin (500 µM), iron-saturated ferrichrome (500 µM), 2,3
dihydroxybenzoic acid (2,3-DHBA, 500 µM), ferric citrate (500 µM ferric nitrate, 1 mM
sodium citrate) or dilution buffer (10 mM HEPES, 150 mM NaCl, 10 mM sodium
bicarbonate; pH 7.4) with ferric citrate serving as positive and dilution buffer serving
as negative control. Plates were incubated at 37°C, 5% CO2 overnight. Growth of
bacteria around a filter disc indicated bacterial utilization of the respective iron
source.
1 Millex -GV, pore size 0.2 µM, Millipore, Eschborn2 Protran BA85, 0.45 µM pore size, 82 mm diameter, Schleicher & Schuell, Dassel3 Circle Filter paper for qualitative analyses, diameter 9 cm, Roth, Karlsruhe4 Schleicher und Schuell, Dassel, Germany
MATER
IALS AND
METH
OD
S
42
Table 1: List of bacterial strains used in this study
MATERIALS AND METHODS
43
Tabl
e 1
cont
inue
d
MATERIALS AND METHODS
44
C.5 Manipulation of nucleic acids
C.5.1 Plasmids
The plasmids used in this study are summarized in Table 2. Restriction endo-
nuclease digests, ligations, generation of blunt ends via Klenow fragment or T4 DNA
polymerase, alkaline phosphatase treatments and agarose gel electrophoresis were
done according to standard protocols and the respective manufacturers' instructions.
Restriction endonucleases, other enzymes, DNA size standards and buffers were
purchased from New England Biolabs, Frankfurt, Germany, unless stated otherwise.
C.5.2 Primers
The primers used in this study are summarized in Table 3. Primers were synthesized
by MWG, Ebersbach, Germany, or Invitrogen, Karlsruhe, Germany.
C.5.3 Isolation of DNA
C.5.3.1 Plasmid DNA
Plasmid DNA was either prepared by alkaline lysis (BIRNBOIM and DOLY 1979)
following standard procedures (SAMBROOK et al. 1989) or by using the JETSTAR®
Midi Plasmid Preparation kit1 according to the manufacturer's instructions.
DNA cleanup following alkaline lysis was performed by phenol-chloroform extraction
according to standard procedures (SAMBROOK et al. 1989) or by using the Gene
Clean® kit2 according to the manufacturer's instructions. Centrifugation steps were
carried out in a microcentrifuge3
C.5.3.2 Total chromosomal DNA of A. pleuropneumoniae
Overnight bacterial cultures on solid medium were harvested by resuspending the
culture in 5 ml PPLO medium and transferred to a 10 ml polypropylene tube; bacterial
cells were recovered by centrifugation4 at 5,000 rpm (SA600 Rotor), 4°C for 10 min,
1 Genomed, Bad Oeynhausen, Germany2 Qbiogene, Heidelberg, Germany3 3 MC-13 Amicon, Heraeus Instruments, Osterode, Germany4 Sorvall RC-5B Refrigerated Superspeed Centrifuge, Du Pont Inst., Bad Homburg, Germany
MATERIALS AND METHODS
45
the supernatant was discarded. Bacterial cells were lysed by the addition of 5 ml lysis
buffer with proteinase K (10 mM EDTA pH 8.0, 1% SDS, 0.5 mg/ml proteinase K);
mixing of the solution was achieved by gentle inversion of the tube. The mixture was
incubated at 55°C for 1 h. RNA contamination was removed by the addition of RNase
to a final concentration 100 µg/ml and further incubation at 37°C for 20 min. DNA was
purified by adding ¼ volume of phenol equilibrated in TE pH 7.8 to the solution and
mixing by careful shaking. Then ¼ volume of chloroform : isoamyl alcohol (24:1) was
added, mixed and centrifuged at 12,000 rpm for 10 min. The top (aqueous) phase
containing DNA was carefully removed using a plastic pasteur pipette and transferred
to a new tube. Chloroform-isoamyl extraction was repeated until no interphase was
visible. The upper phase was transferred into a new tube, and DNA was precipitated
by adding 0.1 volume of 3 M Na-acetate [pH 5.2] and 1 volume of isopropanol. The
DNA thread generated by careful inversion of the tube was collected with a small
pipette tip, washed twice in 70% ethanol for 5 min and finally in 96% ethanol for 5
min. DNA was dissolved in 200 µl A. bidest. overnight at 4°C. 5 µl of DNA were
analysed by gel electrophoresis.
MATER
IALS AND
METH
OD
S
46
Table 2: List of plasmids used in this study
MATERIALS AND METHODS
47
Tabl
e 2
cont
inue
d
MATERIALS AND METHODS
48
Tabl
e 2
cont
inue
d
MATERIALS AND METHODS
49
Tabl
e 2
cont
inue
d
MATER
IALS AND
METH
OD
S
50
Table 3: List of primers used in this study
MATERIALS AND METHODS
51
Tabl
e 3
cont
inue
d
MATERIALS AND METHODS
52
Tabl
e 3
cont
inue
d
MATERIALS AND METHODS
53
Tabl
e 3
cont
inue
d
MATERIALS AND METHODS
54
C.5.4 Polymerase chain reaction
(MULLIS et al. 1968)
PCR was performed in a thermal cycler1 in a 25 or 50 µl total reaction volume using
Taq DNA polymerase2. The mixtures were prepared on ice by addition of the
reagents in the order described in Table 5. In the case of screening deletion mutants
following sucrose counterselection, the DNA template was prepared by boiling a
single colony. Amplification conditions are listed in Table 4. PCR products were
analysed by gel electrophoresis on a 1.5% agarose gel.
C.5.4.1 Preparation of DNA template by colony boiling.
A single colony was touched with a small pipette tip to pick up a very small amount of
bacteria. The material was resuspended in 100 µl TE buffer in a microtiter plate or in
a 1.5 ml reaction tube and boiled in a microwave oven for 8 min at 180 W. 5 µl of this
mixture served as template in a 25 µl PCR reaction. For the negative control, 100 µl
TE buffer was boiled at the same conditions and 5 µl were used in the same volume
of premix.
1 Cyclone Thermocycler, Integra Biosciences, MA, U.S.A. or Crocodile III, Appligene, Illkirch, France2 Invitrogen, Eggenstein, Germany
MATERIALS AND METHODS
55
Table 4: PCR conditions used in this study
primers PCR protocol
all oRN primer pairs 3' 94°C, (30'' 94°C, 1' 54°C, 1' 72°C) x 32, 10' 72°C
RBgl24 3' 94°C, (1' 94°C, 1' 58°C, 3' 72°C) x 35, 10' 72°C
oMut, oDMSADEL,oFDEL, oFHUfs primers 3' 94°C, (30'' 94°C, 1' 54°C, 1' 30'' 72°C) x 32, 10' 72°C
BA7, RE1 3' 94°C, (30'' 94°C, 40'' 55°C, 2' 72°C) x 32, 10' 72°C
urec2, ureX 3' 94°C, (30'' 94°C, 40'' 54°C, 1' 30'' 72°C) x 32, 10' 72°C
Table 5: Components in the PCR reaction
reactioncomponents
stocksolution
finalconcentration
volume:reaction(µl)
volume:reaction(µl)
A. bidest. - - 13.65 27.3
MgCl2 50 mM 1.5 mM 0.75 1.5
PCR Buffer 10x 1x 2.5 5
dNTPs 10 mM 0.2 mM 0.5 1
forwardprimer 5 pmol/µl 0.25 pmol/µl 1.25 2.5
reverseprimer 5 pmol/µl 0.25 pmol/µl 1.25 2.5
Taq DNApolymerase 5 U/µl 0.5 U 0.1 0.2
template - - 5 10
final volume 25 50
Mineral oiloverlay - - 30 40
MATERIALS AND METHODS
56
C.5.5 Representational difference analysis (RDA) of cDNA
(HUBANK and SCHATZ 1994)
C.5.5.1 Preparation of RNA
For induction with bronchoalveolar lavage fluid (BALF), A. pleuropneumoniae cul-
tures were grown to an optical density at 660 nm (OD660) of 0.3. Five ml of BALF,
freshly thawed and prewarmed to 37°C, were added; control cultures were supple-
mented with 5 ml of NaCl (150 mM). Cultures were grown with shaking at 37°C for 30
minutes reaching an OD660 of approximately 0.6. Centrifuge tubes were half filled with
crushed ice and placed on ice until needed. Bacterial cultures were added and cen-
trifuged at 7000 rpm (SA 600 rotor) for 5 minutes. RNA was extracted using the
Nucleospin RNAII kit1 according to the manufacturer's instructions. RNA was
visualized on a 1% formaldehyde gel following standard procedures (SAMBROOK et
al. 1989), and absence of genomic DNA was confirmed by PCR using primers ureX
and ureC2 (Table 3) on 2.5 µl of RNA to which 0.5 µl of DNase free RNase2 (10 U/µl)
had been added. Reverse transcription was performed using 5 µg of RNA from A.
pleuropneumoniae grown in the presence of BALF ('tester') or in the absence of
BALF ('driver'). The RNA was resuspended in 11 µl of RNase-free water, 1 µl of ran-
dom hexamer primer3 (50 pmol/µl) was added. The mixture was heated to 90°C for 2
minutes and then allowed to cool to 42°C over the course of 10 minutes. 4 µl of first
strand buffer, 2 µl of DTT (10mM), 1 µl of dNTPs4 (10 mM each) and one µl of M-
MuLV reverse transcriptase (200 units/µl) were added and incubated at 42°C for 30
minutes. 20 µl of 1x ligase buffer and 0.5µl (200 units) of T4 DNA ligase (400 U/µl)
were added and incubated at 16°C for two hours. The mixture was heated to 95°C for
5 minutes, 1 µl DNAse free RNase was added and incubated at 37°C for 30 minutes.
cDNA was purified using the Gene Clean kit and eluted in a volume of 20.5 µl.
1 Macherey-Nagel, Düren, Germany2 Boehringer Mannheim, Germany3 Amersham Biosciences, Freiburg, Germany4 Roth, Karlsruhe, Germany
MATERIALS AND METHODS
57
C.5.5.2 Second strand synthesis
To 20.5 µl of cDNA, 1 µl EcoPol buffer and 1 µl of random hexamer primer (50
pmol/µl) were added, the mixture was heated to 70°C for 2 minutes and left to cool to
room temperature. 1 µl 10 mM dNTPs, 1.5 µl EcoPol buffer and 1 µl (5 Units) of
Klenow enzyme were added and the reaction was incubated at 37°C for 60 minutes.
The volume of the reaction was adjusted to 500 µl with TE buffer; chloroform-phenol
(Aqua-Roti® Phenol) extraction and overnight ethanol precipitation were performed
according to standard methods (SAMBROOK et al. 1989).
C.5.5.3 Representation of 'tester' and 'driver'
Representation products were generated as follows: 10 µl of cDNA were digested
with DpnII at 37°C for 1 hour in a total volume of 20 µl using the appropriate buffer.
cDNA was purified using the Gene Clean® kit and eluted in a volume of 25 µl. Adap-
tors were generated by mixing 20 µl each of primers RBgl12 and Rbgl24 (100 µM
each) with 10 µl of 10 x ligase buffer and annealed in a thermal cycler using the fol-
lowing program: 94°C, 67°C, 42°C, 37°C, 22°C and 16°C at 10 minutes each. Cor-
rect annealing was confirmed on an 18% TBE-acrylamide gel. 25 µl of the adaptor
preparation were then ligated overnight to 25 µl tester and driver cDNA with the ad-
dition of 2 µl 25 mM ATP and 1 µl of ligase. Amplicons were generated after cleanup
of the ligation products via the Nucleospin Extract kit1 and using 2.5 µl of the respec-
tive preparation in a PCR reaction with primer RBgl24 in a total volume of 50 µl. Two
identical reactions of tester cDNA and ten of driver DNA were set up, heated to 72°C
before the addition of Taq polymerase2, incubated for 5 minutes at 72°C to fill in 5'
overhangs, and followed by 35 PCR cycles (95°C for 1 minute, 58°C for 1 minute,
72°C for 3 minutes). Identical reactions were pooled and subjected to chloroform-
phenol extraction and alcohol precipitation as described above. The precipitation
products were resuspended in 100 µl of A. bidest.
1 Macherey-Nagel, Düren, Germany2 Invitrogen, Eggenstein, Germany
MATERIALS AND METHODS
58
C.5.5.4 Preparation of tester and driver
Tester and driver amplicon preparations were digested with DpnII and purified (Nu-
cleospin Extract kit). Subsequently, adaptors prepared from primers JBgl12 and
JBgl24 were ligated only to 5 µg of tester amplicon DNA, as described above. Tester
and driver were cleaned up using the Nucleospin Extract kit.
C.5.5.5 Hybridization and subsequent PCR
20 µg of driver were mixed with 20 and 200 ng of tester, respectively, subjected to
overnight ethanol precipitation and resuspended in 4 µl PCR buffer. After overlaying
the reaction with mineral oil, it was heated to 95°C, 1 µl of 5M NaCl was added and
hybridisation was allowed to occur for 20 hours at 67°C in a thermal cycler. The vol-
ume was adjusted to 20 µl with A. bidest, and PCR was performed, using 2.5 µl and
0.25 µl of the respective preparations. The resulting PCR products were analyzed in
a 1.5% agarose gel.
C.5.5.6 Isolation of RDA fragments
RDA products between 320 and 700 bp were excised from the gel, purified using the
Gene clean kit, cloned into pCR2.1® using the TOPO cloning kit1 according to the
manufacturer's instructions. The resulting mixture of recombinant plasmids was
transformed into E. coli Top 10 and plated on LB agar supplemented with 100µg/ml
ampicillin. Using a vacuum pump connected to a sterile 1000 µl pipette tip, colonies
were sorted into single wells of a microtiter plate by picking up sterile glass beads (1-
2 mm diameter), touching a bacterial colony and releasing the bead into a well. The
cloned RDA fragments could be amplified from the TOPO 2.1 vector using primers
M13 forward and M13 reverse (Table 3)
C.5.6 Pulsed field gel electrophoresis (PFGE)
C.5.6.1 Isolation of agarose-embedded chromosomal A. pleuropneumoniae DNA
A single A. pleuropneumoniae colony was inoculated into 5 ml supplemented PPLO
broth and incubated at 37°C, 5% CO2 overnight. 2 ml of the overnight culture were
1 Invitrogen, Eggenstein, Germany
MATERIALS AND METHODS
59
transferred to 20 ml supplemented PPLO broth and incubated at 37°C with shaking
(200 rpm) until OD660 = 0.3. The culture was then placed on ice. Chromosomal grade
agarose1 was dissolved at a concentration of 1.2% in A. bidest. in a microwave oven,
and kept at 55°C until use. 5 ml of A. pleuropneumoniae culture were centrifuged at
5,000 rpm at 4°C for 10 min. After removal of the supernatant, cells were washed
once in 5 ml ice-cold PET IV-buffer, recentrifuged, resuspended in 0.5 ml PET IV-
buffer and incubated briefly at 55°C in a waterbath before 0.5 ml of 55°C 1.2% chro-
mosomal grade agarose were added and mixed by repeated pipetting. The suspen-
sion was poured into 100 µl plug molds (Sample CHEF Disposable Plug Mold1).
Agarose was allowed to solidify at 4°C for 10-15 min. The tape from the bottom of the
10-well strip was removed and plugs were removed from the mold by using the tap at
the end of the tape as a tool to push the plugs out of the mold and into a new
polypropylene tube containing 3 ml lysis buffer for five plugs. The tube was incubated
horizontally at 37°C for 2 h after which the lysis buffer was discarded. 3 ml EPS
buffer containing 1% N-laurylsarcosine and 1 mg/ml proteinase K were added and
the plugs were incubated at 55°C overnight. EPS buffer was discarded and the plugs
were washed twice with 3 ml A. bidest. for 15 min by tube-rolling at room
temperature, A. bidest. was discarded. To inactivate residual proteinase K, plugs
were washed twice with 2 ml TE-PMSF for 30 min at room temperature, excess liquid
was removed. Plugs were then washed with 3 ml A. bidest. for 15 min, which was
again removed before washing the plugs with 3 ml TE buffer for 30 min and finally
storing them in 5 ml TE buffer at 4°C .
C.5.6.2 Restriction endonuclease digestion of DNA embedded in agarose plugs
One third of a gel plug was used for each reaction. Prior to digestion, plugs were
equilibrated in 3 volumes of an appropriate restriction endonuclease buffersupplied
by the manufacturer for 1 h at room temperature. New buffer was added, and 10 U of
enzyme were used to cleave the DNA in the plugs overnight at the temperature
appropriate for the respective restriction endonuclease.
1 BioRad Inc., Munich, Germany
MATERIALS AND METHODS
60
C.5.6.3 Pulsed field gel electrophoresis
A 0.8% agarose gel was prepared using 0.5 x TBE buffer, cooled to 55°C and poured
into a gel casting platform1, the gel was allowed to solidify for 5 minutes before
removal of the comb. The digested gel plug was maneuvered into the slot using a
scalpel and a hook formed from a pasteur pipette by heating in a bunsen burner
flame. Bacteriophage lambda concatemers embedded in agarose gel was used as
standard marker. The slots were sealed by filling them up with agarose gel to prevent
buoyancy of the plugs. The gel was placed into the electrophoresis chamber2 and
carefully immersed in cold 0.5 x TBE buffer. PFGE gels were run at 6 V/cm and 12°C
with linear ramped switch times from 10 to 20 sec for 14 h and from 35 to 70 sec for
12 h. After the run, DNA was stained with ethidium bromide in A. bidest (0.2 µg/ml)
for 20 min and destained in A. bidest. for 1 h. Gels were documented on a UV trans-
illuminator and photographed with a polaroid camera or an image documentation
system1.
C.5.7 Construction of A. pleuropneumoniae isogenic deletion mutants
(OSWALD et al. 1999b)
Deletions were introduced into A. pleuropneumoniae by means of an A. pleuropneu-
moniae-Escherichia coli shuttle vector, using the principle of homologous recombina-
tion to exchange an intact gene for the truncated version of the gene contained in the
recombinant plasmid.
C.5.7.1 Transconjugation from E. coli to A. pleuropneumoniae by filter mating
technique
In this study, the truncated gene cloned into the mutagenesis vectors pBMK1 or
pEMOC2 was mobilized from E. coli β2155 (Table 1), a diaminopimelic acid auxotro-
phic donor strain, into the A. pleuropneumoniae recipient. Donor and recipient were
grown on appropriate solid medium and incubated overnight. Cultures were collected
with a sterile cotton swab and resuspended in TNM buffer in separate tubes (E. coli
1 BioRad Inc., Munich, Germany2 CHEF-DR III pulsed-field electrophoresis system, Bio-Rad, Munich, Germany
MATERIALS AND METHODS
61
β2155 must not be resuspended by vortexing, as this may damage the pilus needed
for conjugation). Cell density was determined at OD660. Three nitrocellulose discs1
(0.45 µM pore size, 2.5 cm diameter) were placed onto sterile gel blotting paper in a
petri dish, and aliquots corresponding to 0.1 ml of donor and 0.8 ml of recipient (each
at OD660 = 1) were mixed by careful repeated pipetting and transferred onto each
nitrocellulose disc, allowing the buffer to be absorbed. The discs containing the cul-
ture mixture was then transferred onto gel blotting paper (using sterile forceps)
soaked with prewarmed PPLO broth (with 1% IVX, 1 mM diaminopimelic acid, and 10
mM MgSO4) and incubated at 37°C in 5% CO2 incubator for 7 h. Following incuba-
tion, the filter was placed in a microcentrifuge tube containing 600 µl PPLO broth, the
bacteria were washed from the membrane by vortexing, the suspension was plated
onto a well-dried PPLO agar plate supplemented with IVX and 25 µg/ml kanamycin
or 5 µg/ml chloramphenicol, respectively, and incubated at 37°C in a 5% CO2
incubator for 48 hours.
C.5.7.2 Sucrose counterselection
A single colony of A. pleuropneumoniae confirmed to carry the kanamycin or
chloramphenicol resistance determinant on the chromosome resulting from a plasmid
cointegrate, was inoculated into 1 ml supplemented PPLO broth that had been pre-
warmed at 37°C, 5% CO2 overnight, and incubated at 37°C with shaking (200 rpm)
for 2 h or until the culture was slightly turbid. 400 µl 2.5 x PPLO, 500 µl sucrose
[40%] and 100 µl sterile equine serum were added. The tube was incubated at 37°C
with shaking (200 rpm) for six hours, then 50 µl were plated on supplemented PPLO
agar without kanamycin and incubated overnight at 37°C, 5% CO2.
2.5 x salt-free PPLO broth
Forty-six g of Bacto® Beef Heart For Infusion in 1 l A. dest. were kept at 50°C with
constant stirring for 1 h, boiled in a microwave oven for 3 min and cooled to room
temperature with constant stirring. The suspension was filtered to remove solids, 7.4
1 Millipore, Eschborn, Germany
MATERIALS AND METHODS
62
g of Bacto® Peptone were added, pH was adjusted to 7.4 with 1 M NaOH, and the
solution was sterilized by filtration.
C.5.8 Nucleic acid detection
C.5.8.1 Southern blotting
(SOUTHERN 1975)
DNA was cleaved with the appropriate restriction endonuclease, separated by gel
electrophoresis, and the gel was photographed with a ruler on a UV transilluminator
prior to Southern transfer by capillary blotting onto nylon membrane1 according to
standard procedures (SAMBROOK et al. 1989). DNA crosslinking was achieved by
baking the membrane at 80°C for 60 minutes (in an oven2).
C.5.8.2 Northern blotting
20 µg of RNA were separated on a 1 % formaldehyde gel. The gel was photographed
with a ruler on a UV transilluminator prior to Northern transfer by capillary blotting
onto nylon membrane according to standard procedures (SAMBROOK et al. 1989).
RNA crosslinking was achieved by baking the membrane at 80°C for 120 minutes (in
an oven).
C.5.8.3 Labeling of probes with 32P-dCTP
Probes were generated using either PCR products or purified restriction enzyme
fragments. 20 to 30 ng DNA in 5 µl of A. bidest. were denatured at 100°C for 5 min
and briefly placed on ice. 10,5 µl of A. bidest, 5 µl OLB solution, 1 µl of acetylated
BSA [10 mg/ml], 1 µl Klenow fragment and 2.5 µl of α-32P-dCTP (370 kBq/µl) were
added and the reaction was incubated at room temperature for 4 h. 100 µl of stop
buffer were added to stop the reaction. The labeled DNA probe can be used
immediately or the unincorporated 32P-dCTP can be eliminated using a Sephadex G-
25 column3. The probe was stored in a lead container at –20°C. Immediately prior to
use, the labeled DNA probe was denatured at 95°C for 5 min.
1 Positive membrane, Appligene, Illkirch, France2 Booskamp, Wuppertal, Germany3 NAPTM-5 column, Amersham Biosciences, Freiburg
MATERIALS AND METHODS
63
C.5.8.4 Southern hybridization
Southern hybridization was performed overnight following standard procedures
(SAMBROOK et al. 1989) in a hybridization oven1 at 60°C, using a hybridization
buffer containing 6 x SSC, 0.5% SDS, 5 x Denhardt´s solution and 0.01 M EDTA.
Blots were washed three times for 30 minutes at 60°C (3 x SSC, 0.5% SDS for most
applications; for higher stringency, 1 x SSC, 0.5% SDS was used), and exposed to
an X-ray film2 with intensifying screen at –70°C. The exposure time was adjusted de-
pending on the signal strength.
C.5.8.5 Northern hybridization
Northern hybridization was performed overnight following standard procedures
(SAMBROOK et al. 1989) in a hybridization oven at 55°C. Blots were washed at
55°C once with wash solution I for 20 minutes, then twice with wash solution II for 20
minutes, and were then exposed to an X-ray film with intensifying screen at –70°C.
The exposure time was adjusted depending on the signal strength.
C.5.8.6 DNA colony blotting
Colony blotting was performed using circular nitrocellulose membranes3. Membranes
were placed on colonies, left for five minutes, then removed and placed (colony side
up) on plastic wrap to which 650 µl of NaOH (0.5 M) had been applied, and incubated
fo five minutes. This step was repeated once, then the membrane was placed on
650 µl on buffer containing NaCl (1.5 M) and Tris-HCl (1 M, pH 7.5), and incubated
for five minutes; this step was repeated once, after which the membrane was placed
on 650 µl of Tris-HCl (1 M, pH 7.5) for five minutes, then dried and baked for one
hour at 80°C. Colony blot hybridization was performed as described for Southern
blotting, after careful removal of colony material after prehybridization, at 55°C.
1 Mini hybridization oven, Qbiogene, Heidelberg, Germany2 Kodak X-OMAT® AR or BioMax, SIGMA, Deisenhofen, Germany3 Protran BA 85, Schleicher & Schuell, Dassel
MATERIALS AND METHODS
64
C.5.9 Nucleotide sequencing and sequence analysis
Nucleotide sequencing was done by SeqLab, Göttingen, Germany. Sequence
analyses were performed using the HUSAR program package supplied by Deutsches
Krebsforschungszentrum Heidelberg, Germany (dkfz).
C.6 Manipulation of proteins
C.6.1 Preparation of protein aggregates
(GERLACH et al. 1992a)
In this study, protein aggregates were used to raise antibodies against A.
pleuropneumoniae proteins in rabbits. These protein aggregates were generated by
inserting parts of the respective genes into a matching reading frame of an
appropriate pGEX expression vector (Table 2). Induction of the plasmid's tac
promoter with IPTG results in the production of GST (glutathione-S-transferase)
fusion proteins. These fusion proteins can agglomerate in the cytoplasm of the E. coli
host cell, forming inclusion bodies that can be purified and, upon solubilization, can
be used as antigens to raise antibodies in rabbits.
Protein aggregates were prepared as described by GERLACH et al (1992a). Briefly,
a 250 ml liquid culture of the Escherichia coli strain carrying the expression vector
was incubated to an OD660 of 0.3-0.5, induced with IPTG (1mM) and incubated for 2
hours. Bacteria were harvested by centrifugation (2,600 x g) at 4°C for 10 minutes,
the cell pellet was resuspended in 2.5 ml Tris-HCl (50mM, pH 8.0) with 25% saccha-
rose and frozen at –70°C overnight. After thawing, ¼ volume of Tris-HCl (250 mM,
pH 8.0) containing 10 mg/ml of lysozyme were added and the mixture was incubated
on ice for 10 minutes. Another incubation on ice for 10 minutes was followed by the
addition of 5 volumes 2x RIPA/ TET (mixed 5:4). Then the mixture was sonicated
using the sonicator's mini tip1 4 x 30 seconds at output 3-4.
Aggregate preparations were centrifuged at 15000 g, 4°C for 20 minutes and then
resuspended in 1 ml A. bidest. Two-µl aliquots were run on 10.9% SDS PAGE gels to
confirm the purity of the aggregate preparations.
1 Sonic Cell Disruptor, Branson Sonifer, Branson Power Co., Dannbury, U.S.A.
MATERIALS AND METHODS
65
C.6.2 Determination of protein concentration
Protein concentrations were determined using the Micro BCA® Protein Assay1 in 96
well microtiter plates with bovine serum albumin as the standard [100 µg/ml]. Sam-
ples to be tested were diluted 1.10 and 1:100. The assay was performed according to
the manufacturer's instructions and read in an ELISA reader2 at a wavelength of 550
nm.
C.6.3 Preparation of proteins from A. pleuropneumoniae by whole cell lysis
A single colony was inoculated in 2.5 ml supplemented PPLO, and incubated over-
night at 37°C in 5% CO2. 0.5 ml of overnight culture were transferred to 4.5 ml PPLO-
IVX-0.1% Tween® 80 and incubated with shaking until the cell density at OD660
reached 0.3-0.4. For iron restriction, 2,2 dipyridyl (100 µM final concentration) or
diethylenetriamine-pentaacetic acid calcium trisodium salt hydrate (Na3CaDTPA)
(200 µM final concentration) were added to the culture and grown with shaking for
2 h. One and a half ml bacterial cell culture were harvested by centrifugation at
13,000 rpm for 5 min, and all supernatant was removed. The cell pellet was
resuspended in 50- 100 µl A. bidest. to obtain suspensions of similar optical density,
and 2.5 µl of the cell suspension were used to load each slot of a 15 well comb in a
Bio-Rad Minigel chamber. Prior to loading, cell suspensions were mixed with 10 µl of
2x SDS sample buffer, boiled at 100°C for 3 min and centrifuged briefly. Either
prestained molecular weight standard3 or non-stained molecular weight standard4
were run on the same gel to estimate the molecular mass of the proteins. Samples
were separated on a 10.9% SDS-PAGE gel at 150 V for 65 min. Gels were stained
using Coomassie Blue and documented using an computer based image
documentation system5.
1 Pierce Micro BCA® Protein Assay, Pierce, Rockford, U.S.A.2 MR5000, Dynatech Laboratories Inc., Alexandria, U.S.A.3 Prestained SDS-PAGE Standard Low Range, Bio-Rad, Munich, Germany4 LMW Electrophoresis Calibration Kit, Amersham Biosciences, Freiburg, Germany5 Gel Doc 1000/Multi-analyst; Bio-Rad, Munich, Germany
MATERIALS AND METHODS
66
C.6.4 Preparation of antisera
In this study, rabbit antiserum raised against recombinant A. pleuropneumoniae pro-
teins expressed by E. coli as GST fusions was used to develop Western blots. This
serum was prepared as follows: New Zealand White rabbits1 8 weeks of age were
injected intradermally with approximately 100 µg of protein aggregate that was
solubilized in 5 µl 7 M guanidinium hydrochloride, then added rapidly to 200 µl of
PBS and mixed with 70 µl of Emulsigen®2.
C.6.4.1 Purification of antisera
In order to reduce background signals, antisera were purified by adsorption to
nitrocellulose-bound whole cell lysates of E. coli and A. pleuropneumoniae. The first
round of adsorption used an E. coli DH5αF' strain carrying the plasmid pGEX 5x3,
the second round used the A. pleuropneumoniae isogenic deletion mutant lacking
expression of the protein used to raise antibodies. Sera were used for Western blot
analyses after two rounds of adsorption, at which point background signals were
sufficiently reduced. Further reduction might be achieved by further dilution of sera
prior to purification. A third round of adsorption using an E. coli DH5αF' strain
carrying the respective protein fusion vector was used to demonstrate specificity of
the serum. Specific bands were not visible on blots incubated with the triple-absorbed
sera.
Bacterial strains were grown in 50 ml of appropriate media, induced with IPTG
(1 mM) and incubated for two hours before centrifugation, resuspension in 500 µl A.
bidest., addition of 500 µl SDS-PAGE sample buffer and heating to 100 °C for 5 min-
utes. Twenty cm2 of nitrocellulose were saturated with 500 µl of lysate, washed four
times with washing buffer, blocked for one hour in blocking buffer and incubated with
5 ml of serum (diluted 1:5 in blocking buffer, Appendix I.2) overnight at 4°C on a
rolling incubator3.
1 Harlan-Winkelmann, Borchen, Germany2 MVP Laboratories Inc., Ralston, Nebraska, U.S.A.3 CAT RM5, Ingenieurbüro CAT, M. Zipperer GmbH, Staufen, Germany
MATERIALS AND METHODS
67
C.6.5 Protein detection
C.6.5.1 Western blotting in a tank transfer system
Western blotting was done using the Mini Trans-Blot® system1. Proteins were trans-
ferred either to a nitrocellulose membrane2 or to a polyvinylidene fluoride (PVDF)
membrane3 as described by SAMBROOK et al. 1989 for 30 min at 50 V.
C.6.5.2 Immunoblotting
Immunoblotting was performed according to standard procedures using an alkaline
phosphatase-conjugated goat anti-rabbit IgG antibody, diluted 1:2000, as the conju-
gate, and BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (nitroblue tetra-
zolium) as substrate for visualization of protein bands (SAMBROOK et al. 1989). The
dilutions of antisera were used as indicated in Table 6.
Table 6: Dilutions of antisera used in this study
antiserum dilution
anti-TpbA 1:200
anti-TpbB 1:2000
anti-FhuA 1:2000
anti-DmsA 1:2000
1 BioRad Inc., Munich, Germany2 Protran BA85 0.45µM, Schleicher and Schuell, Dassel3 Immobilone®, Millipore, Eschborn, Germany
MATERIALS AND METHODS
68
C.7 Challenge experiment in the pig
The A. pleuropneumoniae mutants constructed in this study were examined in pigs 7-
9 weeks of age [permit no. 009i-(neu)42502-98/45]. Pigs were infected via aerosol,
thereby mimicking the natural route of infection.
C.7.1 Challenge experiment timeline
day -7: arrival at the facility, blood samples taken for enzyme linked
immunosorbent assay (ELISA)
day 0: aerosol infection
day 7: collection of bronchoalveolar lavage fluid (BALF), blood samples taken
day 14: blood samples taken
day 21: collection of BALF, blood samples taken, pigs subjected to post mortem
examination following euthanasia
C.7.2 Origin and housing of the animals
Thirty-two outbred pigs 8 to 9 weeks of age were purchased from an A. pleuropneu-
moniae-free herd (no clinical symptoms, no serological response in the ApxII-ELISA
(LEINER et al. 1999), randomly assigned to four groups, and cared for in accordance
with the principles outlined in the 'European convention for the protection of verte-
brate animals used for experimental and other scientific purposes' (ETS123). Groups
were housed in separate isolation units with controlled temperature and ventilation.
C.7.3 Aerosol infection chamber
Infections were carried out in an aerosol chamber built by Impfstoffwerk Dessau Tor-
nau1 based on the descriptions of Jacobsen et al. (JACOBSEN et al. 1996) . This
chamber allows the simultaneous infection of four pigs 7-12 weeks of age. The top of
the chamber consists of an acrylic window which allows easy surveillance of the ani-
mals during aerosol exposure. The chamber has two air vents equipped with filters,
one of which is connected to a compressor2 used to exchange the air in the chamber.
1 Dessau, Germany2 KNF Neuberger, Freiburg, Germany
MATERIALS AND METHODS
69
All tubing is made from either autoclaveable silicone or Teflon®. The bacterial sus-
pension is aerosolized via a nozzle1 operated by compressed air2.
C.7.4 Preparation of bacteria for aerosolization
For aerosol infection, the A. pleuropneumoniae strains were grown with shaking in a
200 ml culture (inoculated with 20 ml of an overnight liquid culture) for approximately
3 hours at 37°C to OD660 of 0.4. The culture was placed on ice, diluted 1:300 in ice-
cold NaCl (150 mM), and kept on ice until use for a maximum of 2 hours. Immediately
prior to aerosolation, bacteria were further diluted 1:100 in ice-cold NaCl (150 mM)
resulting in a total living cell count per 13 ml dose (for four pigs) of approximately 1 x
105/ml; upon aerosolization, this dose corresponds to approximately 1 x 102 A.
pleuropneumoniae cells per liter of aerosol in the chamber, a dose which had been ti-
trated for the A. pleuropneumoniae strain AP76 to induce severe but not fatal disease
(TEUTENBERG-RIEDEL et al., unpublished data).
C.7.5 Aerosol infection
Four pigs were put in the chamber at a time. The nozzle was set to "5", achieving a
broad spray for even distribution of the aerosol across the chamber; the valve regu-
lating the flow of the fluid was set to "75" and the challenge dose was aerosolized
over the course of approximately 2 min at a pressure of 2 bar. Ten minutes after
complete aerosolization of the challenge dose, the air in the chamber was exchanged
ten times over a duration of 20 min using a compressor, then the pigs were led back
to their stable.
C.7.5.1 Surveillance of the animals during the experiment
Pigs were examined clinically at least once a day or as needed. Body temperatures
were recorded in charts for each pig along with clinical symptoms like depression,
dyspnea or coughing. To assess the clinical pulmonary condition as noted during
BALF sampling, a clinical score based on the findings of KIPPER (1990) was em-
ployed. The criteria and the score are described in Tables 7 and 8.
1 Model no. 97058, Schlick Duesen, Untersiemau, Germany2 Linde, Hannover, Germany
MATERIALS AND METHODS
70
Table 7: Clinical findings during endoscopy of the porcine lung
physiological findings atthe tracheal bifurcation acute bronchopneumonia chronic
bronchopneumonia
glistening mucosal surface dull mucosal surface
small amounts of seroussecretions
mucous to purulent or bloodysecretions
mucosal blood vesselsinjected to a low-medium
degree
mucosal blood vesselsinjected to a high degree hyperemic mucosa
circular bronchial lumen lumen non-circular due toswelling
deformed bronchiallumina due to activation
of fibroblastscarina not deformed or
swollencarina swollen and displaced
to the left or right
Table 8: Clinical scores as evaluated during endoscopy
Score Amount ofsecretion
Quality ofsecretion
Color ofmucosa
Vascularinjection Mucosal swelling
0 none none light pink low-medium none, carina notswollen, symmetrical
1 low serous hyperemic high
deformed bronchiallumen
orswollen carina
ordeformed bronchial
subdivisions
2 medium mucous
deformed bronchiallumen and
swollen carinaor
deformed bronchialsubdivisions
3 high purulentor bloody
MATERIALS AND METHODS
71
C.7.6 Bronchoalveolar lavage fluid (BALF)1
For the collection of BALF, pigs were anaesthetized by intramuscular application of
azaperone2 (2 mg/kg) followed by an intramuscular injection of ketamine3 (15 mg/kg)
and immobilized in a specially designed hammock. To obtain BALF, a flexible bron-
choscope4 was introduced into the bronchus of the right posterior cranial lobe. The tip
of the bronchoscope was pushed into 'wedge position' as to seal the bronchus.
Twenty ml of isotonic NaCl (prewarmed to 30°C) were injected and recovered by ap-
plying a suction force of 0.2 to 0.5 bar using an especially designed vacuum pump5.
This washing process was repeated five times, and an average of 90 ml of BALF
were obtained. The BALF was kept on ice for up to 2 hours until the bacteriological
status was assessed. BALF intended for use in induction experiments was centri-
fuged at 3000 rpm for 10 minutes to remove cell debris and bacteria, sterility was
confirmed by plating of 100 µl of BALF on Columbia Sheep Blood (CSB ) agar and
supplemented PPLO agar.
C.7.6.1 Assessment of bacteriological status of BALF
One ml of BALF was centrifuged (5000 rpm, 10 min), and the pellet was resuspended
in 60 µl of NaCl (150 mM). Twenty µl were plated on supplemented PPLO agar as
well as on Gassner and CSB agar, respectively. The degree of total bacterial coloni-
zation (growth on CSB agar), as well as colonization by enterobacteria (growth on
Gassner agar) and by A. pleuropneumoniae-like bacteria (minimal growth with dis-
tinct hemolysis on CSB, good growth on supplemented PPLO agar) was assessed as
+++ (confluent), ++ (> 100 colonies), and + (< 100 colonies). In addition, the total
bacterial number as well as the number of A. pleuropneumoniae was assessed by
serial 10-fold dilutions of non-concentrated BALF and plating on CSB and supple-
1 Collection of bronchoalveolar lavage was performed by Dr. I. Hennig and Prof. M. Ganter, Clinic for
Pigs, Small Ruminants, Forensic Medicine and Ambulatory Service, Veterinary School Hannover2 Stresnil®, Janssen GmbH, Neuss, Germany3 Ursotamin®, Serumwerk Bernburg AG, Bernburg, Germany4 Type XP20, Fa. Olympus, Hamburg, Germany5 Endoaspirator, Fa. Georg Paudrach, Hannover, Germany
MATERIALS AND METHODS
72
mented PPLO-agar. Some individual A. pleuropneumoniae-like colonies were sub-
cultured on supplemented PPLO agar and confirmed PCR-analysis using specific
primers.
C.7.7 Post mortem examination
Pigs were euthanized following BALF collection on day 21 post infection by
intravenous injection of 10 ml of Eutha 77®1 per pig.
C.7.7.1 Determination of lung lesion scores
In order to assess lung damage caused by A. pleuropneumoniae infection underlaboratory conditions, HANNAN et al. (1982) developed a simple scheme of lung le-sion mapping and evaluation; by dividing the whole lung into lobes, and assessing atotal possible score of 5 for each lobe (resulting in a maximum score of 35 for theentire lung), individual lesions may be mapped on simplified lung chart in which everylobe is subdivided into triangles. The number of 'affected' triangles is then counted,and the score for this lung lobe calculated as a fraction of five (HANNAN et al. 1982).This scheme has been adapted by the European Pharmacopoeia2 as the referencemethod in vaccine trials for A. pleuropneumoniae.
C.7.7.2 Bacteriological examination of organ samples
The bacteriological examination included surface swabs of affected and unaffected
lung tissue, tonsil, bronchial lymph node, and heart muscle on supplemented PPLO
agar as well as on Gassner and Columbia sheep blood (CSB) agar. Plates were in-
terpreted as described for BALF (C.7.6.1).
C.7.8 Enzyme Linked Immunosorbent Assay (ELISA)
The generalized humoral immune response of pigs was determined in two differentELISAs. In order to assess antibody levels directed against the ApxIIA toxin, a stan-dardized ELISA based on the recombinant A. pleuropneumoniae ApxIIA protein assolid phase antigen was employed (LEINER et al. 1999). In order to assess antibodylevels directed against outer membrane components, an ELISA based on the deter-gent extract of an iron-restricted A. pleuropneumoniae Appwt culture (GOETHE et al. 1 Pentobarbital, Essex Pharma, Munich, Germany2 http://www.pheur.org
MATERIALS AND METHODS
73
2000) as solid phase antigen was used. The detergent extract was diluted 1:50 incarbonate buffer (50 mM; pH 9.6); Polysorb® 96-microwell plates1 were coated with100 µl per well at 4°C for 16 h without subsequent blocking. Plates were washed withPBST (150 mM phosphate-buffered saline [pH 7.2] containing 0.05% Tween® 20)before the addition of serum, conjugate, and chromogen. Sera were initially diluted1:100 and further twofold in the plates in PBST. An internal positive control (pool ofsera taken three weeks post infection from pigs infected with A. pleuropneumoniaeAppwt) and negative control (pool of sera taken from pigs prior to infection) was usedon each plate. Serum dilutions and goat anti-pig peroxidase conjugate were eachincubated for 1 h at room temperature. The ELISA was developed using 2,2-azino-di-[3-ethylbenzithiazoline sulfonate] (ABTS) as substrate. The test was considered validwhen the optical density (OD) of the negative serum at a 1:100 dilution was lowerthan the OD of the positive serum at a 1:12,800 dilution. The titer given is the serumdilution with an OD higher than twice the OD of the negative control serum at a 1:100dilution.
C.7.9 Enzyme Linked Immunosorbent (ELI) spot analyses2
Eighty ml of BALF were centrifuged (400 x g, 10 min), the cells were washed once inPBS and resuspended in 1.5 ml of PBS. Using phase contrast microscopy (500-foldmagnification) and a hemocytometer, the cell numbers were determined for lympho-cytes, red blood cells, and other nucleated cells (including macrophages and granu-locytes). An indirect immunofluorescence staining for lymphocyte subpopulations inthe BALF cells was performed using monoclonal porcine specific antibodies againstCD33, γ/δ T cells4, IgA5, IgG16 and IgM7. Cells from BALF were assayed for antibodysecreting cells (ASC) of the different immunoglobulin isotypes (IgA, IgG1 and IgM)
1 Nunc, Roskilde, Denmark2 ELI spot and FACS analyses were performed by Dr. A. Hoffmann-Moujahid and Prof. Dr. H.-J.
Rothkötter, Dept. of Functional and Applied Anatomy, Medical School Hannover3 8E6; VMRD, Pullman, U.S.A.4 MAC320, R.M.Binns, Babraham, UK5 MCA638, Serotec, Oxford, UK6 MCA635, Serotec, Oxford, UK7 MCA637, Serotec, Oxford, UK
MATERIALS AND METHODS
74
and for A. pleuropneumoniae-specific antibody secreting cells (ASC) of the differentisotypes by ELI spot analysis (ERIKSSON et al. 1998). Nitrocellulose-bottomed 96-well plates1 were coated with an A. pleuropneumoniae antigen preparation (detergentextract of an iron-restricted A. pleuropneumoniae Appwt culture, diluted 1:10,GOETHE et al. 2000) in PBS for 2 h at 37oC. The plates were washed and blockedusing RPMI 1640 containing 5% fetal calf serum. After removal of the block, BALFcells were added and the plates incubated overnight at 37oC in a moist atmosphere(5 % CO2). The cells were removed by intense rinsing (PBS with 0.05% Tween® 20),and monoclonal antibodies against porcine IgA, IgG1 and IgM (see above, all of IgG1isotype) were added for two hours at 37oC. An anti mouse IgG1 alkalinephosphatase-labeled conjugate was used as secondary reagent2. The color reactionwas carried out using alkaline phosphatase buffer (0.1M Tris, 0.15M NaCl, 0.05MMgCl2, pH 9.5) containing nitroblue tetrazolium (NBT; 30 µg/ml) and 5-bromo-4-chloro-3-indolylphosphate (BCIP; 16 µg/ml). The frequency of ELI spots was countedusing a stereo microscope (30-fold magnification) and expressed as number of spotsper 106 lymphocytes. The mean and standard deviation for the ELI spots wascalculated; differences p < 0.05 in the non-parametric Wilcoxon test were taken assignificant.
C.7.10 Statistics
Statistical graphics were created using Plot-It3; the non-parametric Wilcoxon test was
performed using the WinStat® plug-in module4 for Microsoft Excel®. Differences p <
0.05 in the Wilcoxon test were considered significant.
1 MAHB-N45, Millipore, Eschborn, Germany2 Southern Biotechnologies, Birmingham, U.S.A.3 Scientific Programming Enterprises, Haslett, MI, U.S.A.4 R. Fitch Software, Staufen, Germany
RESULTS
75
D Results
D.1 Role of urease and ExbB in A. pleuropneumoniae infection
Two previously constructed isogenic A. pleuropneumoniae deletion mutants
(TONPITAK et al. 2000) were used in a clinical challenge trial in order to evaluate
possible changes in virulence resulting from the deletions of urease and the ExbB
protein in A. pleuropneumoniae. The aerosol infection experiment was conducted
using eight pigs (aged 7-9 weeks) per group. An A. pleuropneumoniae strain lacking
urease expression (App∆ureC) and a strain lacking ExbB expression (App∆exbB)
(TONPITAK et al. 2000) were delivered to the animals in a specially constructed
aerosol chamber. App∆ureC is easily recognized on an agar plate by performing a
urease test as described in section C.4.1, in which urease negative colonies can be
distinguished from wild type bacteria by their yellow color (urease positive colonies
are red). App∆exbB is unable to utilize porcine transferrin as the sole iron source in a
plate bioassay, despite the fact that the expression of the transferrin binding protein
genes tpbB and tbpA situated downstream from the exbB gene remains unaffected
by the deletion (TONPITAK et al. 2000). The results of the individual animals in the
clinical challenge trial are summarized in appendix I.3.
D.1.1 Challenge experiment with A. pleuropneumoniae AP76 and isogenic
deletion mutants App∆∆∆∆ureC and App∆∆∆∆exbB
Three groups of eight pigs were infected with the respective mutant in an aerosol
chamber (C.7.3). Challenge doses as calculated via serial dilutions on PPLO agar
were as follows: 7.4 x 104 (Appwt), 9.7 x 104 (App∆ureC), and 15 x 104 (App∆exbB).
D.1.1.1 Clinical symptoms in infected pigs
One day post infection, pigs in the Appwt and App∆ureC groups began showing
symptoms typical for A. pleuropneumoniae infection (B.1.3), with symptoms in the
App∆ureC group being virtually indistinguishable from those in the wild type group.
Pigs in the App∆exbB group remained clinically healthy.
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D.1.1.2 Bacterial reisolation kinetics and pathomorphological changes in challenged
pigs
BALF was sampled one week before as well as one and three weeks after challenge.
Colonization by bacteria other than A. pleuropneumoniae, as assessed by the total
count of colony forming units (cfu), was highly variable among individual pigs (<10/
ml to 5 x 105/ ml) but no significant differences were found among the three challenge
groups or between the different sampling points. Upon challenge with A.
pleuropneumoniae App∆exbB, no A. pleuropneumoniae could be reisolated from
BALF one or three weeks after challenge. Upon challenge with A. pleuropneumoniae
App∆ureC and Appwt, the challenge strain was reisolated from BALF on day 7 and
on day 21 from the majority of pigs in these challenge groups (Table 9). No consis-
tent difference with respect to the number of A. pleuropneumoniae colonies was ob-
served between the two groups or between day 7 and 21. The correct pheno- and
genotype of isolates was confirmed by urease testing and PCR analyses.
The bacteriological examination of tonsils, lung lymph nodes, heart, pneumonic le-
sions (if present) and intact lung at the end of the challenge experiment (three weeks
after challenge) revealed that all pigs challenged with A. pleuropneumoniae
App∆exbB were culture negative (Table 9). From pigs challenged with A.
pleuropneumoniae Appwt and App∆ureC, A. pleuropneumoniae was consistently
reisolated from pneumonic lesions in pure culture with surface smears showing
dense (++) or confluent (+++) growth in 13 of 16 pigs. From heart and tonsils
reisolation succeeded sporadically with no differences between both groups. The
lymph nodes were culture-positive in four (Appwt) or five pigs (App∆ureC). The
morphologically intact lung tissue was culture positive in four pigs challenged with
Appwt whereas it was culture negative in all pigs challenged with App∆ureC (Table
9).
D.1.1.3 Systemic and local immune response of challenged pigs.
The systemic immune response was determined with two ELISA systems using re-
combinant ApxIIA protein or detergent extract as solid phase antigen (C.7.8). Among
the pigs challenged with A. pleuropneumoniae App∆exbB, none developed a
detectable immune response. In contrast, all pigs challenged with A.
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pleuropneumoniae App∆ureC or Appwt showed a strong humoral immune response
in both systems, and between both groups no significant difference was observed
(Fig. 2). The local immune response was studied based on the cells recovered from
BALF. The total BALF volume (approximately 90 ml) contained ~20 x 106 non-
lymphoid cells (mainly macrophages and granulocytes) and ~ 4 x 106 lymphocytes in
all groups. These cell numbers remained on a comparable level throughout the entire
challenge study. Using FACS analysis, lymphocytes were differentiated before
challenge as well as one and three weeks after challenge into T-cells (CD3+) and
IgM, IgA, and IgG expressing antibody secreting cells (ASC) with no obvious
differences among the groups. Using the ELI-spot assay with an A.
pleuropneumoniae detergent extract as solid phase antigen, A. pleuropneumoniae
specific ASC were determined three weeks after challenge. The number of specific
ASC was clearly increased in pigs challenged with A. pleuropneumoniae App∆ureC
in comparison to the other groups; this increase was significant (p < 0.05) for IgM and
IgG secreting cells (IgM 10 - 210 ASC/106 cells, IgG 40 - 1000 ASC/ 106 cells; Fig.
3).
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Fig. 2: Humoral immune response of pigs challenged with the A. pleuropneumoniae parent strain and isogenic mutants 7 days
before as well as 7 and 21 days after challenge.
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Fig. 3: Local immune response of pigs challenged with the A. pleuropneumoniae parent strain and
isogenic mutants 21 days after challenge. The immune response was assessed by ELI-spot analysis
differentiating between IgM (●), IgA (■), and IgG (▲) antibody secreting cells. Significant differences
are indicated by an asterisk.
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Table 9: Virulence of A. pleuropneumoniae parent and isogenic mutant strains following aerosol challenge
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D.2 Role of transferrin binding proteins in A. pleuropneumoniae infection
The avirulence of the A. pleuropneumoniae ∆exbB strain raised the question whether
this observation was merely due to the abolishment of iron uptake via porcine trans-
ferrin or to the simultaneous inactivation of other iron uptake mechanisms or other
cell functions, caused by the exbB deletion. A strain in which energy dependent iron
uptake mechanisms are impaired but not completely abolished would likely exhibit
reduced virulence and might be a promising candidate for a live vaccine. Therefore,
transferrin binding protein mutants were constructed and examined in the aerosol
infection model. As a vaccine strain should be easily distinguishable from the wild
type by phenotypic means and, in addition, should be limited with respect to shed-
ding, the urease negative A. pleuropneumoniae strain (OSWALD et al. 1999b)
seemed desirable as the recipient, since this strain had been shown to exhibit
decreased persistence in healthy lung tissue while maintaining virulence in acute
infection (D.1.1).
D.2.1 Construction of isogenic mutants lacking transferrin binding protein ex-pression
D.2.1.1 Transconjugation plasmids
Transconjugation plasmids were constructed using the vector pBMK1 (OSWALD et
al. 1999b). The functionality of this suicide A. pleuropneumoniae – E. coli shuttle
vector relies on a kanamycin (Km) resistance determinant for initial selection of
transconjugants and on the levansucrase gene from Bacillus subtilis (sacB) for
counterselection in sucrose-containing media. The expression of the SacB protein
leads to the accumulation of toxic metabolic byproducts (GAY et al. 1983; REYRAT
et al. 1998), thereby causing bacterial death. The plasmid further contains a ColEI
origin of replication (ori) which does not support plasmid replication in A. pleu-
ropneumoniae, a plasmid RP4-derived mobilization function (mob) which allows
plasmid mobilization via conjugation, and a polylinker site next to the Kmr-sacB
cassette. This 'single step transconjugation system' can be used to generate isogenic
mutants that contain the gene carrying the deletion devoid of an antibiotic resistance
determinant.
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D.2.1.1.1 pTF205EM302 for the introduction of the tbpB deletion
The transconjugation plasmid for the introduction of the tbpB deletion,
pTF205EM302, was constructed by ligation of a NsiI-BglII fragment from pTF205/E1
into pTZ18 cut with PstI and BamHI, followed by a 450 bp in-frame deletion by
digestion with NheI and XbaI, and religation. The insert was cloned on a SmaI-
HindIII-fragment into pBluescript SK, removed by digestion with ApaI and NotI and
ligated into pBMK1 (Fig. 4).
D.2.1.1.2 pTF205mut404 for the introduction of the tbpA deletion
The transconjugation plasmid for the introduction of the tbpB deletion,
pTF205mut404, was constructed by ligation of a 2.4 kb NheI-NsiI fragment from
pTF205/405 into pUC19, cutting with XbaI and PstI, transformation into E. coli GS301
(dam) to obtain unmethylated DNA, deletion of 930 bp by digestion with BsaBI, and
religation. The insert was cloned on a HindIII-SmaI fragment into pBluescript SK cut
with HindIII and HincII, removed by digestion with ApaI and NotI and ligated into
pBMK1 (Fig. 5).
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Fig. 4: Construction of pTF205EM302. Arrows denote direction of respective reading frames. Km,
Kanamycin resistance determinant; Ap, Ampicillin resistance determinant. A. pleuropneumoniae gene
fragment in magenta, the fragment to be deleted is highlighted.
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Fig. 5: Construction of pTF205mut404. Arrows denote direction of respective reading frames. Km,
Kanamycin resistance determinant; Ap, Ampicillin resistance determinant. A. pleuropneumoniae gene
fragment in orange, the fragment to be deleted is highlighted.
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D.2.1.2 Construction of isogenic mutants
A. pleuropneumoniae ∆tbpB (App∆tbpB) and A. pleuropneumoniae ∆tbpA
(App∆tbpA) were constructed by homologous recombination upon the introduction of
plasmids pTF205EM302 and pTF205mut404 (Table 2, Fig. 4, Fig. 5), respectively,
using diaminopimelic acid auxotrophic E.coli β2155 as the donor for conjugation into
A. pleuropneumoniae ∆ureC (App∆ureC). Kanamycin resistant colonies were
screened for the presence of the introduced shortened gene via PCR (primers
oMut1r+f, oMut4r+f), and positive clones were subjected to counterselection on
sucrose (C.5.7.2).
A. pleuropneumoniae ∆tbpBA (App∆tbpBA) was constructed by introduction of plas-
mid pTF205EM302, using A. pleuropneumoniae ∆tbpA as the parent strain. Mutants
were confirmed by PCR and subsequent nucleotide sequence analyses (Fig. 6A), as
well as by Southern analyses of genomic DNA. Here, DNA was cut with restriction
enzymes NsiI and KpnI or NsiI and BglII, and tbpB or tbpA specific probes prepared
from PCR products were used to demonstrate successful deletion of the predicted
fragment, respectively. Restriction sites were situated outside of the deletions, re-
sulting in smaller bands in those strains carrying the deletion (Fig. 6B). Expression of
a truncated TbpB protein was shown by Western blotting and immunodetection of
Tbp bands with rabbit antisera (Fig. 6C), and the retained ability of A.
pleuropneumoniae ∆tbpB to utilize transferrin bound iron was demonstrated in a plate
bioassay (Fig. 6D, C.4.2).
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Fig. 6: Analysis of A. pleuropneumoniae tbp mutant strains. A. pleuropneumoniae ∆ureC
(lanes 1), A. pleuropneumoniae ∆tbpB (lanes 2), A. pleuropneumoniae ∆tbpA (lanes 3), and A.
pleuropneu-moniae ∆tbpBA (lanes 4). A, PCR using primers oMut1 and oMut2 (a) or oMut4f and
oMut4r (b); lane N contains a negative control; B, Southern blot analysis using PCR products specific
for tbpB (left) and tbpA (right) as probes; C, Coomassie blue-stained gel (left) and Western blots
developed with serum directed against the TbpB (middle) and TbpA protein (right) of whole cell lysates
obtained from cultures grown under iron-restricted conditions; the solid arrows indicate the position of
the TbpB protein and its truncated derivative, the open arrow indicates the position of the TbpA
protein; D, Plate bioassay with strains cultured on iron-depleted brain heart infusion agar
supplemented via paper disks with porcine transferrin (TF), dilution buffer (B) and ferric citrate (C).
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D.2.2 Challenge experiment with A. pleuropneumoniae ∆∆∆∆ureC and isogenic
deletion mutants A. pleuropneumoniae ∆∆∆∆tbpB, A. pleuropneumoniae ∆∆∆∆tbpA and
A. pleuropneumoniae ∆∆∆∆tbpBA
Four groups of eight pigs were infected with the respective mutant in an aerosol
chamber (C.7.3). Challenge doses for four pigs as calculated via serial dilutions on
PPLO agar were as follows: 9.9 x 104 for the A. pleuropneumoniae ∆tbpBA group,
1.2 x 105 for the A. pleuropneumoniae ∆tbpA group, 1.1 x 105 for the App∆tbpB group
and 1.1 x 105 for the A. pleuropneumoniae ∆ureC group.
D.2.2.1 Clinical symptoms in infected pigs
One day post infection, pigs in the A. pleuropneumoniae ∆ureC group began showing
symptoms typical for A. pleuropneumoniae infection (B.1.3). None of the animals in
the groups challenged with the tbp mutant strains exhibited signs of disease. Two
animals had to be euthanized due to severe respiratory symptoms on days four and
nine post infection, respectively. Another animal died from circulatory failure following
anesthesia and BALF collection on day 7 post infection.
D.2.2.2 Bacterial reisolation kinetics and pathomorphological changes in challenged
pigs
BALF was sampled one week before as well as one and three weeks after challenge.
Colonization by bacteria other than A. pleuropneumoniae, as assessed by the total
count of colony forming units (cfu), was highly variable among individual pigs (<10/ml
to 5 x 105/ml) but no significant differences were found among the four challenge
groups or between the different sampling points. Upon challenge with A.
pleuropneumoniae ∆ureC, the challenge strain was reisolated from BALF on day 7
from two animals (Table 16, appendix I.5). The correct pheno- and genotype of
isolates was confirmed by urease testing and PCR analysis.
A. pleuropneumoniae ∆ureC was consistently reisolated from pneumonic lesions in
pure culture with surface smears showing dense (++) or confluent (+++) growth in 7
of 8 pigs. From tonsils, reisolation succeeded in one animal. The lymph nodes were
culture-positive in three pigs. A. pleuropneumoniae could not be reisolated from ani-
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mals challenged with the tbp mutants. Challenge trial data for individual pigs is listed
in Table 16 (appendix I.35).
D.2.2.3 Systemic immune response of challenged pigs
Pigs in the A. pleuropneumoniae ∆ureC group had antibody titers ranging from 7 to
38 in the ApxII ELISA and 1:3200 to 1:12800 in the detergent wash ELISA. No ani-
mal in any of the groups infected with tbp deletion mutants developed a detectable
humoral immune response against A. pleuropneumoniae (Table 16).
D.3 Identification of genes expressed in vivo
D.3.1 Representational Difference Analysis of cDNA
As it had been shown that transferrin binding protein expression is downregulated
over the course of infection (HENNIG et al. 1999), it was hypothesized that alternate
iron uptake mechanisms would in turn be upregulated at that point to secure the
pathogen's iron supply. In order to identify such genes, as well as other virulence-
related genes that are expressed by A. pleuropneumoniae in vivo, an ex vivo system
using the addition of BALF to the growth medium was used (C.3.2, C.5.5.1). Starting
from total RNA from A. pleuropneumoniae AP76 grown with the addition of BALF as
'tester' and total RNA from A. pleuropneumoniae AP76 grown under standard cultur-
ing conditions as 'driver', fragments obtained after one round of representation were
cloned into the TOPO pCR2.1 vector, and PCR reactions were performed of 170
clones to visualize cloned RDA fragments. In order to identify differentially expressed
genes, those PCR fragments were examined by Southern blotting using radiolabeled
A. pleuropneumoniae chromosomal DNA as well and tester and driver amplicon
cDNA (Fig. 7), 13 differentially expressed DNA fragments from 281 to 597 base pairs
in length were identified. Among these fragments, RN7 and RN26 were overlapping
fragments, and RN29 and RN33 as well as RN35 and RN38 were identical. Frag-
ments were analyzed by nucleotide sequencing and data base homology search.
The origin of the fragments was controlled by a PCR using genomic A.
pleuropneumoniae DNA and primers derived from the respective nucleotide
sequences (Fig. 8, Table 3). The data obtained for the individual fragments are
summarized in Table 10.
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Fig. 7: Southern blotting of RDA fragments (excerpt) showing PCR fragments hybridized to radio-
labeled genomic DNA (A), driver amplicon cDNA (B) and tester amplicon cDNA. Arrows mark frag-
ments selected for sequence analysis.
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Table 10: RDA fragments identified via differential hybridization
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Fig. 8: Amplification of RDA products from genomic A. pleuropneumoniae AP76 DNA using
internal RDA fragment primers; 5- 17, RDA fragment numbers.
D.3.2 Isolation of larger fragments containing RDA fragments
From among the RDA fragments, RN5 and RN 29 were chosen for further characteri-
zation. The decision was made based on the following assumptions: DMSO reduc-
tase (RN5), which in E. coli enables the organism to utilize DMSO as the terminal
electron acceptor, might influence A. pleuropneumoniae survival under the anaerobic
or reduced-oxygen conditions encountered in abscesses, and a ferric compound up-
take receptor (RN29) might play an important role in in vivo iron uptake, particularly
after the downregulation of the transferrin binding proteins one week post infection.
Since the RDA fragments were too small for a sequence-based characterization of
the respective genes, larger gene fragments containing the RDA fragments had to be
identified. A DNA colony blot approach using a sorted A. pleuropneumoniae gene
bank was chosen for this purpose.
D.3.2.1 Construction of a sorted A. pleuropneumoniae genomic library
Total genomic DNA of A. pleuropneumoniae was partially digested with Sau3aI, and
fragments between 2 and 9 kb in size were ligated into pGH433 cut with BamHI or
BglII and pGH432 cut with BglII and transformed into E. coli DH5αF'. Single colonies
were transferred into 96-well microtiter plates using a sterile pipette tip, vacuum
pump and sterile glass beads.
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D.3.2.2 Colony blotting
Transformants from the genomic library were transferred to agar plates from the mi-
crotiter plates using a 96-well replica plater1 and grown at 37°C overnight. Colonies
were transferred onto nitrocellulose, treated according to the protocol described
(C.5.8.6), and hybridized with a pool of probes prepared from PCR products of the
RDA fragments that were selected for further analyses. Positive clones were subcul-
tured and a second round of colony blotting was performed with single probes. For
each RDA fragment, at least one transformant was identified (Table 11).
Table 11: Large fragments containing original RDA fragments
RDA fragment
Name of clone
carrying large
fragment
Gene bank plasmid and
cloning site
RN5 pDM5-116 pGH 433 BglII
RN29 pFH29-4 pGH 433 BglII
RN29 pFH29II-31 pGH 432 BglII
D.3.3 Localization of RDA fragments on the A. pleuropneumoniae genomicmap
In order to determine the relative position of the identified RDA fragments on the ge-
nomic map (OSWALD et al. 1999a), Southern blot analyses of PFGE gels were per-
formed for RN5 and RN29, again using RDA fragment PCR products as probes. RDA
fragment positions were then mapped, results are shown in Fig. 9.
1 Boekel Scientific, Feasterville, PA, U.S.A.
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Fig. 9: Genomic map of A. pleuropneumoniae AP76 with localization of two RDA fragments indi-
cated by arrows, ApaI, AscI and NotI fragments that were used to construct the map are shown in
purple, green and blue, respectively, with fragment names in capitals.
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Characterization of large fragm
ents
Using a variety of restriction endonucleases, physical m
aps of pDM
5-116, pFH29-4
and pFH29-II31 w
ere constructed (Fig. 10).
Fig. 10: Plasmid maps of plasmids pDM5-116, pFH29-4 and pFH29-II31. Colored boxes on mapline indicate the position
of the RDA fragment, arrows denote localization and direction of respective open reading frames and regions of homology.
Vector sequences are not drawn to scale.
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D.3.5 A. pleuropneumoniae dimethylsulfoxide (DMSO) reductase subunitDmsA
D.3.5.1 Sequence analyses
The fragment of 440 bp in length and designated as RN5 contained a continuous
open reading frame (ORF) showing similarity to the catalytic subunit DmsA of
Haemophilus influenzae and E.coli (BILOUS and WEINER 1985, LOOSMORE et al.
1996). Sequence analysis of plasmid pDM5-116 containing RN5 revealed a 2220 bp
open reading frame coding for a protein of 739 amino acids in lengths with a cal-
culated molecular mass of 82 kDa1 bearing 80% identity on the DNA level2 as well as
87% identity and 95% similarity on the protein level3 to the DMSO reductase catalytic
subunit DmsA of Haemophilus influenzae. Similarities to other known genes are
summarized in Table 12. Five bases upstream of the start codon, a putative Shine-
Dalgarno sequence (GGAG) was identified (Fig. 11). 106 bases upstream of the
dmsA start codon lies a putative binding site for the Fnr homologue of A. pleu-
ropneumoniae, HlyX, with the sequence TTGAT----ATCAG, which is a close match to
the proposed consensus sequence for Fnr, TTGAT----ATCAA (GREEN and
BALDWIN 1997). At the N-terminus of the protein sequence, a twin arginine leader
sequence was identified, conforming to the conserved consensus sequence (S/T)-R-
R-x-F-L-K (BERKS 1996) that is typical of proteins that are exported or targeted to
the membrane via the twin arginine translocation pathway (SARGENT et al. 1998).
An alignment of several known DmsA leader sequences is shown in Fig. 12. Using
the SignalIP V1.1 World Wide Web Server4 (NIELSEN et al. 1997a), a signal
peptidase cleavage site was predicted to be situated between AS33 (alanine) and
AS34 (glutamic acid) (Fig. 11). In contrast to the situation in Haemophilus influenzae
(LOOSMORE et al. 1996), no dmsB orf was found immediately downstream of the
1 HUSAR program package - PEPSTATS2 HUSAR program package - blastn3 HUSAR program package - blastp4 http://www.cbs.dtu.dk/services/SignalP
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dmsA orf. The complete nucleotide and protein sequences of A. pleuropneumoniae
dmsA are listed in appendix I.3.
Fig. 11: DmsA signal peptide and upstream nucleotide sequence. Start codon and signal se-
quence highlighted in green, putative HlyX binding site highlighted in yellow, TAT leader consensus
sequence highlighted in red, predicted signal peptidase cleavage site indicated by arrow, SD putative
Shine-Dalgarno sequence.
Fig. 12: Alignment of DmsA TAT leader sequences from several bacteria. Position of the con-
served consensus sequence (S/T)-R-R-x-F-L-K is bold for A. pleuropneumoniae, as well as the twin
arginines for all organisms.
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Table 12: A. pleuropneumoniae dmsA - similarities to known genes.
organism gene identity onDNA level
identity onprotein level -
lowest
identity onprotein level -
highest
accessionnumber(s)
H. influenzae dmsA 80% 27% 87% HI26665P45004
E. coli dmsA 66% 40% 74% ECDMSP18775
S. Typhi dmsA n.i. 40% 74% AB0612
R. sphaeroides dmsA n.i. 22% 61% Q57366
Y. pestis dmsA n.i. 72% 77% AI0403
n.i. not indicated in search results
D.3.5.2 dmsA transcription
In order to verify the RDA result, Northern blot analysis was attempted. 20 µg of total
RNA isolated from A. pleuropneumoniae grown under standard conditions and with
the addition of BALF (as described for the RDA procedure (C.5.5) were separated on
a 1 % formaldehyde gel and subjected to Northern blot analysis, using a PCR
product generated with primers oRN5-1 and oRN5-2 as probe. No specific signal was
observed.
D.3.5.3 A. pleuropneumoniae DmsA expression
DmsA protein expression was examined using a polyclonal rabbit antiserum raised
against a DmsA-GST fusion protein expressed from plasmid pDMP1. Low-level
DmsA expression was detected in A. pleuropneumoniae AP76 under standard
culturing conditions (Fig. 13). Expression was clearly enhanced under anaerobic
conditions (Fig. 14) and a slight enhancement of expression was also seen upon the
addition of ferric sulfate to the culture medium under aerobic conditions, while iron
depletion and addition of ferric citrate had no influence on aerobic DmsA expression
(Fig. 13).
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Fig. 13: Western blot analysis of DmsA expression. A, Coomassie Blue stained SDS-PAGE gel; B,
Western blot; 1, A. pleuropneumoniae AP76 grown under standard conditions; 2, A. pleuropneumo-
niae ∆dmsA grown under standard conditions; 3, A. pleuropneumoniae AP76 grown under iron re-
stricted conditions; 4, A. pleuropneumoniae ∆dmsA grown under iron restricted conditions; 5, A. pleu-
ropneumoniae AP76 grown with the addition of BALF; 6, A. pleuropneumoniae AP76 grown with the
addition of ferric citrate [20 µM]; 7, 50 µM ferric citrate; 8, 20 µM ferric sulfate; 9, 50 µM ferric sulfate.
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Fig. 14: Western blot analysis of anaerobic DmsaA expression. A, Coomassie Blue stained SDS-
PAGE gel; B, Western blot; 1, A. pleuropneumoniae AP76 grown under standard conditions; 2, A.
pleuropneumoniae ∆dmsA grown under standard conditions; 3, A. pleuropneumoniae AP76 grown
under anaerobic conditions; 4, A. pleuropneumoniae ∆dmsA grown under anaerobic conditions.
D.3.5.4 Presence of dmsA in serotype reference strains
A PCR product of identical size was amplified from A. pleuropneumoniae AP76 as
well as from A. pleuropneumoniae biotype 1 reference strains 1-12 using primers
oDMSAdel1 and oDMSAdel2 (Fig. 15).
Fig. 15: PCR using primers oDMSAdel1 and oDMSAdel2. 1-12, A. pleuropneumoniae serotype
reference strains; +, AP76; -, no DNA template.
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D.3.6 A. pleuropneumoniae ferric hydroxamate uptake receptor (FhuA) homo-logue
D.3.6.1 Sequence analyses
The fragment of 440 bp in length and designated as RN29 contained a continuous
open reading frame (ORF) showing 62% identity to the E. coli ferrichrome-iron outer
membrane receptor FhuA gene (fhuA) on the DNA level, and up to 53% identity and
up to 100% similarity on the protein level. Similarities of the full fhuA reading frame to
other known genes are summarized in Table 13. In order to investigate the
expression and function of the FhuA homologue, the complete ORF of A.
pleuropneumoniae AP76 containing RN29 was cloned by identifying overlapping
clones from the genomic library. Two plasmids containing 3100 bp and 4500 bp of A.
pleuropneumoniae DNA and designated pFH29-4 and pFH29-II31, respectively, were
recovered (Fig. 10). Combined, these plasmids contained the complete ORF of the
FhuA-homologue of 1995 bp in length, encoding for a protein 664 amino acids in
length with a calculated molecular mass of 74 kDa, and a partial ORF showing
similarity to E. coli fhuB (30% identity and 56% similarity on the protein level). Using
the SignalIP V1.1 World Wide Web Server, the signal peptidase cleavage site was
predicted to be situated between AS23 (alanine) and AS24 (glutamine) (Fig. 16). The
complete nucleotide and protein sequences of A. pleuropneumoniae fhuA are listed
in appendix I.4.
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Fig. 16: FhuA signal peptide and upstream nucleotide sequence. fhuA start codon and signal
sequence in green highlighted in green, predicted signal peptidase cleavage site indicated by arrow,
SD, putative Shine-Dalgarno sequence, fhuB stop codon highlighted in green.
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Table 13: A. pleuropneumoniae fhuA homologue - similarities to known genes.
n.i. not indicated in search results
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D.3.6.2 fhuA transcription
In order to verify the RDA result, Northern blot analysis was performed. 20 µg of total
RNA isolated from A. pleuropneumoniae grown under standard conditions and with
the addition of BALF (as described for the RDA procedure (C.3.2, C.5.5.1) were
separated on a 1 % formaldehyde gel and subjected to Northern blot analysis, using
a PCR product generated with primers oRN29-1 and oRN29-2 as probe. No specific
signal was observed.
D.3.6.3 Presence of fhuA in serotype reference strains
To investigate the presence and expression of A. pleuropneumoniae fhuA, 12 sero-
type reference strains were analyzed. An identical fragment was amplified from A.
pleuropneumoniae AP76 as well as serotype 4 and 7 reference strains using primers
oFDEL1 and oFDEL2 (Fig. 17). A subsequent Southern blot analysis from genomic
DNA cut with EcoRI and DraI, using the oFDEL1-oFDEL2 PCR product as probe,
revealed that the fhuA gene seems to be unique to A. pleuropneumoniae serotypes 4
and 7 (Fig. 18).
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Fig. 17: PCR using primers oFDEL1 and oFDEL2. 1-12, A. pleuropneumoniae serotype reference
strains; +, AP76; -, no DNA template.
Fig. 18: Southern blot analysis of fhuA from genomic DNA cut with EcoRI and DraI using the
oFDEL1-oFDEL2 PCR product as probe; 1-12 A. pleuropneumoniae serotype reference strains, 13 A.
pleuropneumoniae AP76, 14 A. pleuropneumoniae ∆dmsA, 15 A. pleuropneumoniae ∆fhuA.
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D.3.6.4 A. pleuropneumoniae FhuA expression
FhuA expression was detected in A. pleuropneumoniae AP76 as well as in the sero-
type 4 and 7 reference strains (Fig. 19).
Enhanced expression of the A. pleuropneumoniae fhuA homologue could not be de-
tected upon the addition of BALF, under iron restricted conditions or upon the addi-
tion of ferric citrate (20 or 50 µM) (Fig. 20). Upon the addition of ferric sulfate (20 or
50 µM, Fig. 20), as well as under anaerobic conditions (Fig. 21), a slight decrease in
expression was demonstrated.
Fig. 19: Expression of FhuA in A. pleuropneumoniae serotype reference strains. 1-12, reference
strains; 13, A. pleuropneumoniae AP76
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Fig. 20: Western blot analysis of FhuA expression. A, Coomassie Blue stained SDS-PAGE gel; B,
Western blot; 1, A. pleuropneumoniae AP76 grown under standard conditions; 2, A. pleuropneumo-
niae ∆fhuA grown under standard conditions; 3, A. pleuropneumoniae AP76 grown under iron re-
stricted conditions; 4, A. pleuropneumoniae ∆fhuA grown under iron restricted conditions; 5, A. pleu-
ropneumoniae AP76 grown with the addition of BALF; 6, A. pleuropneumoniae AP76 grown with the
addition of 20 µM ferric citrate; 7, 50 µM ferric citrate; 8, 20 µM ferric sulfate; 9, 50 µM ferric sulfate.
RESULTS
107
Fig. 21: Western blot analysis of anaerobic FhuA expression. A, Coomassie Blue stained SDS-
PAGE gel; B, Western blot; 1, A. pleuropneumoniae AP76 grown under standard conditions; 2, A.
pleuropneumoniae ∆fhuA grown under standard conditions; 3, A. pleuropneumoniae AP76 grown un-
der anaerobic conditions.
D.3.7 Construction of deletion mutants
In order to investigate the functions of DmsA and FhuA in vivo, isogenic deletion
mutants were constructed using a single-step crossover technique (OSWALD et al.
1999b).
D.3.7.1 Construction of pBMKD1 for the introduction of a dmsA deletion into A. pleu-
ropneumoniae
In order to construct a transconjugation vector for the introduction of a dmsA deletion
into A. pleuropneumoniae, a 1988 bp EcoRI-NsiI fragment from pDM5-116 was
cloned into pBluescript to generate plasmid pDM5-118. A 956 bp SwaI-NdeI frag-
ment was deleted from the dmsA reading frame by cutting, blunt ending and religa-
tion, resulting in pDM5-119. A SalI-XbaI fragment from pDM5-119 was ligated into
pBMK1 cut with SalI and XbaI, resulting in transconjugation plasmid pBMKD1 (Fig.
22).
RESULTS
108
D.3.7.2 Construction of pECF2 for the introduction of an fhuA deletion into A.
pleuropneumoniae
For the construction of an fhuA deletion mutant, an improved transconjugation vector,
pEMOC2 (TONPITAK, unpublished), was used. This vector is based on pBMK1, but
contains a chloramphenicol (Cm) resistance determinant1 under the control of an A.
pleuropneumoniae omlA promoter instead of a kanamycin (Km) resistance
determinant, and a 1400 bp spacer between the ApaI and NotI restriction sites.
In order to construct construct a transconjugation vector for the introduction of an
fhuA deletion into A. pleuropneumoniae, a 2200 bp EcoRI-HindIII fragment from
pFH29-4 was cloned into pBluescript, resulting in pFH29-6. A 559 bp BclI-BstEII
fragment from pFH29-6 was deleted from the fhuA reading frame to generate pFH29-
7. An ApaI-NotI fragment from pFH29-7 was cloned into pEMOC2 after removal of
the spacer between the ApaI and NotI restriction sites, resulting in transconjugation
vector pECF2 (Fig. 23)
1 Amersham Bioscience
RESULTS
109
Fig. 22: Construction of pBMKD1. Arrows denote direction of respective reading frames. Km,
Kanamycin resistance determinant; Ap, Ampicillin resistance determinant. A. pleuropneumoniae gene
fragment in light blue, fragment to be deleted is highlighted.
RESULTS
110
Fig. 23: Construction of pECF2. Arrows denote direction of respective reading frames. Km,
Kanamycin resistance determinant; Ap, Ampicillin resistance determinant. A. pleuropneumoniae gene
fragment in green, fragment to be deleted is highlighted.
RESULTS
111
D.3.7.3 Construction and analysis of isogenic dmsA and fhuA deletion mutants
pBMKD1 (Kmr) and pECF2 (Cmr) were transconjugated into A. pleuropneumoniae
AP76. The presence of plasmid cointegrates in the A. pleuropneumoniae genome
was detected by PCR using primers oDMSAdel1/oDMSAdel2 and oFDEL1/oFDEL2,
respectively. Eighty percent of Kmr and all of the Cmr transconjugants contained
plasmid cointegrates. Several transconjugants were selected for counterselection
with sucrose. Ten percent of sucrose resistant colonies showed the expected PCR
profile (Fig. 24). Mutants were verified in a Southern blot analysis, for which genomic
DNA was cut with EcoRI and DraI, blotted, and probed with oDMSAdel1/oDMSAdel2
and oFDEL1/oFDEL2 PCR products, respectively. In A. pleuropneumoniae ∆dmsA,
the DraI restriction site was obliterated by the deletion, resulting in the disappearance
of a 1.2 kb band in the blot. In A. pleuropneumoniae ∆fhuA, two DraI restriction sites
are situated outside the deletion sites, resulting in a smaller band appearing on the
blot (Fig. 25). Absence of gross genomic rearrangements was shown via PFGE (Fig.
26).
Fig. 24: PCR of A. pleuropneumoniae deletion mutants. 1, primers oDMSAdel1 and oDMSAdel2;
a, A. pleuropneumoniae AP76; b, A. pleuropneumoniae ∆dmsA; c, no DNA template; 2, primers
oFDEL1 and oFDEL2; a, A. pleuropneumoniae AP76; b, A. pleuropneumoniae ∆fhuA; c, no DNA tem-
plate.
RESULTS
112
Fig. 25: Southern blot analysis of deletion mutants. A, agarose gel, genomic A. pleuropneumoniae
AP76 DNA cut with EcoRI and DraI; B, oDMSAdel1/oDMSAdel2 PCR product as probe; C,
oFDEL1/oFDEL2 PCR product as probe; a, A. pleuropneumoniae AP76; b, A. pleuropneumoniae
∆dmsA; c, A. pleuropneumoniae ∆fhuA.
Fig. 26: PFGE analysis of A. pleuropneumoniae mutants. 1, A. pleuropneumoniae AP76; 2, A.
pleuropneumoniae ∆dmsA; 3, A. pleuropneumoniae ∆fhuA.
RESULTS
113
D.3.8 Functional analysis of FhuA
A plate bioassay using ferrichrome, 2,3-dihydroxybenzoic acid, porcine transferrin
and ferric citrate as sources of iron did not reveal any differences between the A.
pleuropneumoniae AP76 wild type and ∆fhuA strains, all offered iron sources were
utilized by both strains.
D.3.9 Role of DmsA and the FhuA homologue in A. pleuropneumoniae infec-tion
D.3.9.1 Aerosol infection experiment
A. pleuropneumoniae ∆fhuA (App∆fhuA) and A. pleuropneumoniae ∆dmsA
(App∆dmsA) were used in an aerosol infection model in comparison to the A.
pleuropneumoniae AP76 wild type strain (Appwt). The challenge doses were 1.28 x
105 bacteria for four pigs in the Appwt group, 1.7 x 105 bacteria in the App∆dmsA
group and 0.49 x 105 bacteria in the App∆fhuA group. Body temperatures and clinical
symptoms like depression, inappetence and coughing were monitored daily. Body
temperatures exceeded 40°C in 6 of 8 animals in the Appwt group, in 2 of 8 animals
in the App∆dmsA group and in one animal in the App∆fhuA group one day post in-
fection. Overall, body temperatures in the App∆fhuA groups were significantly lower
than those in the Appwt group on days 2-4 post infection, in the App∆dmsA group,
the difference was significant on day 3 post infection (Fig. 27). One animal in the
App∆fhuA group was euthanized on day 9 post infection, two days after BALF had
been collected, due to severe respiratory symptoms. BALF was sampled one week
before as well as one and three weeks after challenge. The clinical findings from the
endoscopy were documented according to the guidelines delineated in Table 7. The
difference in endoscopical scores between the Appwt and App∆dmsA groups was
statistically significant (p< 0.05) on day 7 post infection (Fig. 28). Upon challenge with
A. pleuropneumoniae App∆dmsa and Appwt, the challenge strains were reisolated
from BALF on day 7 and on day 21 post infection from several pigs in both challenge
groups, while A. pleuropneumoniae could not be reisolated from BALF of animals
challenged with A. pleuropneumoniae ∆fhuA (Table 14). No consistent difference
RESULTS
114
with respect to the number of A. pleuropneumoniae colonies was observed between
the three groups or between day 7 and 21.
Fig. 27: Body temperatures of pigs over the course of eight days, day 0 marking the day of infec-
tion. Asterisks denote statistical significance.
Fig. 28: Endoscopical scores before infection as well as 7 and 21 days after infection. The asterisk
denotes statistical significance (p< 0.05).
RESULTS
115
D.3.9.2 Post mortem examination
The post mortem examination of the pigs on day 21 post infection revealed that
seven of eight animals challenged with Appwt, six of eight animals challenged with
App∆dmsA and three of seven animals challenged with App∆fhuA had lung lesions.
However, the differences in lung scores were not statistically significant. The bac-
teriological examination of tonsils, lung lymph nodes, heart, pneumonic lesions (if
present) and intact lung revealed that only pigs with lung lesions were culture posi-
tive. A. pleuropneumoniae was consistently reisolated from surface smears of pneu-
monic lesions, including abscesses, showing dense or confluent growth. The me-
diastinal lymph nodes were culture-positive in one pig in each group. The morpho-
logically intact lung tissue was culture positive in all pigs with lung lesions (seven of
eight pigs challenged with Appwt, six of eight pigs challenged with App∆dmsA, three
of seven pigs challenged with App∆fhuA, Table 14). The raw data for this challenge
experiment can be obtained from Table 17 in appendix I.5.
D.3.9.3 Systemic immune response
The systemic immune response was determined with two ELISA systems, using de-
tergent extract or recombinant ApxIIA protein as the solid-phase antigen. On day 7
post infection an increase in the serum titer in the detergent wash was seen in the A.
pleuropneumoniae wt group; this increase was significantly higher than that in the A.
pleuropneumoniae ∆fhuA and ∆dmsa mutant groups (Fig. 29). A response towards
the ApxIIA antigen was first observed two weeks after infection. The Appwt group
reacted significantly stronger than the ∆fhu group towards this antigen at two and
three weeks post infection. In the ∆dmsa group, a significantly lower titer than in the
Appwt group was seen in the detergent wash ELISA at this time point (Fig. 29).
RESULTS
116
Fig. 29: Humoral immune response of pigs challenged with the A. pleuropneumoniae parent strain
and isogenic mutants 7 days before as well as 7 and 21 days after challenge. Asterisks denote
statistically significant results (p< 0.05)
RESU
LTS
117
Table 14: Virulence of A. pleuropneumoniae AP76 and isogenic mutant strains following aerosol challenge.
DISCUSSION
118
E Discussion
The elucidation of molecular mechanisms responsible for virulence and persistence
as well as the development of safe and efficacious cross-protective vaccines are, at
present, major goals in A. pleuropneumoniae research. A desirable vaccine would be
a live marker vaccine that i) is easily distinguishable from the wild type strain, ii) pro-
tects against all serotypes, and iii) gives a protective immune response after a single
application. Two approaches for genetic manipulation of the organism have been de-
scribed, i) a random approach using chemical or transposon mutagenesis to obtain
non-hemolytic mutants (INZANA 1991), non-encapsulated mutants (INZANA et al.
1993), urease negative (TASCON CABRERO et al. 1997) or rough mutants (RIOUX
et al. 1998), and ii) a specific approach aimed at the construction of defined isogenic
mutants; here, homologous recombination needs to occur upon the introduction of
suicide vectors containing truncated and therefore non-functional genes. These vec-
tors can be delivered to A. pleuropneumoniae either via electroporation (JANSEN et
al. 1995; WARD et al. 1998; PRIDEAUX et al. 1999) or via transconjugation (MULKS
and BUYSSE 1995; FULLER et al. 1996). Commonly, the isogenic mutants then
carry a permanent antibiotic resistance determinant, a trait that is highly undesirable
in a strain to be used as a vaccine. More recently, a homologous recombination ap-
proach resulting in unmarked isogenic mutants has been developed (OSWALD et al.
1999b) and used successfully to construct mutants lacking urease and ExbB expres-
sion. In the study presented here, these mutants were tested in an aerosol infection
model. In addition, isogenic mutants with deletions in the transferrin receptor con-
sisting of the two transferrin binding proteins TbpA and TbpB were constructed and
and tested in vivo. The third part of this study was intended to identify alternate iron
uptake mechanisms and other host-induced factors.
E.1 Role of urease and ExbB in A. pleuropneumoniae infection
The goal of this experiment was to determine the role of putative A. pleuropneumo-
niae virulence factors in persistence and chronic infection. In our approach using
isogenic mutants, two target genes, the exbB and the ureC genes, were chosen. The
exbB gene was selected as it had been shown previously that a deletion of this gene
DISCUSSION
119
completely inhibited utilization of transferrin-bound iron without preventing expression
of the TbpB protein (TONPITAK et al. 2000) which is known to be a protective anti-
gen (ROSSI-CAMPOS et al. 1992). Since the expression of TbpB protein is par-
ticularly high in acute infection and decreases during the course of disease (HENNIG
1997) and since it had been shown that A. pleuropneumoniae can obtain iron from
siderophores of other bacteria (DIARRA et al. 1996) and, in addition, is able to bind
hemin and haemoglobin (BELANGER et al. 1995), it was hypothesized that the iso-
genic exbB mutant App∆exbB would be attenuated in the acute phase of disease but
would still be able to persist.
The ureC gene was selected since acute A. pleuropneumoniae infection can occur
upon experimental infection using a high challenge dose of a urease-negative A.
pleuropneumoniae mutant (TASCON CABRERO et al. 1997; BOSSE and
MACINNES 2000), and a urease negative field strain has been recovered in one
case of natural infection (BLANCHARD et al. 1993). For other pathogens causing
respiratory tract infections either no effect (in the case of Bordetella bronchiseptica,
MCMILLAN et al. 1999) or a slight decrease in multiplication and persistence (in the
case of Mycobacterium bovis, REYRAT et al. 1996) had been observed. It was hypo-
thesized that urease might support the persistence and shedding of A. pleuropneu-
moniae by locally counteracting the reactive decrease of pH occurring upon infection.
As the RTX-toxin of E. coli is known to be inhibited by subneutral pH-values
(SCHMIDT et al. 1996), a local urease-mediated return to physiological pH might
maintain or restore the toxic efficacy of A. pleuropneumoniae Apx toxin and thereby
impair local defense mechanisms of the host, particularly in the late stage of infec-
tion. Additionally, absence of urease activity serves as a convenient marker.
The results we obtained with A. pleuropneumoniae App∆exbB in part contradicted
this prediction. The complete absence of these mutants in BALF even only one week
after infection and the lack of any specific humoral or local immune response implies
that the ExbBD-mediated uptake of transferrin-bound iron is required for A. pleu-
ropneumoniae virulence. The iron-uptake via exogenous siderophores (DIARRA et
al. 1996) is apparently not sufficient to facilitate colonization of the respiratory tract by
A. pleuropneumoniae or, alternatively, also depends on the ExbBD transporter func-
DISCUSSION
120
tion. This likely dependence on transferrin-bound iron is supported by results with
Neisseria gonorrhoeae showing that transferrin-receptor mutants were unable to
cause infection in human volunteers (CORNELISSEN et al. 1998).
The results obtained in the challenge experiment with A. pleuropneumoniae
App∆ureC confirmed previous results with respect to acute infection (TASCON
CABRERO et al. 1997; BOSSE and MACINNES 2000) and supported the hypotheti-
cal role of urease in chronic infection. Thus, A. pleuropneumoniae App∆ureC could
not be isolated from unaltered lung tissue three weeks after challenge (Tables 9 and
15) and this finding was further supported by the ELI-spot assay showing a signifi-
cantly higher number of A. pleuropneumoniae specific B-cells in the BALF from
App∆ureC-infected pigs in comparison to the parent strain (Fig. 3). This difference
could be due to a more effective antigen uptake and presentation by dendritic cells in
the airways (MCWILLIAM et al. 1996), thereby leading to an increased number of
ASC; this possibility is supported by the urease function hypothesized above. The
similar number of total B- and T-cells does not contradict this potential explanation as
the lytic activity of Apx toxin is concentration-dependent and, therefore, would be ex-
pected to primarily affect A. pleuropneumoniae-specific cells. To substantiate this
hypothesis, however, additional challenge trials using different challenge doses
should be performed.
A major obstacle in preventing A. pleuropneumoniae disease is the serovar-specific
protection induced upon immunization with bacterins. One way to successfully over-
come this problem is the use of attenuated live vaccines (PRIDEAUX et al. 1999;
FULLER et al. 2000). However, licensing of isogenic mutants containing an antibiotic
resistance marker for use in livestock might be difficult to obtain. Therefore, the
feasability of successively introducing multiple mutations without antibiotic markers
into the same parent strain might prove valuable for future A. pleuropneumoniae vac-
cine development. Based on these results, a urease-negative phenotype introduced
as one mutation might be advantageous as it facilitates a simple differentiation from
common A. pleuropneumoniae isolates and, in addition, might reduce shedding of
such a putative live vaccine.
DISCUSSION
121
E.2 Role of transferrin binding proteins in A. pleuropneumoniae infection
As it was shown in the previous challenge experiment (E.1), the elimination of exbB
expression led to the total abolishment of virulence in A. pleuropneumoniae. How-
ever, it was still unclear whether this was due to the impairment of the function of the
transferrin binding proteins or whether the effect was the result of an interference of
the exbB deletion with other iron uptake mechanisms or other ExbB dependent cell
functions. Therefore, impairment of iron uptake via transferrin binding proteins re-
mained an interesting target for subsequent studies. In order to clarify the results
obtained in the exbB challenge trial and in order to explore the potential of transferrin
binding protein deletion mutants for use in a live vaccine, defined A. pleuropneumo-
niae tbpB and tbpA mutants as well as an A. pleuropneumoniae tbpBA mutant were
constructed. Since the TbpB protein is known to be a protective antigen, and also in
order to avoid polar effects, an in-frame mutation was constructed which abolished
transferrin binding activity while prospectively maintaining antigenicity. The results of
clinical evaluation, serology, and post mortem analysis 21 days after challenge
clearly demonstrated a complete lack of virulence of all three A. pleuropneumoniae
tbp-mutants (Table 16). It appears that the tbp mutants are unable to colonize the
porcine lung sufficiently long to induce a detectable humoral immune response at a
dose that, in wild type infection, is sufficient to cause severe but not fatal disease.
This lack of virulence in A. pleuropneumoniae ∆tbpBA indicates that utilization of
transferrin-bound iron is essential for survival of the bacterium in the host and trans-
ferrin binding proteins could thus be classified as virulence factors. These results
confirm the results obtained with N. gonorrhoeae mutants in a human infection
model, where the absence of transferrin binding protein expression led to avirulence
as well (CORNELISSEN et al. 1998). Furthermore, this result strongly implies that the
lack of virulence observed for the A. pleuropneumoniae ∆exbB strain (D.2.2) is
caused by its inability to utilize transferrin bound iron and not by the lack of other
features requiring ExbB function. The lack of virulence of A. pleuropneumoniae
∆tbpA confirms the important role of utilization of transferrin-bound iron for infection.
It correlates well with the function of the TbpA protein seen in vitro in A. pleuropneu-
moniae (Fig. 6D) and in Neisseria spp. (IRWIN et al. 1993; CORNELISSEN and
DISCUSSION
122
SPARLING 1996). The lack of virulence of A. pleuropneumoniae ∆tbpB was unex-
pected, as it was shown that this strain was still able to utilize transferrin-bound iron
in vitro (Fig. 6D). One explanation could be the fact that only TbpB proteins from A.
pleuropneumoniae and Neisseria spp. selectively bind iron-saturated transferrin
(GERLACH et al. 1992a; CORNELISSEN and SPARLING 1996; RETZER et al.
1998) whereas this selectivity is not seen in the TbpA proteins of these species. Also,
it could be argued that the avirulence of this mutant is due to a synergistic effect be-
tween the tbpB and ureC deletions. However, this does not seem probable, as viru-
lence seems to be largely unaffected by the absence of urease alone (D.1). More
likely, it could be hypothesized that in the in vivo situation, the transferrin-dependent
iron acquisition system is inactivated by apotransferrin binding to the TbpA protein in
the absence of the TbpB protein, thereby blocking the attachment site for iron-
saturated transferrin. Alternatively, the TbpB protein might be partly responsible for
the interference with the host transferrin-iron metabolism recently described for
Neisseria spp. (BONNAH et al. 2000), aiding in the downregulation of the host's
transferrin receptor protein. This effect would amount to a decrease of local host
immune response which in turn might be required to facilitate A. pleuropneumoniae
colonization.
E.3 Identification of genes expressed in vivo
Identification of differentially expressed genes, and especially host-induced factors, is
a challenging task for which a variety of approches have been developed more re-
cently. Most importantly, signature-tagged mutagenesis (STM) and in vivo expression
technique (IVET) have been employed, with the latter identifying promoters that are
switched on under in vivo conditions only (FULLER et al. 1999; FULLER et al. 2000).
The STM approach involves the generation of a tagged transposon mutant pool and
subsequent screening in the pig by infection with and subsequent recovery of the
mutants. Mutants that cannot be recovered, carry the tagged transposon in a gene
that is required for in vivo survival (FULLER et al. 2000). Therefore, STM can identify
only genes that are essential in vivo. In the IVET approach, a metabolic mutant re-
quiring exogenous riboflavin has been used for screening in A. pleuropneumoniae.
The mutant is transformed with a library of plasmids that contain a promoterless
DISCUSSION
123
intact copy of the riboflavin genes from Bacillus subtilis as well as a fragment from a
genomic A. pleuropneumoniae library. A fragment containing a promoter that is
switched on in the host may lead to expression of the riboflavin genes in vivo, and
thus complement the deficiency. Recovered mutants are then screened for their
ability to grow on medium without riboflavin, and only mutants requiring riboflavin in
vitro are investigated further (FULLER et al. 1999). Differential Display PCR
(DDPCR) was first described by LIANG and PARDEE (1992). It relies on random
amplification of mRNA from two populations with subsequent runs of both amplicons
on a sequencing gel. This technique did not appear to be applicable to bacterial total
RNA due to the abundance of RNA species other than mRNA in the preparation.
Representational difference analysis was first described by LISITSYN et al. (1993) for
the analysis of DNA sequence differences between closely related populations. This
method is based on the concept of subtractive hybridization, but increases sensitivity
by PCR-based representation, leading to reduction of complexity and size of gene
fragments.
The application of RDA was first described for the detection of transcriptional
differences (cDNA RDA) in eucaryotic systems (HUBANK and SCHATZ 1994) and
was used successfully to identify differentially expressed genes in Ewing's sarcoma
cells (BRAUN 1995). Subsequently, it has been adapted for use with bacterial RNA in
N. meningitidis (BOWLER et al. 1999), where it was used to identify iron-regulated
genes. In S. aureus, genes expressed in biofilm forming bacteria versus planctonic
populations were identified (BECKER et al. 2001), and in M. bovis, increased expres-
sion of mycoseroic acid synthase was detected in intracellular bacteria using this
technique (LI et al. 2001).
In our approach, cDNA RDA was favored over IVET and STM because the effica-
cious use of these methods requires either established target cell culture systems
that are not available for A. pleuropneumoniae, or the use of laboratory animals. As a
suitable small mammal model is not available for A. pleuropneumoniae due to the
strict species specificity of the organism, experiments have to be conducted with
pigs. IVET and STM infection experiments also require biosafety level 2 stables
which are only available in limited number at our facility. Furthermore, the STM
DISCUSSION
124
technique detects only genes essential for growth in vivo that, therefore, cannot be
deleted in the process of live vaccine construction. As it had been shown that the
addition of BALF from pigs infected with A. pleuropneumoniae to culture medium
induced a number of yet unidentified proteins besides the transferrin binding proteins
(HENNIG et al. 1999), this ex vivo approach was considered a suitable and cost-
efficient method of induction for a subsequent cDNA RDA in A. pleuropneumoniae.
In the study presented here, cDNA RDA was performed on A. pleuropneumoniae to
identify alternate iron uptake mechanisms and other genes related to virulence. An ex
vivo approach mimicking in vivo conditions was employed; in this approach, BALF
from pigs infected with A. pleuropneumoniae is added to the medium to obtain in
vivo-like conditions. This approach had been shown to enhance the expression of
several proteins, including that of transferrin binding proteins (TEUTENBERG-
RIEDEL 1998). A major obstacle in performing RDA on bacterial cDNA is the
abundance of rRNA in RNA preparations, as it is not possible to selectively isolate
mRNA from bacteria using standard procedures. Therefore, contamination of the
difference product pool with rRNA transcripts is common. As this could lead to
reduction of rare transcripts over the course of several cycles of subtractive
hybridisation, difference products were cloned after the first round of subtraction.
Cloned RDA fragments were then examined in Southern blot analyses to identify
those fragments that were present in tester cDNA preparations only. Among the
identified genes, an fhuA homologue and a dmsA homologue were selected for
further characterization due to their potential roles in iron metabolism and survival of
A. pleuropneumoniae in abscesses, respectively.
Northern blot analyses were unable to detect dmsA or fhuA transcripts in total RNA
from A. pleuropneumoniae grown with the addition of BALF. In Western blot analysis,
induction of DmsA and FhuA expression by BALF could not be shown, which is likely
the result of a low number of transcripts in conjunction with the limited sensitivity of
the Western blot technique. These results leave the presence of dmsA and fhuA
cDNA in tester cDNA and their absence in driver cDNA as the only indicator of
differential expression. An RT-PCR or RNAse protection assay protocol should be
developed to further substantiate these findings.
DISCUSSION
125
Both dmsA and fhuA are regulated in other organisms. Expression of DmsA is en-
hanced under anaerobic conditions in E. coli (BILOUS and WEINER 1985) and
Rhodobacter capsulatus (MCEWAN et al. 1991). The same could be demonstrated
for A. pleuropneumoniae in this study (Fig. 14), and a consensus sequence of a bind-
ing site for HlyX, the A. pleuropneumoniae analogue of the anaerobic transcriptional
regulator Fnr of E. coli, was identified.
Additionally, the consensus sequence for the twin arginine translocation (TAT)
pathway (BERKS 1996) was identified in the signal peptide sequence of A.
pleuropneumoniae dmsA (Fig. 11). This pathway, also termed "Membrane Targeting
and Translocation" (MTT; SARGENT et al. 1998), is sec-independent and usually
functions to transport folded, cofactor-containing proteins through the cytoplasmic
membrane into the periplasm (BERKS 1996). TAT leader sequences have been
identified in genes of Escherichia coli, Haemophilus influenzae and other species
(BERKS et al. 2000). Four integral cytoplasmic membrane proteins, encoded by tatA,
tatB, tatC and tatE, are involved in TAT translocation in E. coli (BERKS et al. 2000).
The pathway is closely related to the ∆pH-dependent protein import pathway of the
plant chloroplast thylakoid membrane (BOGSCH et al. 1998). The DmsA subunit of
the E. coli DMSO reductase complex is an exception to the TAT transport principle,
as its Tat leader sequence does not target the protein to the periplasm, but to the
inner surface of the cytoplasmic membrane and, at the same time, to stabilize the
DmsAB dimer. Deletion and replacement of the DmsA leader with a TorA
(trimethylamine N-oxide reductase) signal peptide resulted in a five-fold decrease in
specific activity, supporting this hypothesis (SAMBASIVARAO et al. 2000). In some
other bacteria, DMSO reductase activity is located in the periplasm (WEINER et al.
1992). Further studies should determine the location of A. pleuropneumoniae DMSO
reductase. This is the first report of a twin arginine motif in A. pleuropneumoniae, an
MTT mechanism has not been identified in this organism. The organization of genes
encoding the DMSO reductase is different from the organization in E.coli (BILOUS
and WEINER 1988) and Haemophilus influenzae (LOOSMORE et al. 1996) in that
the dmsA gene is not immediately followed by dmsB. Genes encoding DmsB and
DmsC have not been identified in this study.
DISCUSSION
126
In A. pleuropneumoniae infection, DMSO reductase might be advantageous for
survival of A. pleuropneumoniae in abscesses, as it enables respiration rather than
fermentation under anaerobic conditions by using DMSO or a different substrate as
the terminal electron acceptor instead of oxygen. A similar mechanism has been
demonstrated recently for anaerobic nitrate reductase in Mycobacterium (M.) bovis
BCG. Nitrate reductase is an analogue of DMSO reductase in that nitrate serves as
the terminal electron acceptor under anaerobic conditions. An M. bovis BCG mutant
lacking nitrate reductase was attenuated in immunodeficient mice (WEBER et al.
2000; FRITZ et al. 2002).
The FhuA protein is the TonB/ExbBD-dependent ferrichrome receptor of E. coli
(SCHOFFLER and BRAUN 1989; GUNTER and BRAUN 1990) that also serves as
receptor for colicin M and phagesT1, T5 and φ80 (LAZDUNSKI et al. 1998). Similar
receptors are found in many organisms, a common feature being the upregulation of
FhuA protein expression or the respective FhuA homologues under iron-deficient
conditions in E. coli (HANTKE and BRAUN 1975), Vibrio cholerae (ROGERS et al.
2000), Campylobacter jejuni (GALINDO et al. 2001) Rhizobium leguminosarum
(YEOMAN et al. 2000) and Bradyrhizobium japonicum (LEVIER and GUERINOT
1996). However, this could not be demonstrated for A. pleuropneumoniae fhuA.
Neither iron depletion nor supplementation with ferric citrate or ferric sulfate en-
hanced or repressed expression of the FhuA protein. Yet, the addition of ferric sulfate
slightly repressed expression, but the effect was too slight to postulate a regulatory
mechanism (Fig. 20). FhuA expression was also repressed under anaerobic con-
ditions (Fig. 21). This indirectly implies a function for FhuA in uptake of ferric iron
complexes, as under anaerobic conditions, ferric iron is spontaneously reduced to
ferrous iron. In E. coli, the expression of the Fhu proteins is regulated by the Fur
protein (SCHAFFER et al. 1985), but to date, a Fur homologue has not been
identified for A. pleuropneumoniae. Therefore, the regulation of A. pleuropneumoniae
Fhu expression remains unclear and should be the subject of further studies. Also,
the ligand for this probable iron compound receptor could not be identified in this
study. As ligand uptake is expected to be abolished in the deletion mutant A. pleu-
ropneumoniae ∆fhuA, it was concluded that neither ferrichrome, dihydroxybenzoic
DISCUSSION
127
acid nor rhodotorulic acid constitute a ligand, since all of these compounds could be
utilized as the sole iron source by A. pleuropneumoniae wild type and mutant strains
in a plate bioassay (D.3.8). These experiments should be extended to investigate
other iron compounds such as aerobactin, enterobactin or the yet uncharacterized
siderophore produced by certain A. pleuropneumoniae strains (DIARRA et al. 1996),
as well as other iron-binding proteins of the host such as ferritin, hemoglobin or
albumin.
The organization of the fhu operon homologue in A. pleuropneumoniae differs from
the E. coli fhu operon (FECKER and BRAUN 1983) in that fhuA is preceded by fhuB.
In E. coli, the components of the fhu operon appear in the following order: fhuA-fhuC-
fhuD-fhuB. The complete fhu operon was not identified for A. pleuropneumoniae in
this study, but should be elucidated in further experiments. As FhuA function is ExbB
dependent in E. coli, it would be interesting to investigate whether there is a similar
dependence of the A. pleuropneumoniae FhuA on the ExbB protein encoded
upstream of TbpB (TONPITAK et al. 2000).
The in vivo characterization of dmsA and fhuA involved the construction of isogenic
deletion mutants and their use in an animal infection experiment. The construction of
isogenic mutants via homologous recombination, using a suicide vector and a selec-
tion/counterselection procedure with kanamycin and sucrose, respectively (OSWALD
et al. 1999a), resulted in two unmarked deletion mutants, A. pleuropneumoniae
∆dmsA and A. pleuropneumoniae ∆fhuA. In the animal infection experiment, both
mutants were shown to be attenuated in acute disease, as assessed by body tem-
perature diagrams and endoscopy scores (Fig. 27, Fig. 28). In addition, animals in-
fected with the deletion mutants had lower antibody titers than animals infected with
the wild type strain (Fig. 29). There was a striking difference in disease symptoms
between animals in the A. pleuropneumoniae ∆fhuA group, with four animals show-
ing no lung lesions and very low antibody titers. As the challenge dose for the A.
pleuropneumoniae ∆fhuA strain as calculated via serial dilutions was about a third of
the dose of the wild type strain, the experiment should be repeated with a higher
dose in order to exclude dose-dependent effects. However, porcine pleuropneumonia
is highly contagious, therefore, animals of one group should infect each other unless
DISCUSSION
128
the A. pleuropneumoniae ∆fhuA strain is not shed to the same extent as the wild
type. The reisolation data do not allow sound conclusions in this respect. If the repeat
experiment clarifies the results obtained here, an fhuA deletion might be a valuable
trait for a live vaccine.
No consistent difference could be observed between reisolation results from altered
lung tissue (including abscesses) in the A. pleuropneumoniae AP76 wild type and A.
pleuropneumoniae ∆dmsA groups. Therefore, it can be concluded that DmsA
function must be partially compensated by other mechanisms. The dmsA deletion
could be useful in the construction of a vaccine strain in combination with other
deletions.
Several other genes identified in the cDNA RDA experiment should be subjected to
further analyses, especially RN30, RN32, RN36, and RN37 (Table 10). RN30 is
homologous to a H. influenzae nucleoid-associated protein, which is in turn similar to
E. coli YejK, an acidic polypeptide of 335 amino acids for which no function has been
identified, but it is thought to interact with nucleotides (MURPHY et al. 1999). This
protein might be involved in gene regulation. RN32 shows 40% identity to
cyanophycin synthetase of Anabaena variabilis (GenBank accession no. O86109).
Cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is a nitrogen storage polypeptide
of cyanobacteria which is synthetized by cyanophycin synthetase, independent from
ribosomes (BERG et al. 2000). Cyanophycin synthesis has not been described for
any bacterial species outside of cyanobacteria. RN36 is similar to a probable sulfate
adenylate transferase subunit from N. meningitidis (GenBank accession no. F81905)
which in turn is homologous to translation elongation factor Tu. Therefore, RN36
should be examined in regard to its possible function as a translational regulator. RN
37, which resembles H. influenzae L-fuculose phosphate aldolase (GenBank acces-
sion no. P44777) would be an interesting subject because fucose has been found to
increase in the mucins of horses suffering from chronic inflammation of the airways
(JEFCOAT et al. 2001).
In summary, representational difference of cDNA appears to be a valuable technique
for the identification of genes expressed during A. pleuropneumoniae infection in
vivo. It simplifies the investigation or comparison of different stages of the disease by
DISCUSSION
129
using an in vitro model for the induction of gene expression. However, stringent con-
trols are necessary. Future work should aim at the identification of components in
BALF that are capable of inducing gene expression, as this would allow even more
specific investigation of the regulatory systems of A. pleuropneumoniae that are in-
volved in virulence.
SUMMARY
130
F Summary
The role of iron in Actinobacillus pleuropneumoniae infection:Identification and in vivo characterization of virulence-associated genes
Nina Baltes
Actinobacillus (A.) pleuropneumoniae is an obligatory porcine pathogen that causes
a fibrinous and necrotizing pleuropneumonia of significant economic importance. To
date, there are no vaccines available that provide satisfactory cross-serovar protec-
tion and, in addition, allow differentiation between infected and vaccinated animals.
Thus, a live attenuated marker vaccine would be desirable. The ability to acquire es-
sential nutrients in the host is closely related to bacterial virulence. A. pleuropneumo-
niae possesses high-affinity receptors for porcine transferrin which are functionally
dependent on a transmembrane energy coupling mechanism (TonB-ExbBD) for the
uptake of iron from transferrin. In this study, components involved in transferrin-
bound iron uptake were investigated with regard to their contribution to A. pleu-
ropneumoniae virulence, and representational difference analysis of cDNA was used
to identify other virulence-associated genes .
An isogenic exbB deletion mutant of A. pleuropneumoniae that was unable to utilize
porcine transferrin as the sole iron source in vitro was examined in an aerosol chal-
lenge trial. This strain was completely avirulent, which made the construction of iso-
genic mutants lacking expression of either TbpB, TbpA or both transferrin-binding
proteins necessary. In a challenge trial, all mutants were avirulent, thus emphasizing
the crucial role of transferrin bound iron uptake in infection. Furthermore, this result
implies that the avirulence of the exbB deletion mutant is due to its inability to utilize
transferrin-bound iron rather than due to other cell functions requiring the ExbB pro-
tein.
Using an ex vivo model (addition of bronchoalveolar lavage fluid [BALF] to culture
medium) to induce genes normally expressed in the host only, representational
difference analysis was performed on A. pleuropneumoniae cDNA. Ten genes pre-
SUMMARY
131
sent only in cDNA from A. pleuropneumoniae grown BALF were identified. Among
them, the genes for a novel ferric compound receptor unique to serotypes 4 and 7
(fhuA) and the catalytic subunit of dimethylsulfoxide reductase (dmsA) were further
characterized, and isogenic mutants were constructed. A challenge experiment
showed attenuation in acute disease for both strains. Both genes may be useful in
the construction of a novel live vaccine strain carrying multiple deletions.
ZUSAMMENFASSUNG
132
G Zusammenfassung
Die Rolle von Eisen in der Infektion mit Actinobacillus pleuropneumoniae:Identifizierung und in vivo-Charakterisierung virulenzassoziierter Gene
Nina Baltes
Actinobacillus (A.) pleuropneumoniae ist ein obligat pathogenes Bakterium des
Schweines, das eine fibrinös-nekrotisierende Pleuropneumonie von hoher wirt-
schaftlicher Bedeutung hervorruft. Zur Zeit sind keine Impfstoffe verfügbar, die eine
zufriedenstellende serotypübergreifende Immunität vermitteln und die zusätzlich die
Unterscheidung geimpfter und infizierter Tiere erlauben. Daher wäre ein attenuierter
Lebend-Markerimpfstoff wünschenswert. Die Fähigkeit, im Wirtsorganismus an
essentielle Nährstoffe zu gelangen, ist eng verbunden mit bakterieller Virulenz. A.
pleuropneumoniae besitzt hochaffine Rezeptoren für porzines Transferrin, deren
Funktion, die Aufnahme transferringebundenen Eisens, funktionell von einem mem-
branüberspannenden Energieübertragungssystem (TonB-ExbBD) abängt. In dieser
Arbeit wurden einige Komponenten dieses Eisenaufnahmesystems im Hinblick auf
ihre Rolle für die Virulenz von A. pleuropneumoniae untersucht, und es wurde eine
repräsentative Differenzanalyse an cDNA von A. pleuropneumoniae durchgeführt,
um weitere virulenzassoziierte Gene zu identifizieren.
Eine isogene exbB- Deletionsmutante von A. pleuropneumoniae, die in vitro nicht
mehr in der Lage ist, porzines Transferrin als einzige Eisenquelle zu nutzen, wurde in
einem Aerosolbelastungsmodell untersucht. Der Stamm erwies sich als vollständig
avirulent, was die Konstruktion von isogenen Mutanten nötig machte, die entweder
TbpB, TbpA oder beide transferrinbindenden Proteine nicht mehr exprimierten. Alle
diese Stämme waren im Aerosolbelastungsversuch avirulent, was die wichtige Rolle
der Aufnahme von transferringebundenem Eisen in der Infektion unterstreicht. Zu-
dem legt dieses Ergebnis nahe, dass die Avirulenz der exbB-Deletionsmutante auf
die Unfähigkeit, transferringebundenes Eisen zu nutzen, zurückzuführen ist und nicht
auf den Ausfall anderer exbB-abhängiger Zellfunktionen.
ZUSAMMENFASSUNG
133
Mit Hilfe eines ex vivo-Modells (Zugabe von bronchoalveolärer Lavageflüssigkeit
[BALF] zum Kulturmedium) zur Induktion von Genen, die unter normalen Umständen
nur im Wirt exprimiert werden, wurde eine repräsentative Differenzanalyse an cDNA
von A. pleuropneumoniae durchgeführt. Zehn Gene, die nur in BALF-induzierter
cDNA vorhanden waren, wurden identifiziert. Von diesen wurden die Gene eines
neuartigen Eisenrezeptors, der nur in den Serotypen 4 und 7 vorkommt (fhuA) sowie
die katalytische Untereinheit einer Dimethylsulfoxid-Reduktase (dmsA) näher cha-
rakterisiert und isogene Mutanten konstruiert. Im Aerosolinfektionsmodell zeigten
beide Deletionsmutanten eine Attenuierung in der akuten Phase der Krankheit. Beide
Gene könnten sich bei der Konstruktion eines neuartigen Lebendimpfstoffes, der
mehrere Deletionen trägt, als nützlich erweisen.
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APPENDIX
173
I Appendix
I.1 Chemicals
Acrylamide SERVA, Heidelberg2,2'-azino-di (3-ethyl-benzthiazoline-6- sulfonate), ABTS Roth, Karlsruhe
BCIP (5-bromo-4-chloro-3-indolyl phosphate) SIGMA,Deisenhofen
Bisacrylamide SERVA, HeidelbergAgar OXOID, WesselAgarose APPLIGENE, Illkirch, FranceAmmonium persulfate ROTH, KarlsruheAmpicillin SIGMA, DeisenhofenBacto Peptone DIFCO, AugsburgBacto Tryptone DIFCO, AugsburgBacto Beef Heart For Infusion DIFCO, AugsburgBenzyl viologen (1,1�-dibenzyl-4,4�-bipyridiniumdichloride) SIGMA, Deisenhofen
Boric acid SIGMA, DeisenhofenBHI agar DIFCO, AugsburgBromphenol blue SIGMA, DeisenhofenBovine serum albumin (BSA) New England Biolabs, SchwalbachChloramphenicol SIGMA, DeisenhofenCalcium chloride SIGMA, DeisenhofenChloroform ROTH, KarlsruheCitric acid SIGMA, DeisenhofenCoomassie brilliant blue SIGMA, DeisenhofenL-cysteine hydrochloride monohydrate SIGMA, DeisenhofenL-cystine dihydrochloride SIGMA, Deisenhofen
α-32P-dCTP NEN, Boston, MA,U.S.A.
2´-deoxynucleotide 5´-triphosphate (dNTP) ROTH, KarlsruheDiaminopimelic acid SIGMA, Deisenhofendiethylenetriamine-pentaacetic acid calcium trisodium salthydrate (Na3CaDTPA) SIGMA, Deisenhofen
Dimethylformamide SIGMA, Deisenhofen
APPENDIX
174
Dimethylsulfoxide (DMSO) ROTH, Karlsruhe2,2´ dipyridyl SIGMA, Deisenhofen
Ethanol Elch-Apotheke,Hannover
Ethidium bromide SIGMA, DeisenhofenEthylene diamine tetraacetic acid (EDTA) SIGMA, DeisenhofenFicoll (Type 400-DL) SIGMA, DeisenhofenGelatin SIGMA, DeisenhofenD (+) glucose monohydrate MERCK, DarmstadtL-glutamine SERVA, HeidelbergGlycerine ROTH, KarlsruheGlycine SIGMA, DeisenhofenHEPES (N-[2-Hydroxyethyl] piperazine.N´-[2-ethanesulfonic acid]) SIGMA, Deisenhofen
Hydrochloric acid ROTH, KarlsruheIsoamyl alcohol MERCK, DarmstadtIsopropanol ROTH, KarlsruheKanamycin SIGMA, DeisenhofenMagnesium sulfate SIGMA, DeisenhofenMethanol ROTH, KarlsruheMineral oil SIGMA, DeisenhofenNBT (nitroblue tetrazolium) ROTH, KarlsruheNicotinamide dinucleotide (NAD) MERCK, DarmstadtPhenol (Roti®-Phenol, TE equilibrated) ROTH, KarlsruhePhenol red MERCK, DarmstadtPhenyl-methyl-sulfonyl-fluoride (PMSF) SERVA, HeidelbergPolyvinylpyrrolidone SIGMA, DeisenhofenPotassium acetate SIGMA, DeisenhofenPotassium chloride SIGMA, DeisenhofenPPLO broth® DIFCO, AugsburgPPLO agar® DIFCO, AugsburgPotassium dihydrogen phosphate SIGMA, DeisenhofenSarcosyl (N-lauryl-sarcosine) SIGMA, DeisenhofenSodium acetate SIGMA, Deisenhofen
APPENDIX
175
Sodium chloride SIGMA, DeisenhofenSodium deoxycholate SIGMA, DeisenhofenSodium dodecyl sulfate (SDS) SIGMA, Deisenhofendi-sodium hydrogen phosphate SIGMA, DeisenhofenSodium hydroxide SIGMA, DeisenhofenSucrose MERCK, DarmstadtN, N, N´, N´-Tetramethylenediamine (TEMED) SIGMA, DeisenhofenTris (Hydroxymethyl aminomethane) SIGMA, DeisenhofenTween®20 SIGMA, DeisenhofenTween®80 ROTH, KarlsruheUrea SIGMA, DeisenhofenXylene cyanol SIGMA, DeisenhofenYeast extract OXOID, Wessel
APPENDIX
176
I.2 Buffers and solutions
Agarose gel electrophoresis (C.5)
TE: 10 mM Tris (pH 8.0), 1 mM EDTA
10 x TBE: 1 M Tris-borate, 10 mM EDTA (pH 8.0)
50 x TAE: 2 M Tris-HCl (pH 8.0), 1 M acetic acid,
50 mM EDTA (pH 8.0)
6 x DNA loading buffer: 0.25% [w/v] bromphenol blue, 0.25% [w/v]
xylene cyanol, 30% [v/v] glycerin
Conjugation (C.5.7.1)TNM: 1 mM Tris-HCl (pH 7.2), 100mM NaCl,
10 mM MgSO4
ELISA, ELI-Spot analysis (C.7.8, C.7.9)Coating buffer: 100 mM NaHCO3, adjusted to pH 9.6 with
Na2CO3
PBS: 100 mM NaCl, 100 mMNa2HPO4,
100 mM KH2PO4
PBST: PBS + 0.05% Tween®20
Substrate buffer: 0.1 M citric acid (pH 4.25, adjusted with
Na2HPO4) with 0.002% H2O2
Substrate: ABTS (2,2'-azino-di [3-ethyl-benzthiazoline-6-
sulfonate]), 800 mg/l, in substrate buffer
Labeling of nucleic acid probes with 32P-dCTP (C.5.8.3)OLB solution: 100 µl solution A, 250 µl solution B,
150 µl solution C
Solution O: 625 µl 2 M Tris-HCl (pH 8), 125 µl 1M MgCl,
250 µl A. bidest
APPENDIX
177
Solution A: 1 ml solution O, 18 µl mercaptoethanol, 5 µl
100 mM dTTP, 5 µl 100 mM dGTP, 5 µl 100
mM dATP
Solution B: 2 M HEPES (pH 6.6)
Solution C: random hexa-desoxynucleotides in TE buffer
(Amersham Pharmacia)
Stop solution: 20 mM NaCl, 20 mM Tris-HCl (pH 7.5), 2 mM
EDTA [pH 8], 0.25% [w/v] SDS
Northern hybridization (C.5.8.5)Agarose gel: 0.5 g agarose, 40.4 ml A. bidest, 5 ml 10 x
MOPS buffer, 10 ml formaldehyde (37% [v/v])
10 x MOPS buffer pH 7.0: 0.4 M MOPS, 100 mM sodium acetate,
10 mM EDTA
Sample buffer: glycerine 500 µl, 10 x MOPS buffer 500 µl,
formaldehyde (37% [v/v]) 800 µl, 2.5 ml
formamide, 10 mg bromphenol blue,
10 mg xylene cyanol
Hybridizing solution: 0.5 M Na2PO4 (pH 7.2), 7 % SDS, 1 mM EDTA
Wash solution I: 40 mM Na2PO4 (pH 7.2), 5 % SDS, 1 mM
EDTA
Wash solution II: 40 mM Na2PO4 (pH 7.2), 1 % SDS, 1 mM
EDTA
Pulsed field gel electrophoresis (C.5.6)PET IV buffer: 1 M NaCl, 10 mM Tris-HCl (pH 8.0), 10mM
EDTA
Lysis buffer: 1 M NaCl, 10 mM Tris-HCl (pH 8), 0.2 M EDTA,
0.5% N-laurylsarcosine, 0.2% deoxycholate,
2 µg/ml RNase, 1 mg/ml lysozyme
EPS buffer: 0.5 M EDTA, 1% laurylsarcosine, 1 mg/ml
proteinase K
TE-PMSF: 1.5 mM PMSF in TE buffer
APPENDIX
178
PMSF: 17 mg phenylmethylsulfonyl fluoride per
ml isopropanol
Plasmid preparation by alkaline lysis (C.5.1)Solution I: 50 mM Tris (pH 8.0), 10 mM EDTA (pH 8.0),
100 µg/ ml RNase
Solution II: 0.2 M NaOH, 1% [w/v] SDS
Solution III: 3.2 M potassium acetate (pH 5.5)
Protein aggregate preparation (C.6.1)2 x RIPA: 20 mM Tris-HCl (pH7.4), 300 mM NaCl,
2% [w/v] desoxycholic acid,
2% [v/v] Tergitol (NP40)
TET: 100 mM Tris (pH8.0), 50 mM EDTA (pH 8.0),
2% [v/v] Triton X-100
SDS-PAGE (C.6)Acrylamide stock solution: 30% acrylamide, 0.8% bisacrylamide in
A. bidest.
Ammonium persulfate: 10% solution
10.9% separating gel: 3.9 ml A. bidest., 2.5 ml 1.5M Tris-HCl (pH 8.8),
100 µl SDS (10%), 3.8 ml acrylamide stock
solution, 10 µl TEMED, 100 µl ammonium
persulfate [10%]
3.9% stacking gel: 3.05 ml A. bidest., 1.25 ml 0.5 M Tris-HCl
(pH6.8), 50 µl SDS [10%], 10 µl TEMED,
50 µl ammonium persulfate [10%]
10 x SDS-PAGE running buffer: 0.25 M Tris, 2 M Glycine, 1% SDS
Coomassie blue staining solution: 1.25 g Coomassie brilliant blue R250,
225 ml methanol, 50 ml glacial acetic acid,
225 ml A. bidest.
APPENDIX
179
Destaining solution: 300 ml methanol, 100 ml glacial acetic acid,
600 ml A. bidest.
Southern Blotting (C.5.8.1)Denaturing solution: 1.5 M NaCl, 0.5 M NaOH
Neutralizing solution: 1.5 M NaCl, 0.5 M Tris (pH 8.0)
20 x standard saline citrate (SSC): 3 M NaCl, 0.3 M trisodium acetate
Hybridizing solution: 6 x SSC, 0.5% SDS, 5 x Denhardt's solution
50 x Denhardt's solution: 1% [w/v] polyvinylpyrrolidone, 1% [w/v]
Ficoll 400, 1% [w/v] BSA; in A. bidest.
Wash solution: 3 x SSC, 0.5% SDS or 1 x SSC, 0.5% SDS
Western blotting (C.6.5.1)Transfer buffer: 24 mM Tris-HCl (pH 8.0), 110 mM glycin, 20%
[v/v] methanol
Blocking buffer: 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5%
[v/v] Tween® 20, 0.5% [v/v] gelatin
10 x wash buffer: 0.1 M Tris-HCl (pH 8.0), 1.5 M NaCl, 0.5% [v/v]
Tween®20
Substrate buffer: 0.1 M Tris-HCl, 1 M NaCl, 5 mM MgCl
BCIP: 5 mg/ml 5-bromo-4-chloro-3-indolyl phosphate
in dimethylformamide
NBT: 10 mg/ml nitroblue tetrazolium in 70% [w/v]
dimethylformamide
APPENDIX - DMSA
180
I.3 Sequence of A. pleuropneumoniae dmsA
APPENDIX - DMSA
181
APPENDIX - DMSA
182
APPENDIX - DMSA
183
APPENDIX - DMSA
184
APPENDIX - FHUA
185
I.4 Sequence of A. pleuropneumoniae fhuA
APPENDIX - FHUA
186
APPENDIX - FHUA
187
APPENDIX - FHUA
188
APPENDIX - FHUA
189
APPEN
DIX - A
NIM
AL EXPERIM
ENTS
190
I.5 Anim
al experiments
Table 15: Raw data for challenge experiment with A. pleuropneumoniae ∆∆∆∆ureC and A. pleuropneumoniae ∆∆∆∆exbB.
APPENDIX - ANIMAL EXPERIMENTS
191
Tabl
e 15
con
tinue
d
APPEN
DIX - A
NIM
AL EXPERIM
ENTS
192
Table 16: Raw data for challenge experiment with A. pleuropneumoniae ∆∆∆∆tbpA, A. pleuropneumoniae ∆∆∆∆tbpB and A. pleuropneumoniae
∆∆∆∆tbpBA.
APPENDIX - ANIMAL EXPERIMENTS
193
Tabl
e 16
con
tinue
d
APPEN
DIX - A
NIM
AL EXPERIM
ENTS
194
Table 17: Raw data for challenge experiment with A. pleuropneumoniae ∆∆∆∆dmsA and A. pleuropneumoniae ∆∆∆∆fhuA.
APPENDIX - ANIMAL EXPERIMENTS
195
Tabl
e 17
con
tinue
d
APPENDIX
196
I.6 Index of figures
Fig. 1: Energy coupling mechanisms of high-affinity iron uptake systems. ........................................... 33
Fig. 2: Humoral immune response of pigs. ............................................................................................ 78
Fig. 3: Local immune response of pigs .................................................................................................. 79
Fig. 4: Construction of pTF205EM302................................................................................................... 83
Fig. 5: Construction of pTF205mut404. ................................................................................................. 84
Fig. 6: Analysis of A. pleuropneumoniae tbp mutant strains. ................................................................ 86
Fig. 7: Southern blotting of RDA fragments ........................................................................................... 89
Fig. 8: Amplification of RDA products from genomic A. pleuropneumoniae AP76 DNA ....................... 91
Fig. 9: Genomic map of A. pleuropneumoniae AP76 with localization of two RDA fragments ............. 93
Fig. 10: Plasmid maps of plasmids pDM5-116, pFH29-4 and pFH29-II31.. .......................................... 94
Fig. 11: DmsA signal peptide and upstream nucleotide sequence........................................................ 96
Fig. 12: Alignment of DmsA TAT leader sequences from several bacteria. .......................................... 96
Fig. 13: Western blot analysis of DmsA expression. ............................................................................. 98
Fig. 14: Western blot analysis of anaerobic DmsA expression. ............................................................ 99
Fig. 15: PCR using primers oDMSAdel1 and oDMSAdel2 .................................................................... 99
Fig. 16: FhuA signal peptide and upstream nucleotide sequence....................................................... 101
Fig. 17: PCR using primers oFDEL1 and oFDEL2. ............................................................................. 104
Fig. 18: Southern blot analysis of fhuA. ............................................................................................... 104
Fig. 19: Expression of FhuA in A. pleuropneumoniae serotype reference strains .............................. 105
Fig. 20: Western blot analysis of FhuA expression..............................................................................106
Fig. 21: Western blot analysis of anaerobic FhuA expression............................................................. 107
Fig. 22: Construction of pBMKD1 ........................................................................................................ 109
Fig. 23: Construction of pECF2............................................................................................................ 110
Fig. 24: PCR of A. pleuropneumoniae deletion mutants ..................................................................... 111
Fig. 25: Southern blot analysis of deletion mutants............................................................................. 112
Fig. 26: PFGE analysis of A. pleuropneumoniae mutants................................................................... 112
Fig. 27: Body temperatures of pigs...................................................................................................... 114
Fig. 28: Endoscopical scores............................................................................................................... 114
Fig. 29: Humoral immune response of pigs ......................................................................................... 116
APPENDIX
197
I.7 Index of tables
Table 1: List of bacterial strains used in this study ................................................................................ 42
Table 2: List of plasmids used in this study ........................................................................................... 46
Table 3: List of primers used in this study ............................................................................................. 50
Table 4: PCR conditions used in this study ........................................................................................... 55
Table 5: Components in the PCR reaction ............................................................................................ 55
Table 6: Dilutions of antisera used in this study .................................................................................... 67
Table 7: Clinical findings during endoscopy of the porcine lung............................................................ 70
Table 8: Clinical scores as evaluated during endoscopy....................................................................... 70
Table 9: Virulence of A. pleuropneumoniae parent and isogenic mutant strains following aerosol
challenge ....................................................................................................................................... 80
Table 10: RDA fragments identified via differential hybridization .......................................................... 90
Table 11: Large fragments containing original RDA fragments............................................................. 92
Table 12: A. pleuropneumoniae dmsA - similarities to known genes. ................................................... 97
Table 13: A. pleuropneumoniae fhuA homologue - similarities to known genes................................. 102
Table 14: Virulence of A. pleuropneumoniae AP76 and isogenic mutant strains following aerosol
challenge. .................................................................................................................................... 117
Table 15: Raw data for challenge experiment with A. pleuropneumoniae ∆ureC and A. pleuro-
pneumoniae ∆exbB. .................................................................................................................... 190
Table 16: Raw data for challenge experiment with A. pleuropneumoniae ∆tbpA, A. pleuropneu-
moniae ∆tbpB and A. pleuropneumoniae ∆tbpBA. ..................................................................... 192
Table 17: Raw data for challenge experiment with A. pleuropneumoniae ∆dmsA and A. pleuro-
pneumoniae ∆fhuA. ..................................................................................................................... 194
198
Acknowledgements
I wish to thank
Prof. Dr. G.-F. Gerlach for the idea for this work, for his constant guidance and
motivation, for being there for all of his doctoral students, and for making sure that
the cookie jar is never empty for long.
The members of the PhD committee entrusted with my supervision, Prof. Dr. L. Haas
and Prof. Dr. B. Tümmler, for their interest in my work and their valuable discussions,
and Dr. P. Langford.for the evaluation of this thesis.
Dr. I. Hennig-Pauka, Prof. Dr. M. Ganter and the Clinic for Pigs, for doing the
bronchoalveolar lavages.
Prof. Dr. H.-J. Rothkötter and Dr. Astrid Hoffmann-Moujahid (Dept. of Functional and
Applied Anatomy, Medical School Hannover) for performing FACS ad ELI spot
analyses as well as for their help with necropsy duties.
My past and present coworkers at the Institute for Microbiology, in particular: Birgit
Strommenger, Janin Stratmann, Anja Rothkamp (a.k.a. the Daltons), Svenja Thiede,
Walaiporn Tonpitak, Denis Konine, Corinna Winterhoff, Achim Allgaier, Kathrin
Radecker, Ilse Jacobsen, Sabine Jeckstadt, Silvia Kursawe, Kyaw Sunn, Karen
Dohmann and all the other people in this institute who make this place the great
workplace that it is.
Jörg Matusch for never letting the computers gain the upper hand.
Corine Bohan for the Pasteur quote.
Reinhild for the funding of my veterinary studies, which otherwise would not have
been possible. My thanks extend to my grandmother Rosemarie.
My parents for their general relaxedness.
Steffen, for everything.
199
Danksagung
Ich möchte mich bedanken bei
Prof. Dr. G.-F. Gerlach für die Idee zu dieser Arbeit, seine ständige Führung und
Motivation, dafür, dass er jederzeit für alle seine Doktoranden da ist und dafür, dass
er immer dafür sorgt, dass die Keksdose nie lange leer ist.
Meinen Betreuern Prof. Dr. L. Haas und Prof. Dr. B. Tümmler, für ihr Interesse an
meiner Arbeit und die wertvollen Hinweise sowie Dr. P. Langford für die Evaluierung
meiner These.
Dr. I. Hennig-Pauka, Prof. Dr. M. Ganter und der Klinik für Kleine Klauentiere für die
Durchführung der bronchoalveolären Lavage.
Prof. Dr. H.-J. Rothkötter und Dr. Astrid Hoffmann-Moujahid (Abteilung Funktionelle
und Angewandte Anatomie, Medizinische Hochschule Hannover) für die
Durchführung der FACS- und ELI-Spot-Analysen und für die tatkräftige Unterstützung
bei der Sektion.
Meinen jetzigen und ehemaligen Mitarbeitern im Institut für Mikrobiologie
insbesondere: Birgit Strommenger, Janin Stratmann, Anja Rothkamp -auch bekannt
als 'die Daltons'-, Svenja Thiede, Walaiporn Tonpitak, Denis Konine, Corinna
Winterhoff, Achim Allgaier, Kathrin Radecker, Ilse Jacobsen, Sabine Jeckstadt, Silvia
Kursawe, Kyaw Sunn und all den anderen guten Geistern, die dieses Institut zu
einem so schönen Arbeitsplatz machen.
Corine Bohan für das Pasteurzitat.
Jörg Matusch dafür, dass er die Computer jederzeit im Griff hat.
Reinhild für die Finanzierung meines Tiermedizinstudiums, ohne die dieses nicht
möglich gewesen wäre. Auch meiner Oma Rosemarie vielen Dank.
Meinen Eltern für ihre allgemeine Gelassenheit.
Steffen, für alles.