proteomics approach on the identification of … · 2018-01-09 · proteomics approach on the...
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PROTEOMICS APPROACH ON THE IDENTIFICATION OF
VIRULENCE FACTORS OF ENTEROPATHOGENIC AND
ENTEROHEMORRHAGIC ESCHERICHIA COLI (EPEC AND EHEC)
AND FURTHER CHARACTERIZATION OF TWO EFFECTORS:
ESPB AND NLEI
BY
LI MO (M. SC.)
A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE
2006
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ACKNOWLEDGEMENTS
I wish to express my heartfelt gratitude to my main supervisor, Professor Hew Choy
Leong for his care and guidance. His clement character and esteemed research passion
inspirit me through my study. I wish to express my heartful thanks to my co-supervisor,
Associate Professor Leung Ka Yin for his supervision and advice. He had planned
properly and provided many opportunities for my research. I would sincerely thank
Professor Ilan Rosenshine from the Hebrew University, Israel, for his sound instruction
and generous sharing of ideas and experiences.
My Special thanks will also go to my PhD. Committee members. Thank you for spending
so much time in reviewing my thesis and audit my presentation. I would like to take this
opportunity to thank Dr. Lin Qingsong, Dr. John Foo, Ms Wang Xianhui and Ms Kho
Say Tin from the Protein and Proteomics Center for their kind assistance in mass
spectrometry, protein sequencing and data analysis. Further thank goes to Mr. Shashikant
Joshi for his reviewing and suggestions on my thesis.
My appreciation also goes to my previous and present lab members: Dr Yu Hongbing,
Dr Seng Eng Khuan, Dr Srinivasa Rao, Dr Tan Yuan Peng, Dr Yamada, Dr. Li Zhengjun,
Dr. Xie Haixia , Ms Tung Siew Lai, Mr. Zheng Jun, Mr. Peng Bo, Mr. Zhou Wenguang,
Ms Tang Xuhua, Mr. Liu Yang, Mr. Wang Fan, Mr. Chen Liming, for their
encouragement, help, assistance and company.
I would also like to thank my good friends Wang Xiaoxing, Hu Yi, Qian Zuolei, Sheng
Donglai, Luo Min, Tu Haitao, Sun Deying, Alan John Lowton. I appreciate their
friendship and their valuable suggestions, advice and help throughout my study. My
fellow labmates and other friends who have helped me one-way or another during the
course of my project are also greatly appreciated.
Last, but not least, I would like to thank my family members, my father, my mother and
my sister, who are always standing by me to give their utmost support. Their courage and
consolidation is the most powerful strength and is never separated by the distance, which
company me throughout my PhD process.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ····························································································· i
TABLE OF CONTENTS································································································ii
LIST OF PUBLICATIONS RELATED TO THIS STUDY········································ ix
LIST OF FIGURES ········································································································ x
LIST OF TABLES ·······································································································xiii
LIST OF ABBREVIATIONS ······················································································xiv
SUMMARY ··················································································································xvi
Chapter I. Introduction ·································································································· 1
I.1. Escherichia coli-the opportunistic infectious pathogen ······································ 2
I.1.1. Pathogenic E. coli ··························································································· 2
I.1.2. Epidemiology of EPEC ·················································································· 5
I.1.3. Epidemiology of EHEC ················································································· 6
I.2. Pathogenesis and virulence factors of EPEC and EHEC··································· 7
I.2.1. EPEC pathogenesis and virulence factors···················································· 8
I.2.1.1. Attaching and effacing histopathology ·················································· 8
I.2.1.2. Localized Adherence (LA) and Bundle Forming Pili (BFP) ·············· 10
I.2.1.3. EAF plasmids ························································································ 11
I.2.1.4. Invasion·································································································· 12
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I.2.1.5. Flagella··································································································· 13
I.2.1.6. Serine Protease - EspC·········································································· 14
I.2.1.7. Heat-stable enterotoxin (EAST1). ························································ 14
I.2.2. EHEC pathogenesis and virulence factors ················································· 15
I.2.2.1. Shiga toxin ····························································································· 15
I.2.2.2. Intestinal adherence factors ································································· 16
I.2.2.3. Invasion·································································································· 17
I.2.2.4. Flagella··································································································· 18
I.2.2.5. pO157 plasmid······················································································· 19
I.2.2.6. Serine protease ······················································································ 19
1.2.2.6.1. EspP ································································································ 19
I.2.2.6.2. StcE ································································································· 20
I.2.2.7. Heat-stable enterotoxin (EAST1). ························································ 21
I.2.2.8. Iron transport.······················································································· 22
I.2.3. Type Three Secretion System (TTSS) in EPEC and EHEC ····················· 22
I.2.3.1. Pathogenicity island (PAI)···································································· 23
I.2.3.2. Components of TTSS in EPEC and EHEC ········································· 24
I.2.3.2.1. Components of TTSS basal body ·················································· 25
I.2.3.2.2. TTSS translocators········································································· 26
I.2.3.2.3. LEE encoded adhesin····································································· 27
I.2.3.2.4. LEE encoded TTSS regulators ······················································ 29
I.2.3.2.5. Switches of TTSS translocators and effectors ······························ 29
I.2.3.3.6. TTSS chaperons ············································································· 30
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I.2.3.3.7. TTSS secreted Effectors································································· 30
I.3. Objectives ············································································································ 33
Chapter II. Common materials and methods······························································ 35
II.1. Common used medium and buffer··································································· 35
II.2. Bacteria strains and plasmids··········································································· 35
II.3. Tissue culture····································································································· 35
II.4. Molecular biology techniques ··········································································· 36
II.4.1. Genomic DNA isolation·············································································· 36
II.4.2. Cloning DNA fragments and transformation into E. coli cells················ 36
II.4.3. Analysis of plasmid DNA ··········································································· 37
II.4.4. Plasmid DNA isolation and purification ··················································· 37
II.4.5. DNA sequencing ························································································· 38
II.4.6. Sequence analysis ······················································································· 38
II.4.7. Southern hybridization ·············································································· 39
II.5. Protein techniques ····························································································· 40
II.5.1. Preparation of ECPs from E. coli strains ················································· 40
II.5.2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
································································································································ 40
II.5.3. Visualization of protein bands/spots (Coomassie blue staining and silver
staining)·················································································································· 41
II.5.4. Western blotting ························································································· 42
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Chapter III. Comparative proteomics analysis of extracellular proteins of
enterohemorrhagic and enteropathogenic Escherichia coli and their ······················· 44
Abstract······················································································································ 45
III.1. Introduction ····································································································· 46
III.2. Materials and methods ···················································································· 48
III.2.1. Bacterial strains and culture conditions ·················································· 48
III.2.2. Proteins isolation and assay······································································ 49
III.2.3. One- and two-dimensional gel electrophoresis········································ 49
III.2.4. Tryptic in-gel digestion and MALDI-TOF MS analysis························· 50
III.3. Results and discussion ····················································································· 51
III.3.1. ECP production ························································································ 51
III.3.2. Extracellular Proteomes of EHEC and EPEC ········································ 59
III.3.3. Identification of Ler and IHF regulated proteins ··································· 63
III.3.4. Applications and conclusions ··································································· 67
Chapter IV. Characterization of EspB as a translocator and an effector and its
involvement in EPEC autoaggregation········································································ 68
Abstract······················································································································ 69
IV.1 Introduction ······································································································ 70
IV.2. Materials and methods ···················································································· 72
IV.2.1. Bacterial strains and plasmids ································································· 72
IV.2.1. Fractionation of infected HeLa cells ························································ 73
IV.2.2. Invasion assay···························································································· 73
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IV.2.3. Examination of bacterial surface exposed structure by transmission
electron microscopy (TEM) ·················································································· 74
IV.2.4. Phase contrast microscopy ······································································· 74
IV.2.5. Construction of plasmid for expression of EspB-TEM and β-lactamase
based translocation assay······················································································ 75
IV.2.6. Edman N-terminal sequencing ································································· 75
IV.3. Results··············································································································· 76
IV.3.1. Survey of the two forms of EspB······························································ 76
IV.3.2. Mutation of espB did not affect the secretion of other extracellular
proteins··················································································································· 77
IV.3.3. Mutation of espB mutant abolished the translocation of effectors ········ 80
IV.3.4. EspB is translocated into infected HeLa cells·········································· 80
IV.3.5. EspB is involved in the autoaggregation·················································· 83
IV.3.6. EspB mutant does not form the autoaggregation ··································· 83
IV.3.7. ∆espB mutant showed less extracellular filamentous appendages ········· 86
IV.3.8. ∆espB mutant has lower invasion ability ················································· 87
IV. 4. Discussion ········································································································ 89
IV. 5. Conclusion ······································································································· 92
Chapter V. Identification and Characterization of NleI, a New Non-LEE-encoded
Effector of Enteropathogenic Escherichia coli (EPEC) ·············································· 93
Abstract······················································································································ 94
V.1. Introduction ······································································································· 95
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V.2. Material and methods ······················································································· 98
V.2.1. Bacteria strains and culture conditions····················································· 98
V.2.2. Tissue culture conditions············································································ 98
V.2.3. Construction of deletion mutants and plasmids ······································· 98
V.2.4. Flow cytometric analysis ············································································ 99
V.2.5. 2-D SDS-PAGE and proteomics ······························································ 100
V.2.6. Fractionation of infected HeLa cells························································ 100
V.2.7. Expression and Immunoblot analysis······················································ 101
V.2.8. Translocation assay ·················································································· 102
V.2.9. Fluorescence microscopy for observation of translocation, transfection
and actin condensation························································································ 102
V.3. Results ·············································································································· 106
V.3.1. Identification of NleI from EPEC sepL and sepD mutants ···················· 106
V.3.2. NleI is located within a prophage-associated island in EPEC ··············· 107
V.3.3. NleI is a secreted protein and the secretion of NleI is TTSS-dependent113
V.3.4. NleI is translocated into the host cells ····················································· 116
V.3.5. CesT is involved in the translocation but not the stabilization of NleI · 119
V.3.6. NleI is localized in the host cytoplasm and membrane ·························· 119
V.3.7. NleI is regulated by SepD but not regulated by Ler and SepL at the
transcriptional level····························································································· 124
V.3.8. NleI is not involved in the filopodia and pedestal formation ················· 125
V.4. Discussion········································································································· 128
V.5. Conclusion········································································································ 130
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Chapter VI. General conclusions and future directions ··········································· 132
VI.1. General conclusions ······················································································· 132
VI.2. Future directions···························································································· 134
Reference ····················································································································· 137
ix
LIST OF PUBLICATIONS RELATED TO THIS STUDY
1. Li, M., I. Rosenshine, S.L. Tung, X.H. Wang, D. Friedberg, C.L. Hew, and K.Y.
Leung. 2004. Comparative proteomics analysis of extracellular proteins of
enterohemorrhagic and enteropathogenic Escherichia coli and their ihf and ler
mutants. Appl. Environ. Microbiol. 70:5274-5282.
2. Li, M., I. Rosenshine, H.B. Yu, C. Nadler, E. Mills, C.L. Hew, and K.Y. Leung.
Identification and characterization of NleI, a new non-LEE-encoded effector of
enteropathogenic Escherichia coli (EPEC). (Microbes and Infection. 2006.
Accepted.)
3. Li, M., I. Rosenshine, and K. Y. Leung. Characterization of EspB as a translocator
and an effector and its involvement in EPEC autoaggregation. (In preparation)
x
LIST OF FIGURES
Fig. I.1. Characteristic EPEC A/E lesion observed in the ileum after oral inoculation of gnotobiotic piglets.
9
Fig. I.2. Genetic organization of the EPEC / EHEC Locus of Enterocyte Effacement (LEE) pathogenicity island.
10
Fig. I.3. Schematic representation of the EPEC/EHEC type III secretion apparatus.
28
Fig. III.1. Growth and protein secretion of EHEC EDL933 and EPEC E2348/69 in DMEM at 37oC with 5% (v/v) CO2.
52
Fig. III.2. Comparison of ECP production among EHEC EDL933 and EPEC E2348/69 wild type strains and their ler mutants.
53
Fig. III.3. Extracellular proteins from EHEC EDL933. 54
Fig. III.4. Extracellular proteins from EPEC E2348/69. 55
Fig. III.5. Comparative proteome analysis of EHEC EDL933 wild type strain, the ler mutant (DF1291) and ihf mutant (DF1292).
65
Fig. III.6. Comparative proteome analysis of EPEC E2348/69 wild type strain, the ler mutant (DF2) and ihf mutant (DF1).
66
Fig. III.7. One-D SDS PAGE of the ECPs from EHEC EDL933 and EPEC E2348/69 wild type strains, the ler mutants and ihf mutants.
66
Fig. IV.1. Extracellular proteins (ECPs) from EPEC E2348/69 (a) and EHEC EDL933 (b) treated with alkaline phosphatase.
77
Fig. IV.2. Protein secretion of EPEC strains in DMEM at 37oC.
78
xi
Fig. IV.3. Comparative proteomics analysis of ECP from EPEC E2348/69 wild type strain and the ∆espB mutant.
79
Fig. IV.4. Western blot analysis of Triton X-100 insoluble and soluble fractions of HeLa cells after infected with the EPEC wild type and the ∆espB mutant and detected with anti-Tir antibody.
79
Fig. IV.5. Demonstration of the translocation of EPEC EspB proteins into live HeLa cells by using TEM-1 fusions and fluorescence microscopy.
82
Fig. IV.6. Autoaggregation of EPEC in DEM culture is conferred by EspB. 84
Fig. IV.7. Microscopic observation of EPEC demonstration of EPEC E2348/69, ΔespB and ΔespB / pQEespB strains cultured in DMEM.
85
Fig. IV.8. Transmission electron microscopy showed the extracellular filamentous appendages of the EPEC wild type and ∆espB mutant.
86
Fig. IV.9. EPEC espB mutant showed reduced ability to invade HeLa cells. 88
Fig. V.1. Comparative analysis of the extracellular proteomes (ECPs) from the EPEC wild type E2348/69, the sepL::Tn10 mutant, and the ∆sepD mutant.
109
Fig. V.2. Graphic representation of the putative prophage-associated island that contains NleI by comparing the EPEC E2348/69 and the E. coli K12 MG1655 genomes.
110
Fig. V.3. Southern blot analysis of genomic DNA from EPEC E2348/69, Citrobacter rodentium DBS100 (CR), EHEC EDL933, and E. coli K12 MG1655.
111
Fig. V.4. Comparative proteome analysis of the ECPs from EPEC sepL::Tn10 mutant, sepL::miniTn10Kan ∆nleI mutant and sepL::miniTn10Kan ∆nleI / pACAYCnleI mutant.
112
Fig. V.5. Western blot analysis of secreted proteins and total proteins of EPEC E2348/69 wild type and the TTSS secretion mutant escN::TnPhoA expressing pSAnleI-6His with anti-6His antibody.
114
xii
Fig. V.6. NleI is secreted into the culture supernatant and translocated into live HeLa cells in a TTSS-dependent manner.
115
Fig. V.7. Demonstration of the translocation of EPEC effector proteins into live HeLa cells by using TEM-1 fusion proteins.
118
Fig. V.8. NleI is translocated by the EPEC wild type E2348/69 into HeLa cells in a CesT-dependent manner.
121
Fig. V.9. Western blot analysis of HeLa cell fractions after infection of the EPEC E2348/69 wild type and the TTSS secretion mutant escN::TnphoA expressing pSAnleI-6His.
122
Fig. V.10. Confocal microscopy demonstrates that NleI is localized in the cytoplasm and the nuclei of HeLa cells.
123
Fig. V.11. Flow cytometric analysis of the GFP intensity of the EPEC wild type E2348/69, ler::Kan, sepL::Tn10, and ∆sepD mutants, carrying pDN-gfp or pDNnleI-gfp.
126
Fig.V.12. NleI is not affecting actin condensation underneath the EPEC infection sites.
127
xiii
LIST OF TABLES
Table II.1. Bacterial strains and plasmid used in this study
43
Table III.1. Bacteria strains used in this study
48
Table III.2. Summary of the common extracellular proteins of EHEC and
EPEC identified by MALDI-TOF MS
56
Table III.3. Summary of the specific extracellular proteins of EHEC and EPEC respectively identified by MALDI-TOF MS
58
Table IV.1. Bacteria strains and plasmids used in this study 72
Table V.1. Bacteria strains and plasmids used in this study
104
Table V.2. Oligonucleotides used in this study
105
xiv
LIST OF ABBREVIATIONS
1D one-dimensional
2D two-dimensional
aa amino acid
Ampr ampicillin-resistant
BCIP 5-bromo-4-chloro-3-indolyl phosphate
bp base pairs
BSA bovine serum albumin
CFU colony forming units
Cm centimeter(s)
Cmr chloramphenicol-resistant
ºC degree Celsius
DMEM Dulbecco's Modified Eagle Medium
DNA deoxyribonucleic acid
ECP extracellular protein
EDTA ethelyne diamine tetra acetic acid
FBS fetal bovine serum
g gravitational force
HBSS Hank’s balanced salts solution
HEPES N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid
IEF isoelectric focussing
IPTG isopropyl-thiogalactoside
kb kilobase
Kanr kanamycin-resistant
l litre(s)
LB Luria-Bertani broth
LBA Luria-Bertani agar
M molarity, moles/dm3
MALDI matrix-assisted laser desorption ionization
MEM minimal essential medium
xv
mg milligram(s)
min Minute
ml milliliter(s)
mM milli moles/dm3
MOI multiplicity of infection
NBT Nitro blue tetrazolium
orf open reading frame
OD optical density
% Percentage
PAGE Poly acrylamide gel electrophoresis
PBS phosphate buffered saline
PCR polymerase chain reaction
pI isoeletric point
PPD Primary Prtoduction Department
ppm parts per million
PVDF Polyvinylidene difluoride
s second
SDS sodium dodocyl sulfate
Tcr tetracycline-resistant
TE Tris-EDTA
TOF time-of-flight
TTSS type III secretion system
U unit(s)
µg microgram(s)
µl microlitre(s)
wt wild type
v/v volume per volume
w/v weight per volume
X-gal 5- bromo-4-chloro-3-indolyl-B-D-galactopyranoside
xvi
SUMMARY Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are two
closely related human pathogens responsible for sever diarrhea in many countries. The
hallmark of infections caused by EHEC and EPEC is the attaching and effacing (A/E)
lesions on the epithelial cells. EPEC and EHEC utilize the type III secretion systems
(TTSSs) encoded on the locus of enterocyte effacement (LEE) to secrete and translocate
several effectors into host cells. The effectors may subvert the host signaling transduction
pathways and consequently cause diseases.
To further understand the pathogenesis and dissect the virulence of EPEC and EHEC, a
proteomics approach was used to analyze the extracellular protein (ECP) profiles of
EPEC, EHEC and their various mutants. Several common or strain specific virulence
factors identified from the extracellular proteomes of EPEC and EHEC indicate the close
relationship and variations between these two pathogens. Some novel proteins were also
identified by this proteomics approach, such as putative adhesins, secreted proteins and
phage related proteins. These novel proteins provided new candidates for exploiting
potential virulence factors of EPEC and EHEC.
One of the major secreted proteins, EspB, was found to have two dominant forms in
extracellular proteomics profiles of EPEC and EHEC. The two forms of EspB were
shown to remain unmodified at their N-terminus and they were unphosphorylated. With a
translocation signal at N-terminus, EspB was demonstrated to be an effector and to be
translocated into the host cells by TTSSs. By mutagenesis study, EspB was shown to be
xvii
involved in the EPEC aggregation and the ∆espB mutant has a lower invasion ability
when compared to the wild type. Transmission electron microscopy (TEM) showed that
EspB may affect the surface filamentous TTSS composition which may mediate the
bacteria cell interaction.
Furthermore, a putative effector, NleI, was identified from EPEC sepL and sepD mutants
by comparative proteomics study. NleI was found to be encoded outside the LEE region
and to exist in other A/E pathogens including EHEC and Citrobacter rodentium (CR).
The expression of NleI was found to be regulated by SepD at the transcriptional level.
After identifying the secretion and translocation signal at its N-terminal, NleI was
demonstrated to be translocated into HeLa cells in a TTSS dependent manner and the
translocation was facilitated by CesT. Using sub-cellular fractionation and fluorescence
microscopy, NleI was found to be localized in HeLa cell membrane and cytosol without
obvious cytotoxic effect. Fluorescent actin staining (FAS) indicated that NleI did not
affect the filopodia and pedestal formation of HeLa cells during infection.
This study has established a proteomics platform for accelerating the understanding of
EPEC and EHEC pathogenesis and identifying markers for laboratory diagnosis of these
pathogens. The discovery of new secreted proteins and new effectors by comparative
proteomics approach has provided valuable information on new virulence factors in
EPEC and EHEC. These results further supported the notion that EPEC and EHEC may
use multiple virulence factors to exploit the host cells and many factors may coordinate to
subvert different facets of host cellular processes.
1
Chapter I. Introduction
Microbial infection is one of the major causes of disease and mortality in humans.
Although infectious diseases have reduced the threat significantly due to improved
hygiene and sanitation, they are still a major public health problem worldwide. More
attention and efforts have been directed to study the mechanisms of infectious diseases
and to develop the diagnostic methods with particular emphasis on microbes. Among the
wide variety of infectious micro organisms, bacteria are problematic pathogens causing
many morbidity and mortality associated outbreaks around the world. The consequence
elicited by bacterial infections are diverse including food poisoning, diarrhea, meningitis,
lyme disease, pneumonia, tooth ache, anthrax, and even some forms of cancers
(www.who.int). Diarrhea is one of the most important illness caused by enteric bacteria,
with over 2 million deaths occurring each year, particularly among infants younger than 5
years old (www.who.int). Much research has been focused upon the pathogensis of these
bacteria in order to understand their mechanisms of infection and to develop the
diagnostics and therapeutics for these pathogens. Enteropathogenic Escherichia coli
(EPEC) and enterohemorrhagic Escherichia coli (EHEC) are two of the most common
human pathogens which constitute a significant risk to human health worldwide. The
present study attempts to investigate and understand the pathogenesis of EPEC and
EHEC. Approaches for understanding the pathogenesis of EPEC and EHEC, which
involves studying the molecular and cellular basis of microbial pathogenesis and host-
pathogen interaction, are the major focuses.
2
I.1. Escherichia coli-the opportunistic infectious pathogen
Among the members of the normal micro biota of the human intestine, E. coli strains are
the predominant facultative anaerobic inhabitants of the intestines of all animals,
including humans. When aerobic culture methods are used, E. coli strains are the
dominant species found in feces. These organisms typically colonize the gastro-intestinal
tract within a few hours and interact with the hosts after settling down on certain sites.
Once established, E. coli strains may persist for months or years. Resident strains shift
over a long period (weeks to months), and more rapidly after enteric infection or
antimicrobial chemotherapy that perturbs the normal flora. Normally E. coli strains serve
useful functions in animal bodies by suppressing the growth of harmful bacterial species
and by synthesizing appreciable amounts of vitamins. Remaining harmlessly confined to
the intestinal lumen, E. coli strains usually interact with host cells to derive mutual
benefit. Most E. coli live inside the human gastro-intestinal tract without causing any
symptoms of disease. However, a minority of E. coli strains are capable of causing
human illness by several different mechanisms and are called “pathogenic E. coli”.
I.1.1. Pathogenic E. coli
Diseases caused by pathogenic E. coli are numerous and include urinary tract infections
(UTI), meningitis, diarrhea, wound infections, septicemia and even endocarditic disease.
A lot of efforts have been put on to identify pathogenic E. coli and there have been
significant advances in our understanding of the pathogenesis of E. coli infection.
Urinary tract infections are a serious health problem affecting millions of people each
year. Most infections of the urinary tract are caused by one type of bacteria called
3
uropathogenic E. coli (UPEC) and are by far the second most common type of bacteria
infection in the body (Foxman et al., 2000). UTI can range in severity from asymtomatic
through bacteriuria, cystitis and pyelonephritis. UPEC colonizes from the feces or
perineal region and ascend the urinary tract to the bladder. With the aid of specific
adhesins, such as type 1 fimbria, S fimbria, P fimbria and Dr family of adhesins, UPEC is
able to colonize the bladder (Mulvey et al., 2000; Bahrani-Mougeot et al., 2002). To
acquire iron during or after colonization, UPEC produces siderophore and hemolysin and
utilizes the hemoglobin and heme released from lysed erythrocytes (Hantke et al., 2003).
These virulence factors function jointly causing the infection and inflammatory
symptoms in the urinary tract.
Meningitis is the inflammation of the membranes covering the brain and spinal cord.
Though multiple organisms may cause meningitis, E. coli is the leading agent responsible
for around 20% of the neonatal meningitis cases (Health et al., 2003). Meningitis is a
substantial cause of morbidity and mortality in neonates. Typically transmitted via
respiratory droplets, E. coli strains invade the blood stream of infants from the
nasopharynx or gastro-intestinal (GI) tract and are carried to the meninges where they
usually lead to infection of the meninges. The K1 E. coli is considered the major
pathogenic strain of E. coli that causes neonatal meningitis.
Pathogenic E. coli are also well known for their ability to cause intestinal diseases. Six
classes of E. coli that cause diarrhea diseases are now recognized: enterotoxigenic E. coli
(ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAggEC), diffusely
4
adherent E. coli (DAEC), EHEC and EPEC (Nataro and Kaper, 1998). Each class falls
within a serological subgroup and manifests distinct features in pathogenesis. ETEC is an
important cause of diarrhea in infants and travelers in developing countries or regions of
poor sanitation. It was first isolated from piglets and was further identified as a human
pathogen too. ETEC colonizes the surface of the small bowel mucosa and causes diarrhea
through secretion of heat-labile (LT) and heat-stable (ST) enterotoxins. EIEC closely
resembles Shigella in their invasive mechanisms and the nature of clinical illness they
produce. EIEC penetrates and multiplies within epithelial cells of the colon causing
widespread cell destruction. The clinical syndrome is similar to Shigella dysentery and
includes a dysentery-like diarrhea with fever. The distinguishing feature of EAggEC
strains is their ability to attach to tissue culture cells in an aggregative manner. These
strains are associated with persistent diarrhea in young children. DAEC displays a
diffusive adherence phenotype to cultured HEp-2 cells. They are responsible for some
diarrhea cases in children of ages from 1-5 years. Their surface fimbria and outer
membrane protein may mediate the diffusive adherence phenotype of DAEC. EPEC and
EHEC are two major pathotypes of diarrhea E. coli. They can induce watery diarrhea or
bloody diarrhea, and thus, are distinct from other diarrhea E. coli groups. These two
pathogenic E. coli affect the infants and adults world wide and have become the well
recognized human pathogens. Our present work is mainly focused on EPEC and EHEC
and the epidemiology and pathogenesis of these two pathogens will be further discussed
in the following paragraphs.
5
I.1.2. Epidemiology of EPEC
EPEC refers to an important category of diarrheagenic E. coli that were first identified in
epidemiological studies in the 1940s and 1950s as causes of epidemic and sporadic
infantile diarrhea (Levine and Edelman, 1984). EPEC is an established aetiological agent
of human diarrhea and remains an important cause of infant mortality in developing
countries (Nataro and Kaper, 1998). The most notable feature of the epidemiology due to
EPEC is the striking age distribution seen in persons infected with this pathogen. EPEC
infection is primarily a disease of infants younger than 2 years and the infection can often
be quite severe, and many clinical reports emphasize the severity of the disease (Bower et
al., 1989; Rothbaum et al., 1982). Several outbreaks of diarrhea due to EPEC have been
reported in healthy adults, presumably due to ingestion of a large inoculum from a
common source (Viljamen, et al., 1990; Hedberg et al., 1997). Sporadic disease has also
been seen in some adults with compromising factors such as diabetics, achlohydria
(Levine and Edelman, 1984).
The transmission of EPEC is through fecal-oral route with contaminated hands and
materials. The reservoir of EPEC infection is thought to be symptomatic or asymptomatic
children or adult carriers (Levine and Edelman, 1984). Numerous studies have
documented the spread of infection through hospitals, nurseries, and day care centers
from an index case (Bower et al., 1989, Wu and Peng 1992). Epidemiologic studies in
several countries have shown high backgrounds of asymptomatic carriage. In
symptomatic patients, EPEC can be isolated from stools up to 2 weeks after cessation of
symptoms (Hill et al., 1991). Although animals such as rabbits, pigs, and dogs have
EPEC-like organisms associated with disease (Zhu et al., 1994; Nakazato et al., 2004),
6
the serotypes found in these animal pathogens are usually not human serotypes (Krause et
al., 2005; Leomil et al., 2005).
I.1.3. Epidemiology of EHEC
EHEC is the causative agents of hemorrhagic colitis, a bloody diarrhea that usually
occurs without fecal leukocytosis or fever. Although more than 25 serotypes of EHEC
have been isolated from hemorrhagic colitis cases, O157:H7 is the predominant EHEC
serotype isolated in outbreaks of hemorrhage colitis (Griffin 1995). EHEC O157:H7 was
the cause of several large outbreaks of disease in North America, Europe, Japan and some
developing countries (Griffin and Tauxe 1991, Yoshioka et al., 1999). Numerous
epidemiological studies have demonstrated an association between hemorrhagic colitis
caused by EHEC and the subsequent development of a life-threatening systemic
complication referred to as the hemolytic uremic syndrome (HUS) (Boyce et al., 1995).
HUS most commonly presents as acute renal failure accompanied by the swelling and
detachment of glomerular endothelial cells and hemolytic anemia with schistocytosis.
HUS following EHEC infection is now recognized as the most common cause of
pediatric acute renal failure in many developed nations. Patients with EHEC infections
are also at increased risk of neurological abnormalities such as seizures, cortical
blindness and coma (Siegler 1995).
EHEC can be transmitted by food and water and from person to person. Most cases are
caused by ingestion of contaminated foods, particularly foods of bovine origin. A very
7
low infectious dose for EHEC infection has been estimated from outbreak investigations.
EHEC has a wide reservoir of various animals, including cattle, sheep, goats, pigs, cats,
dogs, chicken and gulls (Beutin et al., 1993; Griffin and Tauxe 1991; Johnson et al., 1996;
Wai et al., 1996). The most important animal host other than human is cattle. High rates
of colonization of Shiga-like toxins (Stxs) positive EHEC have been found in bovine
herds in many countries.
I.2. Pathogenesis and virulence factors of EPEC and EHEC
Pathogenic E. coli strains differ from those that predominate in the enteric flora of
healthy individuals in that they are more likely to express virulence factors which are
molecules directly involved in pathogenesis but ancillary to normal metabolic functions.
In addition to their roles in disease processes, these virulence factors presumably enable
the pathogenic strains to explore niches unavailable to commensal strains, and thus to
spread and persist in the bacterial community. EPEC and EHEC colonize the intestinal
mucosa, subvert the intestinal epithelial cell signal transduction and demolish the
function of host cells, and subsequently cause diarrhea diseases. These two enteric E. coli
are closely related pathogens sharing many similarities in their virulence determinants.
Many similar virulence factors of EPEC and EHEC distinguish them from non-
pathogenic E. coli strains. Though EHEC strains are considered to have evolved from
EPEC strains through acquisition of bacteriophages encoding Shiga-like toxins (Stxs)
(Reid et al., 2000; Wick et al., 2005), there are still differences in their pathogenesis
independent of Stxs activity. To expound the difference and similarity of EPEC and
EHEC, their pathogenesis and virulence factors will be separately discussed below.
8
I.2.1. EPEC pathogenesis and virulence factors
Although EPEC was the first E. coli to be associated with human disease, it was not until
the late 1980s and early 1990s that the mechanisms and bacterial gene products used by
EPEC to induce the attaching and effacing lesions and diarrhea disease started to be
unraveled.
I.2.1.1. Attaching and effacing histopathology
The typical histopathology changes caused by EPEC infections is the attaching and
effacing (A/E) lesion, which can be observed in intestinal biopsy specimens from patients
or infected animals and can be reproduced in cell cultures (Polotsky et al., 1977; Moon,
et al., 1983; Andrade, et al., 1989; Ulshen and Rollo, 1980). This striking phenotype is
characterized by effacement of microvilli and intimate adherence between the bacterium
and the epithelial cell membrane (Fig. I.1.). Marked cytoskeletal changes including the
accumulation of polymerized actin, are seen directly beneath the adherent bacteria, where
the bacteria sit upon a pedestal-like structure (Fig. I.1.; Taylor et al., 1986; Jerse et al.,
1990). These pedestal structures can extend up to 10 mm out from the epithelial cell in
pseudopod like structures (Moon et al., 1983). It is observed that the composition of the
A/E lesion contained high concentrations of polymerized filamentous actin (Knutton et
al., 1989). This observation led to the development of the fluorescent-actin staining (FAS)
test which enabled the screening of clones and mutants, resulted in the identification of
the bacterial genes involved in producing this pathogenic lesion. Similar A/E lesions are
seen in animal and cell culture models of EHEC, Citrobacter rodentium (CR), Hafnia
alvei and a variety of E. coli strains isolated from animals. Thus, EPEC strains are the
9
prototype of an entire family of enteric pathogens that produce A/E lesions on the
epithelial cells. The proteins which are responsible for A/E lesion are encoded in Locus
of Enterocyte Effacement (LEE) pathogenicity island (PAI) (Fig. I.1.) This LEE PAI
composed of 41 genes which are organized in five major polycistronic operons (LEE1-
LEE5). Many virulence features of the A/E pathogens are related to this LEE PAI which
will be discussed in detail in the type III secretion system (TTSS) section (I.2.3).
Fig. I.1. Characteristic EPEC A/E lesion observed in the ileum after oral inoculation of gnotobiotic piglets. Note the intimate attachment of the bacteria to the enterocyte membrane with disruption of the apical cytoskeleton. Note the loss of microvilli and the formation of a cup-like pedestal to which the bacterium is intimately attached (highlighted by circle). Reprinted from Baldini et al., 1983b.
10
Fig. I.2. Genetic organization of the EPEC / EHEC Locus of Enterocyte Effacement
(LEE) pathogenicity island. (This figure was reprinted from Deng et al., 2004)
I.2.1.2. Localized Adherence (LA) and Bundle Forming Pili (BFP)
In the early 1980s, investigators reported that EPEC strains adhere to tissue culture cells
in densely packed three-dimensional clusters on the surface of the host cells (Baldini et
al., 1983). This particular pattern of EPEC strains adherence was known as localized
adherence (LA). This pattern of adherence is so characteristic of and specific to EPEC
strains that it can be used as the basis for diagnostics (Cravioto et al., 1991). It is found
that the factor mediating this LA of EPEC was certain rope-like fimbriae called bundle
forming pili (BFP) (Giron et al., 1991). Strains of E. coli produce various types of
adherence pili, some of which may be involved in pathogenicity (Hacker 1992). The BFP
produced by EPEC are 50 to 500 nm wide and 14 to 20 μm long (Giro’n et al., 1993).
The BFP of EPEC exists as bundles and intertwines with BFPs of other bacterial cells to
11
create three dimensional networks. The genes encoding BFP share 41% identity with the
toxin co-regulated pilus (tcp) gene of V. cholerae (Donnenberg et al., 1992). However,
several of the genes are unique in EPEC in which homologues are barely found in other
species. In addition, the protein sequence of BFP shared homology and other
characteristics with the type IV pili of Pseudomonas aeruginosa, Neisseria gonorrhea
and Moraxella bovis (Giro’n et al., 1997). Recently, Khursigara and co-workers (2001)
have identified that BFP binds to the lipid phosphatidylethanolamine, and it has been
proposed that this could be responsible for BFPs interaction with both the host and other
bacterial cells. BFP may be partially or wholly responsible for the LA phenotype by the
recruitment of bacteria in the environment of the host cell (Giron et al., 1993). A recent
data also indicated that the structure of BFP and the intact function of BFPs are required
for the full virulence of EPEC (Bieber et al., 1998).
I.2.1.3. EAF plasmids
The genes coding for BFP of EPEC are located in a large plasmid called EPEC adherence
factor (EAF) plasmid (Baldini et al., 1983). A number of EPEC strains contain a large,
50-70 MDa plasmid and share extensive homology as EAF plasmid among EPEC strains
(Nataro, et al., 1987). Plasmid encoded regulators (Pers) are encoded in a cluster of perA,
perB, perC genes at the downstream of the bfp gene cluster in the same EAF plasmid.
These perABC genes encode transcriptional activators that positively regulate several
genes in the chromosome and on the EAF plasmid. PerA is homologue to an AraC family
of bacteria regulators and activate the expression of the down stream gene perC. The
production of PerC can increase the expression of the chromosomal eae (E. coli attaching
12
and effacing) and espB (E. coli secreted protein B) genes, as well as that of genes
encoding 20 kD, 33 kD and 50 kD outer membrane proteins (Gomez-Duarte et al., 1995).
The BFP structural gene bfpA is also under the control of per operon since it is described
that bfpTVW regulatory gene cluster which regulates bfpA expression is in fact allelic
with perABC (Zhang and Donnenberg, 1996).
The importance of the EAF plasmid was investigated by many groups and it is reported
that this large plasmid is involved in the LA on the human epithelia cells and the
adherence to piglet intestinal epithelial cells (Baldini et al., 1983). The plasmid cured
derivative did not process this adherence property and transformation of this plasmid to
non-adherent E. coli. strain enabled this strain to adhere to HEp-2 cells (Baldini et al.,
1986). The crucial importance of EAF plasmid in human disease was tested by using the
wild type E2348/69 strain and the plasmid-cured derivatives and the results indicated that
the plasmid was necessary for full virulence of EPEC in volunteers (Levine et al., 1985).
I.2.1.4. Invasion
Several investigators have shown that EPEC strains are capable of entering a variety of
epithelial cell lines (Andrade et al., 1989; Miliotis 1989). Furthermore, some published
photographs of animal and human EPEC infections showed apparently intracellular
bacteria (Moon, 1983). However, unlike the intracellular pathogens such as Shigella spp.,
EPEC strains do not multiply intracellularly or escape from phagocytic vacuoles and thus
do not appear to be specifically adapted for intracellular survival. The invasion phenotype
is very useful in studying the molecular genetics of EPEC because many of the genes
13
involved in invasion are also involved in forming the A/E lesion. Donnenberg et al.
(1990) have used TnphoA and the gentamicin protection assay to isolate mutants deficient
in cell entry. bfpA or dsbA loci affect the EPEC invasion property which are also required
to produce functional BFP fimbriae. intimin and espB mutants were also deficient in
intimate adherence as well as invasion. sep/esc genes encoding the TTSS proteins were
also deficient in invasion. These results indicate that there is significant overlap between
genes responsible for the invasion process and genes involved in producing attaching and
effacing lesion.
I.2.1.5. Flagella
The EPEC flagellum consists of components that facilitate the export of flagellar proteins
and flagellar assembly (Aldridge and Hughes, 2002). Flagella are major organelles that
are important in the pathogenesis of EPEC. For EPEC, flagella are sufficient to induce
interleukin (IL)-8 releases in T84 cells via activation of the Erk and p38 pathways (Giron
et al., 2002). EPEC flagella are important for adherence and the mutation of fliC reduces
adherence. Purified flagella bind to the epithelia cells and block the binding of EPEC
(Zhou et al., 2003). Epidemiological studies of EPEC infection have revealed that EPEC
strains isolated throughout the world belong to a restricted number of O antigen
serogroups, and notably to a limited number of flagellar (H) antigen types (Nataro and
Kaper, 1998). Giron and co-workers (2002) investigated the involvement of flagella as an
adhesin of EPEC and the biological relevance of flagella expression by bacteria adhering
to cultured epithelial cells. They demonstrated that the flagella produced by EPEC
contribute to the adherence properties of the bacteria and that a molecule secreted by
14
eukaryotic cells induces their expression. Data also showed that there exists a molecular
relationship between flagella, virulence-associated TTSS, Per, and quorum sensing.
However, Cleary and co-worker (2004) observed the adhesion of wild-type EPEC strain
E2348/69 and a set of isogenic single, double and triple mutants to differentiated human
intestinal Caco-2 cells and they found no evidence that flagella contribute to EPEC
adhesion.
I.2.1.6. Serine Protease - EspC
EspC is a large serine protease with 110 kD molecular weight and is secreted by EPEC
and related strains of E. coli. It is homologous to members of the autotransporter protein
family, which includes IgA proteases of Neisseria gonorrhoeae and Haemophilus
influenzae, Tsh protein produced by avian pathogenic E. coli, SepA of Shigella flexneri,
and AIDA-I of DAEC (Stein et al., 1996). This heat-labile factor blocks lymphocyte
proliferation and production of interferon-γ, IL-2, IL-4 and IL-5 in response to a variety
of stimuli (Klapproth et al., 1996; Malstrom and James 1998). EspC produces an
enterotoxic effect, which is indicative of internalization and cleavage of the calmodulin-
binding domain of fodrin and resulting in actin cytoskeleton disruption. Recently, it has
been observed that EspC does not function in a manner similar to the homologous Pet
protein, a serine protease specific to enteroaggregative E. coli (Navarro-Garcia et al.,
2004).
I.2.1.7. Heat-stable enterotoxin (EAST1).
15
Many EPEC strains produce a low-molecular-weight heat-stable protein called EAST1
which is encoded by astA gene (Savarino et al., 1996). The EPEC strain E2348/69 was
found to contain two copies of the astA gene, one in the chromosome and another one in
the EAF plasmid. The significance of this toxin in EPEC pathogenesis is unknown. Some
EAF-negative EPEC-like organisms responsible for outbreaks of disease in adults also
contained the astA gene (Viljanen et al., 1990). More frequently isolation of EPEC strains
containing astA from adults other than from EPEC strains lacking astA might yield
insights into the striking age distribution seen with infections due to EPEC.
I.2.2. EHEC pathogenesis and virulence factors
Initially, EHEC was distinguished from other strains of E. coli by its serotype, namely
O157:H7. But subsequently, it was found to produce Shiga toxin (Stx), which has
become the defining feature of this pathotype. An inflammatory response is also induced
in response to EHEC intimate adherence, the A/E histopathology.
I.2.2.1. Shiga toxin
Shiga toxins, also known as verotoxins and Shiga-like toxins, occur in two major
antigenic groups, namely Stx1 (VT-I) and Stx2 (VT-II) (Paton and Paton 1998). Shiga
toxin was first identified in S. dysenteriae (O’ Brien et al., 1992) where it is
chromosomally encoded. But the genes for its production are readily transmitted between
E. coli strains by toxin-encoding bacteriophages. The EHEC that are most commonly
associated with human disease, are lysogenized with λ-like bacteriophage that encode the
structural genes for Stx1 and/or Stx2 (Strockbine et al. 1986). The Stx family is
16
composed of Shiga toxin (Stx), and a group of closely related toxins elaborated by EHEC,
which are designated Stx2, Stx2c and Stx2e (Scotland and Smith 1997). The production
of Stx1 from EHEC and S. sysenteriae is repressed by iron and reduced temperature, but
expression of Stx2 is unaffacted by these factors (O’ Brien and Holmes 1987). Although
there is antigenic heterogeneity among the members of the Stx family, all the biologically
active toxins characterized to date share an AB5 molecular configuration; in other words,
a single enzymatically active A-subunit protein in noncovalent association with a
pentamer of receptor-binding B-subunit proteins.
EHEC can produce severe or even fatal renal and neurological complications as a result
of the translocation of Shiga toxins across the gut (O’ Brien and Holmes 1987).
Production of a potent Stx is essential for many of the pathological features as well as the
life-threatening sequelae of EHEC infection. In contrast to advances in the genetics and
biochemistry of Stxs, the precise role of the toxins in the pathogenesis of hemorrhagic
colitis and HUS has yet to be fully elucidated. The myriad of metabolic events may lead
to intravascular coagulation, which results in thrombocytopenia, microangiopathic
hemolytic anemia, with erythrocyte fragmentation and renal failure. However the
pathogenesis is a multi-step process, involving a complex interaction between a range of
bacterial and host factors (O’ Brien et al., 1996).
I.2.2.2. Intestinal adherence factors
It is generally assumed that the colon and perhaps also the distal small intestine are the
principal sites of EHEC colonization in humans, although this has not been demonstrated
17
directly (Boyce et al., 1995). Within EHEC strains belonging to serotype O157:H7, there
is heterogeneity in adherence, and this may reflect differences in mechanisms. Some
strains adhered in a diffuse fashion, with bacteria distributed evenly over the surface of
the epithelial cells. Other strains formed tight clusters or microcolonies at a limited
number of sites on the epithelial surface. The existence of intestinal adherence factors of
Stx-producing E. coli strains of serotypes O157:H7 is suggested by the isolation of
serotypes other than O157:H7 that lack the eae gene but are still associated with bloody
diarrhea or HUS in humans, was reported (Dytoc et al., 1994; Scotland et al., 1994).
Type 1 fimbriae were suggested to be involved in the adherence of some EHEC strains
(Durno et al., 1989). The best-characterized EHEC adherence phenotype, however, is
intimate or attaching and effacing (A/E) adherence. This property is exhibited by a
subgroup of EHEC strains, which is highly similar to EPEC (refer to EPEC A/E lesion).
The genes which are responsible for this phenotype will be discussed in more detail
below in the TTSS section (I.2.3).
I.2.2.3. Invasion
EHEC strains have also been examined for their capacity to invade epithelial cells. It is
found that EHEC strains differ from other enteric pathogens (e.g., salmonellae, shigellae,
and EPEC) in that they are unable to efficiently invade HEp-2 and Henle 407 cells
(Oelschlaeger et al., 1994). However, O157:H7 EHEC strains were taken up by T24
bladder cells and HCT-8 cells, although individual EHEC strains varied in their invasive
capacity. The invasion process was dependent upon both bacterial protein synthesis and
host cell microfilaments (Oelschlaeger et al., 1994). However, McKee and O’Brien (1995)
18
reported that the level of uptake of O157:H7 EHEC by HCT-8 cells was significantly
lower than that of EPEC or Shigella flexneri strains and no greater than that of a
commensal E. coli strain. In a recent report, Luck and co-workers (2005) compared the
adherence properties of LEE-negative EHEC against LEE positive strains in vitro and
reported that a number of EHEC LEE-negative serotypes were internalized by epithelial
cells compared with EHEC O157:H7 that remained extracellular. Invasion was shown to
be dependent on an intact actin cytoskeleton and microtubule function, along with active
GTPases, but was not dependent on the activity of tyrosine kinases. The study suggested
that LEE negative EHEC strains may use a mechanism of host cell invasion to colonize
the intestinal epithelium which may compensate for the lack of the LEE and hence the
inability to form A/E lesions (Luck et al., 2005).
I.2.2.4. Flagella
The EHEC flagella are likely to be similar to EPEC which are major organelles that are
important in the pathogenesis of EHEC. However, the role for EHEC O157:H7 flagella is
equivocal. Purified H7 antigen did not inhibit adherence of EHEC O157:H7 to HEp-2
cells, whereas purified H6 flagella of EPEC have been demonstrated to inhibit in vitro
adherence (Giron et al., 2002). The different flagellin structures of EHEC O157:H7 and
EPEC O127:H6 may drive the different binding and tropism property. EHEC flagella
have been shown to play a role in persistence in chickens and seemed to be required for
long-term persistence in animal (Best et al., 2005). Given the importance of type III
secretion for the colonization of EHEC and EPEC and the structural relationship of the
19
TTSS with flagella, a cross-talk between TTSS and flagella system is likely to exist
during the colonization process.
I.2.2.5. pO157 plasmid
E. coli O157:H7 and most other EHEC strains associated with human disease possess a
94 - 104 kb plasmid called pO157 (Schmidt et al., 1994; Schmidt et al., 1996). The role
of this plasmid in pathogenesis is not certain since the majority of animal studies
performed with EHEC strains lacking this plasmid show no difference when compared
with the plasmid-containing parental strain. Sequence analysis of pO157 plasmid from
EHEC led to the detection of genes encoding enterohemolysin, catalase-peroxidase and
putative adherence factors in this plasmid (Schmidt et al., 1995; Brunder et al., 1996).
The two serine proteases, EspP and StcE/TagA, are also encoded in this O157 plasmid
(Brunder et al., 1997; Lathem et al., 2002). A gene cluster for a type II secretion system
is found in this plasmid and StcE/TagA is secreted by this system (Lathem et al., 2002).
This plasmid also encodes a hemolysin of the RTX family (Schmidt et al., 1995, Bauer
and Welch 1996). In a collection of O111 :H- EHEC strains from Germany, this
hemolysin was expressed in 88% of strains isolated from patients with HUS but in only
22% of patients with diarrhea without HUS (Beutin et al., 1993). Epidemiological
evidence suggests a stronger correlation of the presence of this plasmid with the
development of HUS rather than diarrhea (Schmidt and Karch, 1996).
I.2.2.6. Serine protease
1.2.2.6.1. EspP
20
A putative virulence factor encoded on pO157 is the extracellular serine protease EspP
(Brunder et al., 1997). The espP gene encodes a 1,300-amino-acid protein, which is
subsequently subjected to N- and C-terminal processing during the secretion process and
come out with a mature form of apparent size of 104 kDa. EspP has homology
(approximately 70%) to EspC, a 110-kDa EPEC secreted protein, and to a lesser degree
to immunoglobulin A1 (IgA1) proteases of H. influenzae and Neisseria spp. The region
of homology includes the serine-containing proteolytic active site. EspP did not exhibit
IgA1 protease activity in vitro but was capable of cleaving pepsin, an activity which was
inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride. EspP was also
able to cleave human coagulation factor V. Thus, it is suggested that the secretion of
EspP by EHEC colonizing the gut could result in exacerbation of hemorrhagic disease
(Brunder et al. 1997). Similar report from Djafari and co-workers (1997) also showed
that EspP is cytotoxic for Vero cells. A role for EspP in pathogenesis is also consistent
with the presence of antibodies to the protease in sera from five of six children with
EHEC infection but not in sera from age-matched controls.
I.2.2.6.2. StcE
StcE, or Tag A, is a secreted protease of C1 esterase inhibitor from EHEC (Lathem et al.,
2002). It is secreted via the type II secretion apparatus and is a metalloprotease that
specifically cleaves C1 esterase inhibitor (a member of the serine protease inhibitor
family), having no protease activity on other serine protease inhibitors, extracellular
matrix proteins or universal protein targets. StcE is encoded in the pO157 plasmid of
EHEC O157:H7 (Lathem et al., 2002) together with EspP. The bacterial protease is
21
positively regulated by the LEE-encoded regulator - Ler ( Elliott et al., 2000; Li et al.,
2004) and resulting pathology may include localized proinflammatory and coagulation
responses, causing tissue damage, intestinal oedema and thrombotic abnormalities
(Lathem et al., 2002). A result of StcE cleavage of C1-INH is the enhancement of C1-
INH ability to inhibit complement-mediated lysis of host ovine erythrocytes (Grys et al.,
2005). This is due to direct interaction between StcE, host cells and C1-INH. Thus,
through cleavage at the N-terminal domain, StcE treated C1-INH also provides a
significantly increased serum resistance for E. coli K12, showing a StcE induced
protection system against complement for the infecting bacteria and infected host cells
(Lathem et al., 2004). Through protease activity on glycoproteins and the resultant
cleavage from the host cell, StcE helps block the clearance of EHEC O157 through
glycoprotein destruction and contributes to the intimate adherence of the bacteria to host
cell surfaces (Grys et al., 2005).
I.2.2.7. Heat-stable enterotoxin (EAST1).
Another virulence factor that might contribute to the pathogenesis of the watery diarrhea
often seen during the early stages of EHEC infection is the enterotoxin EAST1 which is
encoded by astA gene. This is a 39-amino-acid enterotoxin that was initially recognized
in certain strains of EAggEC and is distinct from the heat-stable toxins produced by
ETEC (Savarino 1991). Other Stx-producing EHEC strains including O26:H11 and non-
O157/O26 strains also carry the astA gene (Savarina et al., 1996). In these strains, there
were two copies of astA, which were located on the chromosome. Enterotoxicity for
rabbit ileal tissue was confirmed by testing culture ultrafiltrates (10 kDa) of three of the
22
astA1 O157:H7 strains in Ussing chambers. The significance of EAST1 in the
pathogenesis of disease due to EHEC is unknown but it could possible account for some
of the non-bloody diarrhea frequently seen in persons infected with these strains.
I.2.2.8. Iron transport.
EHEC O157:H7 contains a specialized iron transport system which allows this organism
to use heme or hemoglobin as an iron source (Law and Kelly 1995). A 69-kDa outer
membrane protein encoded by the chuA (E. coli heme utilization) gene is synthesized in
response to iron limitation, and expression of this protein in a laboratory strain of E. coli
was sufficient for utilization of heme or hemoglobin as the iron source (Torres and Payne
1997). A gene homologous to chuA is also present in strains of S. dysenteriae I, but not in
other Shigella spp. or in other Shiga toxin-producing E. coli strains such as those of
serotype O26:H11 (Mills and Payne 1995). The growth of E. coli O157:H7 is stimulated
by the presence of heme and hemoglobin, and the lysis of erythrocytes by one or more of
the hemolysins reported for this pathogen could release these sources of iron, thereby
aiding infection.
I.2.3. Type Three Secretion System (TTSS) in EPEC and EHEC
TTSS is machinery by which bacteria inject many effector proteins across bacteria and
eukaryotic cells membranes to reach host cells target. It is a common virulence
mechanism which is conserved in many human, animal and plant pathogens. The genes
encoding a functional TTSS are usually residing in a pathogenicity island (PAI). EPEC
and EHEC also utilize TTSS to deliver virulence factors (effector proteins) into
23
eukaryotic cells. The filamentous TTSSs have high similarity in between EPEC and
EHEC which are both encoded in PAI of LEE. These filamentous TTSSs also confer the
capacity to form A/E lesions in EPEC and EHEC which are the major virulence factor in
these two pathogens. The following sections will review the common features of the
chromosomal PAI of LEE and the filamentous TTSS complex in EPEC and EHEC.
I.2.3.1. Pathogenicity island (PAI)
The term ‘pathogenicity island’ (PAI) is used to depict a cluster of virulence-associated
genes that are collocated on the bacteria chromosome of pathogenic bacteria but are
absent from non-pathogenic varieties of the same species (Hacker and Kaper, 2000).
Newly emerging data also suggest that PAIs may exist in plasmids or bacteriophage
genomes (Hacker and Kaper, 2000). The PAIs were first described in human pathogens
of the E. coli family Enterobcteriaceae (Hacker and Kaper, 1990), but are also present in
plant pathogens (Jackson et al., 1999). And they have recently been found in the genomes
of various pathogens of humans, animals and plants (Hacker and Kaper 2000). PAIs
usually occupy relatively large genomic region, covering more than 10-200 kb DNA
regions. They often show evidence of having been acquired from other bacteria. This
evidence includes the PAIs DNA consist regions that differ from the core genome in G+C
content in general and in different codon usage; flanked by small directly repeated
sequence; associated with transfer RNA genes and carry cryptic or functional genes
encoding mobility factors at their termini, such as integrase, transposases insertion
sequences or parts of these elements (Kaper et al., 1999). Together, these observations
suggest that PAIs may be transferred horizontally between bacteria by processes
24
involving bacterial viruses or plasmids. The ongoing processes of rearrangement, deletion
and transfer of PAIs have suggested a strong impact on microevolution and adaptation of
pathogenic microbes during the infectious process. The widespread occurrence of the
PAIs reveals that these genetic elements have been adapted after the long evolutionary
journey. Virulence factors encoded on PAIs represent the entire spectrum of bacterial
virulence factors, from adhesins to invasins, from secretion system to toxins, and from
modulins to host defense avoidance mechanisms.
I.2.3.2. Components of TTSS in EPEC and EHEC
The PAI that contributes to the adhesive ability of EPEC and EHEC is termed the LEE
because EPEC and EHEC require it to produce A/E lesions on enterocytes (Frankel et al.,
1998). The LEE is absent from the non-pathogenic laboratory strain of E. coli K-12, but
when LEE is transferred to this strain, it was bestowed A/E capacity upon epithelial cells
(Jarvis et al., 1995). Nucleotide sequence analysis of the LEE region of EPEC and EHEC
revealed the presence of 41 open reading frames (ORFs), most of which are organized
into five polycistronic operons named LEE1 through LEE5. Generally, the LEE genes
encode: (i) the components of the type III secretory pathway basal body; (ii) a series of
translocator proteins termed E. coli-secreted proteins (Esp), which are secreted via this
pathway; (iii) an outer membrane protein adhesin known as intimin that is encoded by the
eae gene; (iv) three global regulators which regulate the expression of most of the LEE
genes; (v) two molecular switches which control the secretion of translocators and
effectors; (vi) chaperon proteins for translocators and effectors; (vii) effector proteins
which can be secreted and translocated into host cells. Functions of some of the proteins
25
encoded by these genes in LEE region have been assigned while some genes still
remained cryptic.
I.2.3.2.1. Components of TTSS basal body
The TTSS apparatus of EPEC and EHEC are multicomponent organelles assembled by
approximately 20 proteins (Fig. I.2; Garmendia et al., 2005). Components of the basal
body are broadly conserved among virulence TTSS and flagellar systems (Aizawa 2001;
Tampakaki et al., 2004). EscC and EscV are the main components of the outer and inner
membrane ring structures, respectively (Hueck 1998; Gauthier et al., 2003). EscC
belongs to the secretin family of proteins, which are found in all TTSSs and are involved
in the transport of molecules across the outer membrane by formation of a large homo-
multimeric annular complex. EscV is predicted to have seven transmembrane domains
and to form the EPEC and EHEC inner membrane ring structures. The presence of a
putative signal sequence indicates that the protein is likely to be directed to the inner
membrane through the sec pathway, as observed for EscV homologues in other TTSSs.
EscJ, which also contains a sec-dependent signal sequence, is highly conserved within
TTSSs (Hueck 1998). EscJ and its homologues are lipoproteins proposed to span the
periplasm, serving as a bridge between the inner and outer membrane protein rings (Deng
and Huang 1999). Those protein shares sequence similarity within the domain of the
flagellar protein FliF that is believed to line the central pore of the inner membrane ring
and proposed to form part of the proximal rod structure (Ueno et al., 1994). EscJ is
required for the secretion of more distal apparatus components across the outer
membrane, as an escJ mutant failed to assemble functional TTSS machinery (Crepin et
26
al., 2005). EscN is a cytoplasmic protein which binds to inner membrane components of
TTSS (Gauthier et al., 2003). It is believed to be the energy source for the TTSS export
machinery since it shares homology with the F0F1 ATPase and has a putative ATP-
binding site (Hueck 1998). It is suggested that the EscN ATPase interacts with the TTSS
basal components and facilitates protein secretion or translocation (Akeda and Galan
2005; Pozidis et al., 2003).
I.2.3.2.2. TTSS translocators
The translocators such as EspA, EspB and EspD are TTSS secreted proteins that are
required for the translocation of the effector proteins into the host cytoplasm (Frankel et
al., 1998; Fig. I.1). EspA is localized in the TTSS needle tip by the addition of new
subunits at the tip to form a helical tube with a hollow central channel, connecting the
bacterial needle complex to the host cell and thereby forming a conduit through which
effector proteins are transported (Daniell et al. 2003; Crepin et al., 2005). EspB and EspD
are inserted into the membrane of infected cells where they are thought to form
translocation pores in the host cell membrane (Wachter et al., 1999; Ide et al., 2001;
Shaw et al., 2001). EspD is required for polymerization of EspA filaments and cell
attachment and hemolysis (Knutton et al., 1998; Kresse et al., 1999; Daniell et al., 2001).
Besides playing structural role in host membrane to help other effectors’ transportation,
EspB can be detected in cytoplasm of infected cells, which implies that EspB may also
have an additional function as an effector (Wolff et al., 1998). EspA, EspB and EspD are
essential for the pathogenesis of EPEC and are regarded as virulence factors (Abe et al.,
1998; Donnenberg et al., 1993; Tacket et al., 2000).
27
I.2.3.2.3. LEE encoded adhesin
Intimin is an adhesin expressed by EPEC and EHEC which is required for tight binding
to host cells (Jerse et al., 1990). This adhesion is encoded by eae gene located in LEE5
operon and exported via the general secretory pathway. Intimin is highly conserved
among EPEC and EHEC strains with overall 83% protein identity (Yu et al., 1992). The
homology of intimin comes from the N-terminus, while the C-terminus of intimin
contains the receptor-binding residues exhibit diversity (Frankel et al., 1994; Oswald et
al., 2000). After exported from bacteria cytosol, intimin is inserted into the bacterial outer
membrane where it interacts with its receptors Tir and form intimate attachment. Besides
the interaction with Tir, intimin can also bind to host generated receptors (Reece et al.,
2001). It was shown that tissue tropisms for the γ subtype which expressed by EHEC
O157:H7 was follicle-associated in humans and cattle. While α subtype expressed by
EPEC P127:H7 shows a more broad specificity (Phillios and Frankel, 2000; Phillips et al.,
2000; Naylor et al., 2003). The role of intimin in human and animal disease was tested
and results indicated that intimin is essential for full virulence of EPEC and EHEC
(Donnenberg et al., 1993a; Donnenberg et al., 1993b; Deng et al., 2004).
28
Fig. I.3. Schematic representation of the EPEC/EHEC type III secretion apparatus. The basal body of the TTSS is composed of the secretin EscC, the inner membrane proteins EscR, EscS, EscT, EscU, and EscV, and the EscJ lipoprotein, which connects the inner and outer membrane ring structures. EscF constitutes the needle structure, whereas EspA subunits polymerize to form the EspA filament. EspB and EspD form the translocation pore in the host cell plasma membrane, connecting the bacteria with the eukaryotic cell via EspA filaments. The cytoplasmic ATPase EscN provides the energy to the system by hydrolyzing ATP molecules into ADP. SepD and SepL have been represented as cytoplasmic components of the TTSS which may serve as switches for TTSS. (This figure is reprinted from Garmendia et al., 2005)
29
I.2.3.2.4. LEE encoded TTSS regulators
The central positive LEE encoded regulator (Ler) is encoded in the LEE1 operon and is
primarily an anti-repressor which is essential for the expression of most of the LEE genes
(Bustamante et al., 2001; Elliot et al., 2000). Ler is an auto-regulator which will also
regulate the expression of itself in order to maintain proper amount in the bacterial cells
for regulation net work (Berdichevsky et al., 2004). GrlA and GrlR are another two
recently identified LEE-encoded regulators which are highly conserved in all A/E
pathogens (Deng et al., 2004). GrlA acts as a positive regulator for LEE expression
including ler. With the reciprocally positive regulation between each other, GrlA and Ler
form a positively transcriptional regulatory loop to maintain an appropriate
spatiotemporal response during the infection (Barba et al., 2005). GrlR functions as a
negative regulator for LEE (Deng et al., 2004, Lio et al., 2004) and it would control the
production of Ler and other TTSS proteins when it is highly expressed.
I.2.3.2.5. Switches of TTSS translocators and effectors
LEE also encodes two switches responsible for translocators and effectors secretion.
SepL, is a soluble cytoplasmic protein and associates with the bacteria membrane fraction,
which is required for the A/E effect (Kresse et al., 2000; O’Connell et al., 2004). It is
encoded in the LEE4 operon which contains espA, espD and espB genes. sepD, also
known as rorf6, is localized within the LEE2 operon in between escC and escJ. It is
found that SepD is a required component of the EPEC TTSS and will interact with SepL
to form a membrane bound complex (Creasey et al., 2003; O’Connell et al., 2004). SepL
30
and SepD are showed to be essential for the secretion of TTSS translocators but not
effectors (Deng et al., 2004). It is postulated that SepL and SepD form a molecular switch
that controls the secretion of translocators and effectors as a conserved feature in all A/E
pathogens, including CR, EHEC and EPEC (Deng, et al., 2005).
I.2.3.3.6. TTSS chaperons
To day, five LEE-encoded chaperones have been identified for EPEC and EHEC. CesAB
is the chaperon for translocator proteins EspA and EspB (Creasey et al., 2003a). CesAB
stabilizes and interacts with EspA and EspB specifically in bacterial cytosol prior to the
secretion. CesD is an inner membrane bound protein and serves as a bivalent chaperon
for translocator protein EspB and EspD (Wainwright and Kaper, 1998). CesD2 is another
chaperon for EspD which appears to be required for proper secretion of EspD (Neves et
al., 2003). CesF is the chaperon for effector protein EspF, which is necessary for the
stability and translocation of EspF and for EPEC to reduce transepithelial electrical
resistance (TER) (Elliott et al., 2002). CesT is a LEE encoded TTSS chaperon for multi-
effectors (Abe et. al., 1999; Creasey et. al., 2003b; Thomas et. al., 2005). It plays a vital
role in stabilization and recruitment of LEE and non-LEE encoded effectors and assures
their secretion efficiency.
I.2.3.3.7. TTSS secreted Effectors
TTSS secreted effector proteins are translocated into host cells by the functional TTSS
for the purpose of modulating signal transduction pathways and cellular processes
(Hueck 1998). Genes encoding translocated effectors have been found both within and
31
outside the LEE pathogenicity islands that encode the TTSS. To date, 6 EPEC LEE-
encoded TTSS-translocated effectors have been identified: Tir (EspE), EspF, EspG, EspH,
Map and SepZ (EspZ) (Kenny et al., 1997; McNamara et al., 2001; Elliott et al., 2001;
Tu et al., 2003; Kenny and Jepson 2000; Kanack et al., 2005). EPEC and EHEC TTSS
translocate effector protein, Tir, into host cells (Kenny et al., 1997). After translocation,
Tir is inserted into the host cell plasma membrane and serves as a bacterial receptor for
the bacterial outer membrane protein intimin (Jerse et al., 1990; Kelly et al., 1990). In the
plasma membrane, Tir adopts a hairpin-loop topology featuring a central extracellular
domain that binds intimin (Luo et al., 2000). The interaction between Tir and intimin
leads to the actin polymerization and the intimate attachment of EPEC and EHEC to
hosts plasma membranes and formation of an actin-rich pedestal, beneath the adherent
bacterium (Moon et al., 1983; Knutton et al., 1989). Mitochondria associate protein (Map)
was translocated by TTSS and targets to the mitochondria (Kenny and Jepson, 2000). It
has been suggested to disrupt mitochondria normal functions and initiate filopodium
formation immediately upon interaction with the host cell via the GTPase Cdc42 (Jepson
et al., 2003). In constrast, Alto and co-workers (2006) recentely found that Map mimics
Cdc42 rather than using Cdc42 for filopodia formation. In contrast, EspH represses
filopodium formation and enhances the formation of actin-rich pedestals (Tu et al., 2003).
EspG is a 44-kDa protein homologous to VirA, a translocated protein of S. flexneri that
triggers host microtubule destabilization. It is indicated that EspG together with another
homologue EspG2 can disrupt microtubules network, enhance perturbation of the tight
junction barrier and even alter epithelial paracellular permeability (Tomson et al., 2005;
Matsuzawa et al., 2005). EspF disrupts intestinal barrier function and therefore
32
potentially contributes to EPEC diarrhea. EspF also plays a role in epithelial cell
apoptosis. An interesting feature worth to be noted is all the LEE-encoded effectors
except Tir are dispensable for A/E lesion formation.
In addition, A/E pathogens secrete a number of non-LEE-encoded effectors. Newly
identified effectors located outside of the LEE locus include Cif, NleA/EspI, Tccp/EspFu,
EspJ and EspG2 (Marches et al., 2003; Gruenheid et al., 2004; Mundy et al., 2004;
Campellone et al., 2004; Garmendia et al., 2004; Dahan et al., 2005; Shaw et al., 2005;
Matsuzawa et al., 2005). Cif is carried on a lambdoid phage and triggers an irreversible
cytopathic effect in HeLa cells, which is characterized by the progressive recruitment of
focal adhesions, assembly of stress fibers, and arrest of the cell cycle. EspI/NleA is
carried within prophage CP-933P, localized to the Golgi, and is required for full
virulence in the C. rodentium model. TccP/EspFu is an EHEC O157 effector that is
carried on prophage CP-933U and translocated into host cells, where it displays an Nck-
like activity. EspJ is a TTSS secreted effector protein encoded by prophage CP-933U of
attaching and effacing pathogens that modulates the infection dynamics of clearance of
the pathogen from the host's intestinal tract (Dahan et al., 2005). In addition to these
effectors, Deng and co-worker (2004) recently identified 7 new non-LEE-encoded
effector proteins NleB, NleC, NleD, NleE, NleF, NleG from CR by using systematical
mutagenesis and proteomics approaches. These Nles have been shown to be secreted by
CR in sepD and sepL mutants and their secretion are TTSS dependent. Although some of
the Nles have been shown to be translocated into host cells (Marches et al., 2005), their
functions have yet to be further characterized.
33
I.3. Objectives
It is reported that several virulence factors from EPEC and EHEC perturb different facets
of the host physiology, resulting in the cumulative effect of diarrhea. The objective of this
study was to use proteomics approach to identify the virulence factors of EPEC and
EHEC. The virulence factors from EPEC and EHEC were characterized using approaches
in molecular biology, biochemistry and cellular biology to provide an in-depth
understanding of the EPEC and EHEC pathogenesis, which may be analogous diseases in
human and animals.
Secreted or surface exposed proteins from pathogens may play essential roles in the
processes of pathogen-host interaction, thus contributing to the virulence. Initially, a
proteomics approach was used to compare the ECPs between the EPEC and EHEC wild
type and their respective ler and ihf mutants in order to identify virulence factors from
EPEC and EHEC (Chapter III). The comparative proteomics profiles were used to depict
the close relationship and differences between EPEC and EHEC. Extracellular virulence
factors, including known virulence determinants and potential virulence proteins, were
studied in an integrative manner to reveal the pathogenicities of EPEC and EHEC.
EspB was one of the dominant secreted protein identified from EPEC and EHEC ECP
proteomes and was reported to be a virulence factor. Thus, EspB was further
characterized in Chapter IV. This includes the observation and survey of different forms
34
of EspB, as well as the investigation of the property of EspB as an effector and a
translocator and the involvement of EspB in the EPEC aggregation formation.
Effector proteins are key virulence factors used by A/E pathogens to subvert host cell
signaling pathway. Since SepL and SepD constitute a molecular switch for controlling
the secretion of translocators and effectors, the sepL and sepD mutants were used to
identify effectors from A/E pathogens. In Chapter V, a novel effector - NleI was
identified from EPEC sepL and sepD mutants by comparative proteomics. The
translocation property and sub-cellular localization of NleI were further characterized.
35
Chapter II. Common materials and methods
II.1. Common used medium and buffer
Luria-Bertani (LB) broth from Difco laboratories was used for the normal maintenance
and propagation of E. coli for molecular microbiology procedures. Dulbecco’s modified
eagle medium (DMEM) with L-glutamine, 25 mM HEPES buffer and pyridoxine
hydrochloride but without phenol red (Gibco BRL) was used for culturing HeLa cells,
EPEC and EHEC strains to induce extracellular protein (ECP) secretion.
Phosphate buffered saline (PBS) was used for washing and resuspension of the bacteria.
PBS contained 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4 and 1.4 mM KH2PO4, and
the pH was adjusted to 7.2.
II.2. Bacteria strains and plasmids
The bacterial strains and plasmids used in this thesis are listed in Table II.1. E. coli
strains were grown on LB agar (Difco) plates or in LB broth (Difco) or DMEM
supplemented as needed with ampicillin (Amp, 100 µg/ml), chloramphenicol (Chl, 30
µg/ml), kanamycin (Kan, 50 µg/ml), and streptomycin (Str, 50 µg/ml). Cultivation of
bacteria in DMEM was carried out at 5% (v/v) CO2 atmosphere, 37oC. Stock cultures of
E. coli were maintained at -80°C as a suspension in supplemented LB broth containing
25% (v/v) glycerol.
II.3. Tissue culture
36
HeLa cells were cultured in DMEM (Gibco BRL) supplemented with 10% heat-
inactivated fetal bovina serum (FBS) and penicillin (200 IU/ml) and streptomycin (100
µg/ml). The cells were grown in cell culture flasks (100 ml) at 37oC in a humidified
atmosphere of 5% CO2. Before experiment, Hela cells were washed with PBS or DMEM
to remove FBS and antibiotics.
II.4. Molecular biology techniques
II.4.1. Genomic DNA isolation
E. coli strains were grown in LB broth cultured at 37°C overnight. Bacterial genomic
DNA was extracted using QIAGEN Genomic DNA Purification Kit according to the
manufacturer’s protocols. After the mini-preparation, the purified genomic DNA was
dissolved in TE buffer (10 mM Tris-HCl, and 1 mM EDTA, pH 7.5) and stored at -20°C
for future use.
II.4.2. Cloning DNA fragments and transformation into E. coli cells
PCR amplified DNA fragments were cloned into the pGEM-T Easy vector system
(Promega, USA) following the manufacturer’s instructions and were transformed into E.
coli JM109 or E. coli DH5α. E. coli competent cells were prepared and transformed as
described by Sambrook and co-workers (1989). After transformation, bacteria were
recovered in LB broth and plated on LB agar plates containing ampicillin,
isopropylthiogalactoside (IPTG, Bio-Rad), 5-bromo-4-chloro-3-indolyl-β-D
galactopyranoside (X-gal, Bio-Rad), to allow for blue-white colony selection.
37
II.4.3. Analysis of plasmid DNA
To check the potential clones with target DNA fragments inserted in plasmids, the boiling
lysis procedure was used as described by Holmes and Quingley (1981). Briefly, 20 μl of
overnight bacterial culture was obtained and spun at 8000 × g for 2 min. The bacterial
pellet was resuspended in 3 μl of sterile water, followed by addition of 8 μl of STET
solution [0.1 M NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), 5% Triton X-
100, 0.8 μg/μl lysozyme and 10 μg/μl RNase A]. Subsequently, the tubes were placed on
boiling water bath for 30 s and allowed to cool down to room temperature. One µl of 10 ×
buffer of appropriate restriction enzyme and 0.1 - 0.2 units of restriction enzyme was
added and the tubes were incubated at appropriate temperature for 30 min. The raw
digested DNA samples were analyzed by gel electrophoresis using 1% (w/v) agarose gel
(Seakem®, BioWhittaker Molecular Applications) followed by staining in ethidium
bromide. Clones containing the right insert in the plasmids were chosen for performing
further experiments. Alternatively, for low copy plasmids, PCR amplification was used to
screen those desired clones by using primers specific for the inserted DNA.
II.4.4. Plasmid DNA isolation and purification
E. coli strains containing desired plasmids were cultured in LB broth (with appropriate
antibiotics) and incubated in an orbital shaker (Forma scientific) with 225 rpm shaking at
37°C overnight for plasmid isolation. Plasmid DNA was isolated using the QIAGEN
Plasmid Mini Purification Kit (QIAGEN Spin columns, QIAGEN GmbH). The
concentration and quality of the plasmid DNA were determined by measuring the OD260
and OD280 using a spectrophotometer (Shimadzu UV-1601, Japan).
38
II.4.5. DNA sequencing
The ABI PRISM BigDye Terminator version 3.1 Cycle Sequencing Ready Reaction Kit
was used to perform sequencing PCR reaction (Applied Biosystems). The sequencing
reaction mix consisted of: 1 µl of autoclaved water, 100 to 200 ng of the plasmid DNA or
40 to 50 ng of PCR product, 0.5 µl of 2.5 µM primer and 1 µl of BigDye. Sequencing
PCR reaction was carried out in GeneAmp PCR system 2400 or 2700 (Applied
Biosystems) using the conditions: 1 min at 96 oC, 25 cycles of denaturation 96oC, 10 s,
annealing 50oC, 5 s, extension 60oC, 1 min 45 s. The PCR product was precipitated by
NaAc solution [1 µl of NaAc (pH 3.6), 2.5 µl of water and 15.5 µl of 100% ethanol].
After centrifugation at 14000× g for 20 min, the pellet obtained was washed with 500 µl
of 70% ethanol twice. Later the pellet was air dried and stored at -20°C. Prior to
sequencing, 12 µl of Hi-Di™ Formamide (Applied Biosystems) was added to dissolve
the pellet and the sample mixture was loaded into 96 well sequencing plate. DNA
sequencing was carried out on ABI PRISM® 3100 genetic analyzer using the dye
termination method.
II.4.6. Sequence analysis
Sequence assembly and further editing were carried out with Vector NTI DNA analysis
software (InforMax). BLASTN, BLASTP and BLASTX sequence homology analyses
and a protein conserved-domain database analysis (CDD search) were performed using
the BLAST network server of the National Center for Biotechnology Information
(NCBI) (http://www.ncbi.nlm.nih.gov/BLAST; Altschul et al., 1990).
39
II.4.7. Southern hybridization
Corresponding DNA probe was labeled by nick translation with biotin 14-dATP using
the BluGene Non-Radioactive Nucleic Acid Detection System (Invitrogen).
Approximately, 5 - 10 µl of PCR product or 1 - 2 µg of plasmid DNA was digested using
appropriate restriction enzymes and incubated with the labeling mixture at 37 oC for 6 -
12 h according to manufacturer’s protocol. Approximately 2 - 4 µg of genomic DNA of
EPEC, EHEC or CR strains was digested with appropriate restriction enzymes prior to
Southern analysis. The digested DNA were resolved by electrophoresis on 1% agarose
gel and stained with ethidium bromide. After washing, DNA in the gel was transferred to
a Hybridization Transfer Membrane (Gene Screen Plus, Perkin Elmer) for overnight at
room temperature. The next day, the transferred DNA was cross-linked to a membrane
using UV-transilluminator (SPS-TF, Vilber Lourmat) for 5 min and air-dried. The nylon
membrane with DNA was incubated with the pre-hybridization solution (DIG Easy Hyb,
Roche Diagnostics GmbH) and further incubated with the hybridization solution which
contained heat denatured, biotin labeled probe in the hybridization bags (Invitrogen) for
overnight at 42°C. After hybridization, the membrane was washed and blocked with DIG
Wash and Block Buffer Set (Roche Diagnostics GmbH) and incubated with alkaline
phosphatase (AP) conjugated anti-digoxigenin antibody for 1 h in blocking solution.
After wash with washing solution for 3 times, the hybridization result was visualized by
incubating membrane with 10 ml detection buffer containing 200 µl of NBT / BCIP stock
solution (Roche Diagnostics GmbH) in the dark. The membrane was observed once in 5 -
10 min till the appearance of bands and wash with TE buffer to terminate the reaction.
40
II.5. Protein techniques
II.5.1. Preparation of ECPs from E. coli strains
To prepare the extracellular proteins (ECPs) from E. coli strains, bacteria were cultured in
LB broth overnight. Then the overnight LB cultures were diluted at 1:50 into DMEM and
incubated for 6 to 9 h according to strains at 37 oC in a 5% (v/v) CO2 atmosphere. Bacterial
cells were removed from the culture by centrifugation (5500 × g, 10 min, 4 oC) and the
supernatant was filtered through a 0.22 µm low protein binding filter (Millex, Millipore).
The ECP fraction was isolated by TCA precipitation (Shimizu et al., 2002) and the protein
pellet was washed with –20 oC acetone thrice and air-dried. The protein pellet was
solubilized in ReadyPrep Reagent 3 [5 M urea, 2 M thiourea, 2% (w/v) CHAPS, 2% (w/v)
SB 3-10, 40 mM Tris, and 0.2% (w/v) Bio-Lyte 3/10 ampholyte; Bio-Rad]. Insoluble
materials were removed by centrifugation at 18 000 × g for 10 min at room temperature and
protein samples were stored at –20 oC until analysis. Protein concentration was determined
by using the Bio-Rad protein assay kit with bovine serum albumin as the standard.
II.5.2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
Ten to fiften % polyacrylamide gels were used for protein separation based on molecular
weight property. Normal SDS-PAGE was performed according to a standard protocol
(Sambrook et al., 1989). Briefly, different percentage of acrylamide resolving gel
solution was poured into the gap between two glass plates and the 4% stacking gel was
layered on top of the resolving gel and a clean Teflon comb was immediately inserted.
After the stacking gel had polymerized, the Teflon comb was carefully removed and the
wells were washed with milli-Q water to remove any traces of unpolymerized acrylamide.
41
Prior to loading the protein samples, the sodium-dodecyl (SDS) gel-loading buffer (50
mM Tris-HCl, 100 mM DTT [Bio-Rad], 2% [w/v] SDS, 0.1% [w/v] bromophenol blue
(Bio-Rad), 10% glycerol) was added and the samples were boiled for 5 min. After
loading the samples, the electrophoresis apparatus was attached to an electric power
supply and a constant current of 5 mA per gel was applied. After the dye front has moved
into the resolving gel, the current was increased to 15 mA per gel.
II.5.3. Visualization of protein bands/spots (Coomassie blue staining and silver
staining)
50% methanol (v/v), 10% acidic acid solution (v/v) with 0.1% (w/v) Coomassie Brilliant
Blue R-250 (Bio-rad) (Meyer and Lamberts, 1965; Sambrook et al., 1989) was used to
stain the 1D or 2D SDS-PAGE for 1 h or overnight and de-stained with 20% methanol
(v/v) and 10% acetic acid (v/v).
For more sensitive detection of proteins in the gel, silver staining (Blum et al., 1987) was
performed. The gel was first fixed in 50% (v/v) methanol and 10% (v/v) acetic acid for at
least 30 min, followed by 15 min in 50% (v/v) methanol. After that, the gel was washed 4
times (5 min each) with milli-Q water, followed by treated with 0.02% (w/v) fresh
prepared sodium thiosulfate solution for 1 min, and washed another 2 times (1 min each)
with milli-Q water. Freshly prepared silver nitrate (0.2 μg/ml) (Merck) solution was then
added and the gel was stained for 25 min. After staining, the gel was washed twice (1 min
each) with milli-Q water and developed in 3% [w/v] sodium carbonate, 0.025% [v/v]
formalin [Sigma] solution for 5 - 10 min till the desired patterns appreaed. The
42
developing was stopped by 1.4% (w/v) EDTA for 10 min and washed with mili-Q water
twice.
II.5.4. Western blotting
Proteins from EPEC, EHEC or HeLa cells were resolved using a 12.5% SDS-PAGE gel.
Proteins from SDS-PAGE were electro-blotting transferred onto a PVDF membrane
(Bio-Rad) according to the manufacture instructions in a semi-dry transfer system (Trans-
Blot SD cell, Bio-Rad). The membranes were then blocked with 5% milk solution and
then incubated in appropriately diluted primary antibody for 1 h or overnight. After wash
with PBS-T for three times, the membrane was incubated with appropriate secondary
antibody solution (goat anti-rabbit IgG or goat anti-mouse IgG secondary antibody
conjugated with alkaline phohpatase (AP) or horseradish peroxidase (HRP). Positive
bands were visualized by using bromocholoroindolyl phosphate (BCIP) and nitroblue
tetrazolium (NBT) as substrates for AP-conjugated secondary antibodies. Alternatively,
the enhanced Chemiluminescence Detection Kit (Pierce) was used as substrate to give a
more sensitive detection for HRP conjugated secondary antibodies, and followed by
exposure on to a Kodak film (Kodak).
43
Table II.1. Bacterial strains and plasmid used in this study
Strain or plasmid Genotype and/or relevant property Source / Reference
E. coli strains
DH5α SupE44 ∆lac U169 (ø80lacZ∆M15)
hsdR17 recA1 endA1 gyrA96 thi-1
relA1; Amps
Hanahan, 1983
MC1061(λpir) (λpir), thi thr-1 leu6 proA2 his-4 argE2
lacY1 galK2 ara14 xyl5 supE44, λpir
Rubires et al., 1997
SM10(λpir) thi thr leu tonA lacY supE recA::RP4-2-
Tc::Mu Kmr λpir
Miller and
Melakanos, 1988
JM109 Cols, Amps Promega
S17-1(λpir) thi pro hsdR hsdM+ recA [RP42-Tc::Mu-Km::Tn7
(Tpr Smr)Tra+]
Simon et al., 1983
BL21-DE3
E2348/69 Wild type EPEC O127:H6, Smr J. Kaper
EDL933
Wild type EHEC O157:H7, Slt1, Slt2 negative
derivative
J. Leong
Plasmids
pGEM®-T Easy vector Cloning vector; Ampr Promega
pACYC184 Tetr, Cmr Amersham
pRE112 Suicide vector; R6K ori sacB Cmr Edwards et al., 1989
44
Chapter III. Comparative proteomics analysis of extracellular proteins
of enterohemorrhagic and enteropathogenic Escherichia coli and their
ihf and ler mutants
Part of this chapter has been published in:
Li, M., I. Rosenshine, S.L. Tung, X.H. Wang, D. Friedberg, C.L. Hew, and K.Y. Leung.
2004. Comparative proteomics analysis of extracellular proteins of enterohemorrhagic
and enteropathogenic Escherichia coli and their ihf and ler mutants. Appl. Environ.
Microbiol. 70:5274-5282.
45
Abstract
EHEC and EPEC are closely related human pathogens responsible for food-borne
epidemics in many countries. In this study, a proteomics approach was used to analyze
the extracellular protein profiles of EHEC EDL933 and EPEC E2348/69, and their ihf
and ler mutants. The proteins were separated by using two-dimensional (2-D) SDS
PAGE followed by matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF MS) and subsequently by searching among protein database.
Fifty-nine major protein spots from their extracellular proteomes were identified,
including six proteins of unknown functions. Twenty-six of them were conserved
between EHEC EDL933 and EPEC E2348/69 while some of them are strain specific
proteins. Four common extracellular proteins (EspA, EspB, EspD and Tir) were regulated
by both IHF and Ler in EHEC EDL933 and EPEC E2348/69. TagA in EHEC EDL933,
EspC and EspF in EPEC E2348/69 were present in the wild type strains but absent in
their respective ler and ihf mutants, while FliC was overexpressed in the ihf mutant of
EPEC E2348/69. These results showed the relation and divergence of EHEC and EPEC
in an integrated view from their secretion proteom. And that proteomics was
demonstrated to be a powerful platform technology for accelerating the understanding of
EHEC and EPEC pathogenesis and identifying markers for laboratory diagnosis of these
pathogens.
46
III.1. Introduction
EHEC and EPEC are closely related human pathogens (Donnenberg and Whittam, 2001).
They can colonize the intestinal mucosa and induce diarrhea disease in a wide range of
animals including rabbits, dogs, piglets, cattle and humans. EHEC, especially those of
serotype O157:H7 which produce Shiga-like toxin (Stx), is a common cause of diarrhea,
hemorrhagic colitis, and hemolytic uremic syndrome, while EPEC is the most common
bacterial cause of infant diarrhea (Nataro and Kapper, 1998). Both have been implicated in
food-borne outbreaks in many countries and cause diarrhea by colonizing the intestinal
mucosa (Nataro and Kapper, 1998; Paton and Paton, 1998). EHEC and EPEC are
distinguished from other pathogenic E. coli strains by their ability to produce a
characteristic histopathological feature known as the A/E lesion on the mucosa (Frankel et
al., 1998; Nataro and Kapper, 1998). The AE lesion is characterized by destruction of the
microvilli and induction of actin-based pedestal formation underneath the eukaryotic
membrane at the site of attachment (Donnenberg et al., 1997). EHEC and EPEC secrete
many ECPs and some of them are characterized as virulence factors such as EspA, EspB,
EspC and Tir. A TTSS is conserved in EHEC and EPEC which is necessary for the
formation of AE lesions (Jarvis et al., 1995; Jarvis and Kapper, 1996). TTSS is encoded in
a chromosomal pathogenicity island designated as LEE, which is a major secretion
apparatus for secreting virulence factors which interact directly with the host (Kenny and
Finlay 1995; McDaniel et al., 1995). The LEE encoded regulator (Ler) activates most of the
genes within the LEE region and is central to the process of AE lesion formation (Mellies et
al., 1999). The integration host factor (IHF) is a histone-like protein which can bind to
specific DNA consensus sites and bend the DNA to form a nucleoprotein complex (Nash et
47
al., 1996; Rice et al., 1996). The IHF is involved in a wide variety of cellular processes,
including directly activating the expression of the ler transcriptional unit, and Ler in turn
mediates the expression of the other LEE genes (Goosen and van de Putte, 1995; Friedberg
et al, 1999). The expression of both IHF and Ler is needed to elicit the actin rearrangement
associated with AE lesions.
Proteomics offers a powerful platform technology for the study of protein expression and
identification. This is a useful tool for the analysis of the disease process and bacteria-
host interactions at the protein level. Recent advances in micro-sequencing and mass
spectrometry, combined with the availability of the complete genome sequence allowing
the identification of proteins from either the sequences of single peptides or even the
masses of a number of peptides (Pappin et al., 1993). The DNA sequences of two EHEC
genomes were determined recently (Hayashi et al., 2001; Perna et al. 2001), and the
EPEC genome sequence will be completed soon (refer to Sanger Institute website,
http://www.sanger.ac.uk/Projects/Microbes). Therefore, EHEC and EPEC are ideal
organisms for proteomics analysis.
Here we use the proteomics approach to analyze the extracellular proteomes of EHEC
and EPEC in order to give further insight into the pathogenesis and divergence of these
two emerging pathogens. Comparative proteomics analysis among the wild type strains,
the ler and ihf mutants were used to identify putative virulence factors from EHEC and
EPEC.
48
III.2. Materials and methods
III.2.1. Bacterial strains and culture conditions
The wild type strains of EHEC, EPEC and their mutants used in this study are listed in
Table III.1. EHEC ler and ihf mutants were constructed by using the Lambda Rad system
as described earlier (Friedberg et al, 1999; Yona-Nadler et al., 2003). E. coli strains were
routinely cultured at 37oC in Luria-Bertani (LB) broth without shaking. When required,
media were supplemented with ampicillin (Amp, 100 µg/ml), kanamycin (Kan, 50 µg/ml),
and streptomycin (Str, 100 µg/ml).
Table III. 1. Bacterial strains used in this study
Strain Description Reference or source
E2348/69 Wild type EPEC O127:H6, Smr J. Kaper
EDL933
Wild type EHEC O157:H7, Slt1, Slt2 negative derivative
J. Leong
DF2 EPEC E2348/69 ler::kan Friedberg et al. (1999)
DF1 EPEC E2348/69 ihfA::kan Friedberg et al. (1999)
DF1291 EDL933 ler::Kan This study
DF1292 EDL933 ihf::Kan This study
49
III.2.2. Proteins isolation and assay
To prepare the ECPs of EHEC and EPEC, overnight cultures in LB were diluted at 1:50
into DMEM and incubated for 9 and 6 h, respectively at 37 oC in a 5% (v/v) CO2
atmosphere. Bacterial cells were removed from the culture by centrifugation (5500 xg, 10
min, 4 oC) and the supernatant was filtered through a 0.22 µm low protein binding filter
(Millex, Millipore). The ECP fraction was isolated by TCA precipitation (Shimizu et al.,
2002) and the protein pellet was washed with –20 oC acetone thrice and air-dried. The
protein pellet was solubilized in ReadyPrep Reagent 3 [5M urea, 2M thiourea, 2% (w/v)
CHAPS, 2% (w/v) SB 3-10, 40 mM Tris, and 0.2% (w/v) Bio-Lyte 3/10 ampholyte; Bio-
Rad] and were stored at –20 oC until analysis. Protein concentration was determined by
using the Bio-Rad protein assay kit with bovine serum albumin as the standard.
III.2.3. One- and two-dimensional gel electrophoresis
ECPs in the amount of 10 to 30 g that gave similar profiles of background proteins were
used to generate the extracellular proteomes of different strains for comparative analysis.
One-dimensional (1-D) SDS PAGE was carried out following a standard protocol
(Sambrook et al., 1989). Two-dimensional (2-D) SDS PAGE was performed with the
EttanTM IPGphor™ Isoelectric Focusing System (Amersham) according to the
manufacturer’s instructions. Dry gel strips were rehydrated for 12 h at room temperature
with 8 M urea, 2% (w/v) CHAPS, 0.5% IPG buffer, 50 mM DTT, and a trace amount of
bromophenol blue. For the first dimension, the ECP samples were separated using 18 cm
rehydrated Immobiline DryStrips with a non-linear gradient pH 3-10 (Amersham) using a
cup-loading system and focused at 500 V for 1.5 h, 4000 V for 2 h, 8000 V for 40000 Vh.
50
Following isoelectric focusing, IPG strips were reduced, alkylated and detergent-
exchanged. 2-D SDS PAGE was carried out in 12.5% sodium dodecyl sulfate-
polyacrylamide gels (20 × 20 cm) and the proteins were visualized with silver staining.
Computer-assisted gel analysis using PD-QUEST Version 7.1.0 (Bio-Rad) was
performed on images captured with a Molecular Dynamics Personal Densitometer (Bio-
Rad). ECP samples were isolated from at least three independent cultures. More than
three separate gels of each sample were analyzed. Protein spots that displayed dominant
and consistent patterns were selected for further identification.
III.2.4. Tryptic in-gel digestion and MALDI-TOF MS analysis
The protein spots of interest were excised from the 2-D gels and digested with porcine
sequencing grade modified trypsin (Promega) following the procedure described by
Shevchenko and coworkers (1996). The extracted peptides were resuspended in 0.1%
(v/v) trifluoroacetic acid (TFA) and 50% (v/v) acetonitrile (ACN). The peptide mixture
(0.5 μl) was spotted on a MALDI target plate simultaneously with 0.5 μl of matrix
solution [a saturated solution of α–cyano-4-hydroxycinnamic acid dissolved in 0.1% (v/v)
TFA and 50% (v/v) ACN]. Peptide mass fingerprint maps of tryptic peptides were
generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) using a Voyager DE-STR Biospectrometry work station mass
spectrometer (Applied Biosystems). All the spectra were obtained in reflectron mode
using an accelerating voltage of 20 kV and a delayed extraction of 150 ns. Mass
calibration was performed using a peptide mixture (angiotensin I) 1296.6853 [M+H]+ and
ACTH (18-39 clip) 2465.1989 [M+H]+ as an external standard. The internal calibration
51
using two peptides arising from trypsin autoproteolysis 842.51[M+H]+ and 2211.10
[M+H]+ was carried out wherever possible. Peptide masses were searched against the
NCBInr database using the MS-Fit program (http://prospector.ucsf.edu) and the Mascot
software (http://www.matrixscience.com) setting a mass tolerance of 50 ppm (internal
calibration) or 200 ppm (external calibration). Proteins with a minimum of four matching
peptides and the sequence coverage exceeding 20% with the match proteins were
considered positive.
III.3. Results and discussion
III.3.1. ECP production
The wild type strains EHEC EDL933, EPEC E2348/69, and their respective ler mutants
(DF1291 for EHEC and DF2 for EPEC) and ihf mutants (DF1292 for EHEC and DF1 for
EPEC) were grown in DMEM for the production of ECPs. EPEC E2348/69 grew slightly
faster in the early log phase and produced greater amounts of ECPs than EHEC EDL933
in DMEM (Fig. III.1). The ECP production of the wild type strains EHEC EDL933 and
EPEC E2348/69 at 6 h was higher than their respective ihf and ler mutants (Fig. III.2)
suggesting that the wild type strains produced more extracellular proteins in the
supernatants. Nine-hour culture for EHEC strains and six-hour culture for EPEC strains
were used for the optimum production of ECPs to construct the extracellular proteomes.
52
02
46
810
1214
1618
20
0 1.5 3 4.5 6 7.5 9 10.5 12
Culture time (h)
ECP
conc
entra
tion
(µg/
ml)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
OD
600n
m
Fig. III.1. Growth and protein secretion of EHEC EDL933 and EPEC E2348/69 in DMEM at 37oC with 5% (v/v) CO2. Growth of EHEC EDL933 ( ) and EPEC E2348/69 ( ) was measured using the optical density at 600 nm. Open bar, ECP production of EHEC EDL933; solid bar, ECP production of EPEC E2348/69. Results are expressed as means ± SEM from three independent experiments.
53
0
2
4
6
8
10
12
EDL933 DF1291 DF1292 E2348/69 DF2 DF1
E. coli strain
ECP
conc
entra
tion
(µg/
ml)
Fig. III.2. Comparison of ECP production among EHEC EDL933 and EPEC E2348/69 wild type strains and their ler mutants (DF1291 for EHEC and DF2 for EPEC) and ihf mutants (DF1292 for EHEC and DF1 for EPEC). EHEC and EPEC strains were cultured in DMEM at 37oC with 5% (v/v) CO2 for 6 h. Results are expressed as means ± SEM from three independent experiments.
54
Fig. III.3. Extracellular proteins from EHEC EDL933. Proteins were separated by 2-D SDS PAGE using IPG of pH 3-10NL. Proteins highlighted were identified by peptide mass mapping using MALDI-TOF MS. Numbers represent the spot identifications listed in Tables III.2 and III.3.
22
24
26
27 28
20
23
19
14
15
1
86
9
7
43
2
12
5
10 11
17
2529
18
16
21
13
MW
124
49
20
35
28
(kDa
80
7
pH 3 pH 10
55
Fig. III.4. Extracellular proteins from EPEC E2348/69. Proteins were separated by 2-D SDS PAGE using IPG of pH 3-10NL. Proteins highlighted were identified by peptide mass mapping using MALDI-TOF MS. Numbers represent the spot identifications listed in Tables III.2 and III.3.
pH 3 pH 10
8
5
7
6
12
30
23
3
2
9
4
31
1
14
22
32
10
11 17
19
2120
24 25
16 15
13
18
26
124
49
20
35
28
MW (kDa)
80
7
56
Table III. 2. Summary of the common extracellular proteins of EHEC and EPEC identified by MALDI-TOF MS Spot no. (from EHEC
or EPEC) Sequence
coverage (%) Protein MW(Da)/
pI (Caculated) Matched
E. coli stain NCBI
Access# Matched protein name in EHEC and other E coli databases ler / ihf
regulated 1-EHEC 45% 58022.5 / 5.01 EDL933 15804222 Translocated intimin receptor Tir + / + 1-EPEC 27% 56843.5 / 5.16 E2348/69 2665524 + / + 2-EHEC 44% 69115.0 / 4.83 EDL933 15799694 Hsp70 (DnaK) - / - 2-EPEC 39% 69115.0 / 4.83 EDL933 15799694 - / - 3-EHEC 30% 57319.6 / 4.81 EDL933 15804735 Hsp60 (GroEL) - / - 3-EPEC 27% 57319.6 / 4.81 EDL933 15804735 - / - 4-EHEC 31% 61046.4 / 5.95 EDL933 15801469 Oligopeptide transport periplasmic binding protein - / - 4-EPEC 38% 61046.4 / 5.95 EDL933 15801469 - / - 5-EHEC 33% 60331.5 / 6.21 EDL933 15804087 Dipetide transport periplasmic binding protein - / - 5-EPEC 35% 60331.5 / 6.21 EDL933 15804087 - / - 6-EHEC 47% 45610.7 / 5.32 EDL933 15803300 Enolase - / - 6-EPEC 42% 45610.7 / 5.32 EDL933 15803300 - / - 7-EHEC 43% 43232.1 / 5.24 EDL933 15804570 Protein chain elongation factor EF-Tu - / - 7-EPEC 54% 43232.1 / 5.24 EDL933 15804570 - / - 8-EHEC 45% 41130.3 / 5.08 EDL933 15803460 Phosphoglycerate kinase - / - 8-EPEC 20% 41118.2 / 5.08 K12 16130827 - / - 9-EHEC 26% 39083.2 / 5.32 EDL933 15804216 EspD + / + 9-EPEC 37% 39473.4 / 5.13 E2348/69 1668720 + / +
10-EHEC 24% 41123.6 / 4.97 EDL933 15801009 Hypothetical protein - / - 10-EPEC 21% 41123.6 / 4.97 EDL933 15801009 - / - 11-EHEC 27% 38791.0 / 5.24 EDL933 15801238 Spermidine/putrescine periplasmic transport protein - / - 11-EPEC 22% 38791.0 / 5.24 EDL933 15801238 - / - 12-EHEC 46% 32630.6 / 5.33 EDL933 15804215 EspB + / + 12-EPEC 68% 33140.8 / 5.24 E2348/69 1169453 + / + 13-EHEC 40% 42383.9 / 6.02 EDL933 15803459 Fructose-bisphosphate aldolase class II - / -
57
13-EPEC 27% 42383.9 / 6.02 EDL933 15803459 - / - 14-EHEC 21% 45344.7 / 6.03 EDL933 15803076 Serine hydroxymethyltransferase - / - 14-EPEC 20% 45344.7 / 6.03 EDL933 15803076 - / - 15-EHEC 45% 35532.5 / 6.61 EDL933 15802193 Glyceraldehyde-3-phosphate dehydrogenase A - / - 15-EPEC 45% 35532.5 / 6.61 EDL933 15802193 - / - 16-EHEC 41% 38668.1 / 9.30 EDL933 15801691 Putative receptor - / - 16-EPEC 39% 38686.1 / 9.30 EDL933 15801691 - / - 17-EHEC 29% 37200.8 / 5.99 EDL933 15800816 Outer membrane protein 3a - / - 17-EPEC 36% 37200.8 / 5.99 EDL933 15800816 - / - 18-EHEC 34% 36030.3 / 5.64 EDL933 15802270 Putative adhesin - / - 18-EPEC 28% 36030.3 / 5.64 EDL933 15802270 - / - 19-EHEC 22% 34905.8 / 5.89 EDL933 15800491 Unknown protein encoded by prophage CP-933K - / - 19-EPEC 26% 34905.8 / 5.89 EDL933 15800491 - / - 20-EHEC 38% 26995.0 / 5.07 EDL933 15802999 Phosphoribosylaminoimidazole-succinocarboxamide synthetase - / - 20-EPEC 35% 26995.0 / 5.07 EDL933 15802999 - / - 21-EHEC 50% 26971.8 /5.64 EDL933 15804508 Triosephosphate isomerase - / - 21-EPEC 32% 26971.8 /5.64 EDL933 15804508 - / - 22-EHEC 41% 28556.4 / 5.85 EDL933 15800464 Phosphoglyceromutase 1 - / - 22-EPEC 53% 28556.4 / 5.85 EDL933 15800464 - / - 23-EHEC 44% 20574.1 / 4.78 EDL933 15804217 EspA + / + 23-EPEC 35% 20468.9 / 4.52 E2348/69 1018995 + / + 24-EHEC 34% 20761.4 / 5.03 EDL933 15800320 Alkyl hydroperoxide reductase C22 protein - / - 24-EPEC 54% 20761.4 / 5.03 EDL933 15800320 - / - 25-EHEC 51% 23104.6 / 5.95 EDL933 15804445 Protein disulfide isomerase I - / - 25-EPEC 41% 23118.6 / 5.95 CFT073 26250621 (Thiol:disulfide interchange protein dsbA precursor ) - / - 26-EHEC 47% 18266.0 / 4.73 EDL933 15802950 PTS system, glucose-specific IIA component - / - 26-EPEC 33% 18266.0 / 4.73 EDL933 15802950 - / -
58
Table III. 3. Summary of the specific extracellular proteins of EHEC and EPEC respectively identified by MALDI-TOF MS Spot no. (from
EHEC or EPEC) Sequence
coverage ( %) Protein MW(Da)/
pI (calculated) Matched
E. coli strain NCBI
Access # Matched protein name in EHEC and other E coli databases ler / ihf
regulated
27-EHEC 30% 141758.4 / 6.36 EDL933 3822134 EspP - / - 28-EHEC 28% 99548.5 / 6.39 EDL933 10955349 TagA + / +
29-EHEC 43% 24691.8 / 6.11 EDL933 15802406 Hypothetical protein - / -
30-EPEC 30% 140860.9 / 5.61 E2348/69 1764164 EspC + / + 31-EPEC 27% 20977.7 / 10.61 E2348/69 2865308 EspF + / + 32-EPEC 24% 17879.9 / 6.96 EDL933 15802663 Putative superinfection exclusion protein B of prophage CP-933V - / -
33-EPEC 23% 52982.7 / 4.51 E2348/69 5107855 FliC - / +
59
III.3.2. Extracellular Proteomes of EHEC and EPEC
ECPs from EHEC EDL933 and EPEC E2348/69 were subjected to 2-D SDS PAGE,
consistent and dominant extracellular protein spots were excised for the tryptic in-gel
digestion, MALDI-TOF MS analysis and a protein database search. Fifty-nine of them
were identified (Tables III. 2 and 3) which covered most of the prominent proteins. A
total of 29 and 30 proteins were identified for EHEC EDL933 and EPEC E2348/69,
respectively (Figs. III.3 and 4). Peptide mass fingerprints generated from the EHEC
EDL933 protein spots were assigned to the complete genome database of EHEC EDL933
for protein identification. Since the genome sequence of EPEC E2348/69 is not available,
peptide mass fingerprints generated from the EPEC E2348/69 protein spots were then
compared to a limited library of ORFs of EPEC E2348/69 and other E. coli genomes
database such as EHEC EDL933, K-12 and CFT073 (Tables III. 2 and 3).
Among these 59 proteins in the EHEC EDL933 and EPEC E2348/69 extracellular
proteomes, 26 proteins were produced in both strains (Table III. 2). Three of the Esp
proteins (EspA, EspB, and EspD) appeared as the most prominent protein spots
distributed from the acidic to neutral pH range (pH 3-6) in both EHEC EDL933 and
EPEC E2348/69 extracellular proteomes. EspB and EspD are components of the TTSS
translocon and are required for the delivery of the translocated intimin receptor (Tir) via
the TTSS needle complex with a filamentous extension formed by EspA (Ebe et al., 1998;
Kenny et al., 1997; Knutton et al., 1998; Wolff et al., 1998). High molecular weight heat
shock proteins Hsp60 (GroEL) and Hsp70 (DnaK), which may function as chaperons in
the type II and type IV secretion systems with less specificity for the target preprotein
60
(China and Goffaux ,1999), were accumulated in the acidic region in the EHEC EDL933
and EPEC E2348/69 extracellular proteomes. In Legionella pneumophila and
Helicobacter pylori, Hsp60 and Hsp70 were reported to be involved in colonization,
attachment and invasion (Bernard and Goffaux, 1999). The identification of similar heat
shock proteins in the extracellular proteomes of EHEC and EPEC may suggest their
participation in pathogenesis. One protein from EHEC EDL933 and EPEC E2348/69,
which has high similarity to an unknown protein encoded by prophage CP-933K in
EHEC, and another protein from EPEC E2348/69, which is homologous to a putative
superinfection exclusion protein B of prophage CP-933V in EHEC, were identified in the
respective proteomes. Many phage proteins have been reported as virulence factors
(Boyd and Brussow, 2002) which implicate these two putative prophage-encoded
proteins may play important roles in EHEC and EPEC pathogenesis.
Several enzymes that are involved in different metabolic processes were found in the
extracellular proteomes of EHEC EDL933 and EPEC E2348/69. These were nucleotide
metabolic enzymes such as phosphoribosylaminoimidazole-succinocarboxamide synthase,
enzymes in the glycolytic pathway such as glyceraldehyde-3-phosphate dehydrogenase,
phosphoglycerate kinase, triosephosphate isomerase, phosphoglycerate mutase, and
enolase, and some amino acid metabolism enzymes such as serine
hydroxymethyltransferase, and one detoxification enzyme alkyl hydroperoxide reductase
C22. Furthermore, some outer membrane, periplasmic and cytosolic proteins were also
detected in the extracellular proteomes.
61
Uncommon extracellular proteins, EspP, TagA (also called StcE, 24), and a hypothetical
protein were found only in EHEC EDL933. EspP is a secreted serine protease which
cleaves pepsin and human coagulation factor V and was proposed to be a contributing
factor in mucosal hemorrhage in patients (Brunder et al., 1997). TagA is an extracellular
metalloprotease and was recently reported as a protease that specifically cleaved C1
esterase inhibitor (C1-INH) (Lathem et al., 2002). These two proteases are encoded in the
EHEC large plasmid pO157 and are regarded as the virulence factors of EHEC. EspC,
EspF, a hypothetical protein, and a putative superinfection exclusion protein B of
prophage CP-933V were found in EPEC E2348/69. EspC is a secreted immunoglobulin
A protease-like protein encoded within a second pathogenicity island in the EPEC
chromosome and was regarded as an enterotoxin (Millies et al., 2001). We also identified
EspF in EPEC E2348/69 which induces epithelial cell death but the mechanism remains
unknown (Crane et al., 2001). The genome of EHEC EDL933 also contains the espF
gene and the encoded protein shares 93.4% identity at the amino acid level with the EspF
of EPEC E2348/69. The reason for not detecting EspF in the ECPs of EHEC EDL933
may be due to the low level of secretion of EspF and its extremely basic pI.
The EHEC and EPEC are very similar pathogens. They colonize the intestinal mucosa,
subvert intestinal epithelial cell function, and produce the characteristic AE lesion via
their secreted and membrane proteins (Jarvis et al., 1995; Jarvis and Kapper, 1996). From
the ECP proteomics profiles, twenty-six of these proteins, 90% and 87% of the total
identified proteins respectively, are common in EHEC EDL933 and EPEC E2348/69,
62
which demonstrate the close relationship between these two pathogens. At the same time,
there are several proteins (such as TagA, EspP and EspC) which are exclusively produced
by EHEC EDL933 or EPEC E2348/69. This shows there are variations between these
two E. coli strains. Our proteome profiles of EHEC EDL933 and EPEC E2348/69 have
demonstrated both the similarity and divergence of these two pathogens. The variability
in proteins that may interact with the host suggests that these variable proteins may be
subject to natural selection for evasion of the host immune system or serve to spread a
particular strain in a given population (Donnenberg and Whittam, 2001).
The most prominent proteins in the EHEC EDL933 and EPEC E2348/69 extracellular
proteomes are mainly secreted proteins such as EspA, EspB, EspC, EspD, EspF, EspP,
TagA and Tir. The heat shock proteins (such as Hsp60 and Hsp70) which help protein
refolding and prevent protein degradation have been reported earlier to be released into
the extracellular fraction (Pugsley, 1993). Though some outer membrane, periplasmic
and cytosolic proteins are detected in our extracellular proteome profiles, this is probably
due to the lysis of the bacteria in the culture or the formation of blebs (Yaron et al., 2000).
Some protein spots could not be identified possibly due to their low molecular weights
which would not produce enough peptides for the fingerprint analysis. Despite these
limitations, our results show that the coupling of silver staining and MALDI-TOF
analysis is a sensitive method for protein detection and the extracellular proteomes we
generated are reliable and reproducible.
63
III.3.3. Identification of Ler and IHF regulated proteins
The extracellular proteomes of EHEC EDL933 and EPEC E2348/69 give an integrated
overview of the ECP profiles and can be used as a reference for the global analysis of
protein expressions in pathogenic E. coli (such as EHEC and EPEC) under various
culture conditions, and for comparison between wild types and mutant strains.
Comparison of extracellular proteomes among the wild type strains, and the ler and ihf
mutants revealed that four common protein spots namely EspA, EspB, EspD, and Tir
were present only in the wild type strains of both EHEC EDL933 and EPEC E2348/69
while absent in the ler and ihf mutants (Figs. III.5 and 6). These four proteins are TTSS
secretion proteins which are highly conserved in EHEC EDL933 and EPEC E2348/69
with a small degree of divergence: espB (74.01 % identity), espA (84.63 % identity),
espD (80.36 % identity), and tir (66.48 % identity) (Perna et al., 1998). They are
regulated by Ler and IHF at the transcription level (Beltrametti et al., 1999; Mellies et al.,
1999; Friedberg et al., 1999), and their expression is dependent upon culture conditions,
growth phase and host contact (Rosenshine et al., 1996; Hiroyuki et al., 2002; Yoh et al.,
2003). EspF is also co-transcribed with espADB and regulated by Ler (Elliott et al., 2000).
However, EspF was identified only in EPECE2348/69 and not in EHEC EDL933.
The differences in protein profiles of the wild type strains and the ler and ihf mutants
could be attributed to the lost functions of Ler and IHF which resulted in the decreased
protein secretion for the ler and ihf mutants. On the other hand, there are other Ler and
IHF regulated TTSS proteins that have not been identified here. This may be due to
higher translocation levels compared to secretion levels such as for EspH (Tu et al.,
64
2003), or due to their extreme acidic or basic isoelectric points, which would allow them
to migrate beyond our normal IPG gel strip range and thus not be visualized by 2-D SDS
PAGE.
We also identified one flagella component protein, FliC that is solely present in EPEC
2348/69 ihf mutants (Fig. III.6). This proteomics result indicates that ihf depressed the
expression of FliC, which is consistent with a report that IHF mediates the repression of
flagella in EPEC (Yona-Nadler et al., 2003).
Comparison of 1-DE profiles of ECPs of EHEC EDL933 and EPEC E2348/69, and their
respectively ler and ihf mutants showed that a protein band of around 110 kDa was
regulated by Ler and IHF (Fig. III. 7). Elliott and coworkers (2000) also reported that Ler
regulated a 110 kDa protein in both EPEC E2348/69 and EHEC EDL933. This protein
was identified as EspC in EPEC using an anti-EspC antibody while the identity of the
equivalent 110 kDa protein in EHEC remained unclear. In the extracellular proteome of
EHEC EDL933, TagA was found to be regulated by Ler with a similar molecular weight
but a different isoelectric point than EspC (ca. 110 kDa) (Figs III. 5 and 7). The
expression level of Tag A was much lower in the ler mutant than the wild type indicating
that TagA was regulated by Ler. By using the proteomics approach, we have identified
this cryptic110 kDa protein as TagA in EHEC EDL933, which could not be identified by
Elliott and coworkers using an anti-EspC antibody (Elliott et al., 2000).
65
Fig. III.5. Comparative proteome analysis of EHEC EDL933 wild type strain, the ler mutant (DF1291) and ihf mutant (DF1292). Proteins were separated by 2-D SDS PAGE using IPG of pH 3-10NL. Proteins highlighted with circles were detected only in the wild type strain and were missing in the ler and ihf mutants. Numbers represent the spot identifications listed in Tables III.2 and III.3.
DF1292pH 3 pH 10
DF1291pH 3 pH 10
23
1
28
9 12
EDL933 pH 3 pH 10
49
20
35
28
MW (kDa)
80 124
66
Fig. III.6. Comparative proteome analysis of EPEC E2348/69 wild type strain, the ler mutant (DF2) and ihf mutant (DF1). Proteins were separated by 2-D SDS PAGE using IPG of pH 3-10NL. Proteins highlighted with circles were detected only in the wild type strain and were missing in the ler and ihf mutants. The triangle highlighted a protein that was present only in the ihf mutant. Numbers represent the spot identifications listed in Tables III.2 and III.3.
Fig. III.7. One-D SDS PAGE of the ECPs from EHEC EDL933 and EPEC E2348/69 wild type strains, the ler mutants (DF1291 for EHEC and DF2 for EPEC) and ihf mutants (DF1292 for EHEC and DF1 for EPEC). Only the high molecular weight bands were showed. The ca.110 kDa protein bands were present in EHEC EDL933 and EPEC E2348/69 but not in their respective ler and ihf mutants.
~110kDa EDL933 DF1291 DF1292
E2348/69 DF2 DF1~110kDa
DF1
33
pH 3 pH 10 pH 3 pH 10 DF2
pH 3 pH 10 E2348/69
1
12 9
23
30
31
MW (kDa)
80
35
49
28
20
124
67
III.3.4. Applications and conclusions
The ability of pathogenic bacteria to cause disease in a susceptible host is determined by
multiple factors acting individually or together at different stages of infection. Proteomics
can provide an integrated view of the gene products of certain bacteria for such global
analysis. The secretion of virulence factors is a common feature of many pathogenic
bacteria, thus it is important to generate extracellular proteomes to facilitate studies in
bacterial pathogenesis. For the first time, the extracellular proteomes of EHEC EDL933
and EPEC E2348/69 were provided and a proteomics approach was used to analyze the
extracellular proteins of these two closely related human pathogens. These proteome
profiles can help link the virulence determinants in an integrated manner, and also
facilitate the characterization of changes in mutants. In this study, several virulence
proteins regulated by Ler and IHF have been confirmed, and some interesting features
were found for further investigation.
EHEC serotypes other than O157:H7 and EPEC are not routinely identified in most
clinical microbiology laboratories. Thus the characteristic proteins of EHEC and EPEC,
as identified in their extracellular proteomes, could also be used as new diagnostic tests
for these pathogens. For example, EspA, EspB, EspD and Tir are common in EHEC
EDL933 and EPEC E2348/69, while EspP and TagA are specific for EHEC EDL933 and
EspC is specific to EPEC E2348/69. In addition, many other diarrhea-causing strains of E.
coli cannot be easily isolated in the clinical microbiology laboratory and a proteomics
analysis of these strains may reveal biomarkers that could be used in their diagnosis.
68
Chapter IV. Characterization of EspB as a translocator and an effector
and its involvement in EPEC autoaggregation
The results from this chapter are included in the following manuscript:
Li, M., I. Rosenshine, and K. Y. Leung. Characterization of EspB as a translocator and
an effector and its involvement in EPEC autoaggregation. (In preparation)
69
Abstract
EspB is a major secreted protein in EPEC and EHEC when cultured in a minimal
medium. It is shown to be an essential virulence factor of EPEC and EHEC and is
implicated to have roles in multiple functions. In this study, different experimental
approaches were used in an attempt to identify the roles of EspB in EPEC and EHEC.
Two forms of EspB were identified in the extracellular proteome of EPEC and EHEC. It
was found that these two forms of EspB were not due to the cleavage of N-ternimal
signal region nor were due to the phosphorylation of residues of EspB. 2-D SDS PAGE
of the ECP of ∆espB strain showed that the ∆espB mutant did not affect the secretion of
other extracellular proteins. However the ∆espB mutant was shown to be defective in the
translocation of effectors, such as Tir. Using β-lactamase reporter system, EspB was
shown to contain the typical secretion and translocation signal in the first 20 residues.
Translocation was demonstrated for EspB showing that EspB is an effector. EspB was
found to be involved in the autoaggregation of EPEC and a ∆espB mutant displayed
diffuse growing pattern in liquid culture medium. Transmission electron microscopy
(TEM) showed that the bacterial cells surface of ∆espB was smooth without protrusive
filamentous organelles. Moreover, the ∆espB mutant displayed less invasion ability when
compared to the EPEC wild type in HeLa cells.
70
IV.1 Introduction
EPEC and EHEC are important causal agents of food born diarrhea in developed and
developing countries. They both can cause A/E lesions in the colonized intestine
enterocytes which is characteristic of EPEC and EHEC (Frankel et al., 1998). EPEC and
EHEC use TTSS to colonize the host gastro-intestinal track and secrete a lot of proteins
to overcome peristaltic clearance and thus gain access to host-derived nutrients. EspA,
EspB and EspD are three major TTSS dependent secretion proteins (Galan 2001; Collmer
et al., 2002), which have been posited to play several functions in the process of infection.
EspA is a main component of the filamentous structure localized in the TTSS needle tip
and formed a hollow tube through which effector proteins are transported (Daniell et al.
2003; Crepin et al., 2005). EspD is a hydrophobic protein with acidic pI and is secreted
by EPEC and EHEC. It has two putative transmembrane domains and has been observed
in the host cell membrane (Wachter et al., 1999). It is hypothesized that EspD monomers
can interact with each other through a coiled-coil domain to form the main structure of
the translocation pore in the host membrane. EspD is also known to be required for the
polymerization of EspA filaments (Knutton et al., 1998; Kresse et al., 1999).
EspB is a major secreted protein of EPEC and EHEC and is a key virulence factor of
EPEC and EHEC in humans and animal models (Abe et al., 1998; Tacket et al., 2000).
As being an approximately 38 kD protein, EspB has one putative transmembrane domain
and three putative coiled-coil domains. EspB is hypothesized to be inserted into the
membrane of infected cells which together with EspD are thought to form a translocation
pores in the host cell membrane (Wachter et al., 1999; Ide et al., 2001; Shaw et al., 2001).
It is demonstrated that EspB protein is needed for the full pore formation and the
71
subsequently hemolytic effect (Warawa et al., 1999). EspB, EspD and EspA together can
form a filamentous protein translocation apparatus through which effector proteins were
translocated.
Besides acting as an essential component of the translocation apparatus in host membrane
to help other effectors’ transportation, EspB itself seems to be translocated and can be
detected in cytoplasm of infected cells implying that EspB may also have an additional
function as an effector (Taylor et al., 1998; Wolff et al., 1998). Recently, EspB from
EHEC was found to bind to the cytoskeleton protein β-catenin in HeLa cells (Kodama et
al., 2002). Other authors have found that the EspB of EPEC can interact with α-1-
antitrypsin and that this interaction plays a role in pore formation (Knappstein et al.,
2004). Luo and Donnenberg (2006) used random mutated espB to identify the different
domains involved in effector translocation and as a component of the translocation pore.
They found that the principle role of EspB is to form a pore in the host cell membrane, as
determined by hemolytic activity, which can be separated from its role in protein
translocation.
Since several functions have been attributed to EspB, more roles were assigned to this
protein. Using proteomics approach, two forms of EspB were identified in ECP profiles
of EPEC and EHEC. The involvement of EspB in the EPEC aggregation was also
observed. The present study aims to characterize the possible duel roles of EspB to
provide complete functional features of EspB.
72
IV.2. Materials and methods
IV.2.1. Bacterial strains and plasmids
The bacterial strains and plasmids used in this study are listed in Table IV.1.
Table IV. 1. Bacteria strains and plasmids used in this study
Strains or plasmids Description Reference or source
Strains
E2348/69 Wild type EPEC O127:H6, Smr J. Kaper
EDL933
Wild type EHEC O157:H7, Slt1, Slt2 negative derivative
J. Leong
∆espB E2348/69 in-frame deletion of espB This study
Plasmids
pQE31 coliE1; lacI ; Ampr Qiagen
pQEespB Residues 1-193 of EspB fused to TEM-1 This study
pCXespB Residues 1-193 of EspB fused to TEM-1 This study
pCXespB20 Residues 1-21 of EspB fused to TEM-1 This study
73
IV.2.1. Fractionation of infected HeLa cells
Infection of HeLa cells with EPEC strains and fractionation of HeLa cells infected with
EPEC strains were prepared as described previously (Tu et al., 2003). Briefly, HeLa cells
(5 x 106 cells / 100 mm dish) were infected with 100 µl of overnight EPEC wild type and
∆espB mutant culture. After 5 h infection, HeLa cells were washed 3 times and lyzed by
1% Triton X-100. Centrifugation was applied to precipitate all insoluble components
including cell membrane, nuclei, large cytoskeleton complexes, and attached bacteria.
Cytosolic proteins in the supernatant were recovered as the soluble fraction.
IV.2.2. Invasion assay
Invasion of HeLa cells was monitored by the gentamicin protection assay as previously
described (Rosenshine et al., 1996), with small modification. Briefly, one day before
infection, HeLa cells were seeded into 24 wells plate using 2-3 x105 cell / well. Bacterial
colonies were inoculated into 4 ml LB liquid medium and incubated at 37oC overnight
without shaking. On the day of infection, bacteria were subcultured into DMEM using
1:50 dilution. Before infection, HeLa cells were washed with PBS for 2-3 times to
remove the antibiotic and serum. After incubating bacterial culture in 37 oC, 5% CO2
incubator for 1 h, 1 ml/well bacterial culture were added into HeLa cell culture plate and
incubate at 37oC in a CO2 incubator for 3.5 h. After infection, the bacterial culture was
removed and HeLa cells were washed with PBS. HeLa cells were further incubated with
1 ml DMEM containing 100ug/ml gentamicin for additional 2h. 2 hours later, the DMEM
with gentamicin was removed and the HeLa cells were washed with PBS for three times.
After wash, the HeLa cells were lysed in 1 ml lysis solution containing 1% TritonX-100
74
and 10 mM MgSO4. The number of viable intracellular bacteria was quantified by plating
onto LB plates supplemented with 30ug/ml streptomycin and CFU counting. The
invasion rates were calculated from the mean of two wells in triplicate experiments.
IV.2.3. Examination of bacterial surface exposed structure by transmission electron
microscopy (TEM)
The glutaraldehyde/ruthenium red/uranyl acetate method was used to stain the cells
(Mustaftschiev et al., 1982). A drop of bacterial culture was placed on a coated grid for 1
min and blotted with filter paper. The grid was then floated on the top of an equal-volume
mixture of 1% glutaraldehyde in 0.2 M cacodulate buffer (pH 7) with specimen slide
down for 1 min. The grid was picked up and blotted with filter paper. A drop of 1%
ruthenium red in water was then added. After the incubation for 2 min, the grid was
blotted again and stained with 0.05% uranyl acetate for 1 min. The grid was then blotted.
TEM observation was performed after the grid was dried.
IV.2.4. Phase contrast microscopy
After culturing EPEC wild type, ∆espB mutant and ∆espB / pQEespB strains in DMEM
for 6 h, bacteria culture were stained with Crystal Violet and transfered to glass slides
covered with coverslips and examined under Axiovert 25 CFL inverted phase-constrast
microscope (Carl-Zsiss, Germany) at 100× magnification. Photographs were taken using
a digital camera.
75
IV.2.5. Construction of plasmid for expression of EspB-TEM and β-lactamase based
translocation assay
Plasmids pCXespB and pCXespB20, encoding the TEM-1 fusion with EspB or the first
20 residues of EspB, were obtained by ligating the full espB gene and the first 60 bp of
espB gene with β-lactamase gene. Translocation of EspB and the first 20 amino acids of
EspB were assayed with the β-lactamase reporter system as described by Charpentier and
Oswald (2004). Briefly, overnight LB cultures of the EPEC wild type and the mutants of
escN::TnphoA carrying pCX341, pCXespB and pCXespB20 were diluted 1:50 in DMEM
and incubated at 37oC in a 5% CO2 atmosphere for 2 h. The preactivated bacteria at an
MOI of 100:1 were added to HeLa cells grown on cover slips in 24-well culture plates.
Infections were allowed to proceed for 1h and then induced with 1 mM IPTG for an
additional 1h. Monolayers were then washed three times with Hanks’ balanced salt
solution (HBSS) (Invitrogen), loaded for 1 h with 1 μM CCF2/AM (Invitrogen) and
washed twice with HBSS (Charpentier and Oswald, 2004). The cover slips were mounted
to slides and were observed on a fluorescence microscope (Olympus, BX60) with a 4’,6’-
diamidino-2-phenylindole (DAPI) filter set (340- to 380-nm excitation and 425-nm long-
pass emission).
IV.2.6. Edman N-terminal sequencing
The regions of the two spots of EspB were excised from 2-D gel and transferred onto
PVDF membrane and visualized by commassie blue staining. Amino-terminal
sequencing of the two EspB protein samples were performed by automated Edman
degradation using the ABI Procise Protein Sequencing System (USA). An on-line
76
reverse-phase HPLC (PTH-C18 column) with an on-line 785A phenylthiohydantoin
(PTH)-amino acid 140C analyser were used to identify the PTH amino acids.
IV.3. Results
IV.3.1. Survey of the two forms of EspB
Using the proteomics approach, two dominant EspB spots were found in both EHEC
EDL933 and EPEC E2348/69 extracellular proteomes (Figs. III.3 & 4). EspB was shown
to have multiple functions (Tacket et al., 2000). It is possible that EspB may be present in
different forms via modifications for different functions. N-terminal sequencing was
carried out to examine the possible modification of the two spots of EspB. However, the
first 20 residues of the N-terminal of the two EspB spots were identical which suggested
that no cleavage was observed (data not shown). It was found that EspB has serine and
threonine rich domains, which may be phosphorylated to produce two forms in the
supernatant. Therefore, the ECPs of EHEC and EPEC were treated with alkaline
phosphatase and 2-D gels were used to check the possible phosphorylation modification.
However the 2-D gels showed no detectable de-phosphorylation (Fig. IV.1.) and the two
forms of EspB were still present in the 2-D gels. These data suggested that the two forms
of EspB proteins may not be due to the N-terminal processing or phosphorylation of
certain residues of EspB.
77
Fig. IV.1. Extracellular proteins (ECPs) from EPEC E2348/69 (a) and EHEC EDL933 (b) treated with alkaline phosphatase. Proteins were separated by 2-D SDS PAGE using IPG of pH 4-7L. Proteins highlighted were the two major forms of EspB identified by peptide mass mapping using MALDI-TOF MS.
IV.3.2. Mutation of espB did not affect the secretion of other extracellular proteins
EspB is one of the dominant proteins in the ECPs of EHEC EDL933 and EPEC E2348/69,
which is central to EPEC and EHEC interactions with host cells in vitro (Foubister et al.,
1994; Tacket et al., 2000; Kodama et al., 2002). It is found that EPEC ∆espB mutant
produced much less ECPs when compared to the wild type (Fig. IV.2.). The
complemented strain ∆espB/pQEespB has higher ECP secretion than ∆espB mutant
suggesting the partial complementation of the EspB. To further test the reduced secretion
of the ∆espB was due to the lower amount of EspB or other ECPs, 2-D SDS PAGE was
used to get an integrated view of the extracellular proteome of the ∆espB mutant. Fig.
IV.3. showed that only the EspB protein was missing in the 2-D profile of the ∆espB
mutant when compare to the EPEC wild type. Such result indicated that the ∆espB
mutant may not affect the secretion of other ECPs and EspB is the dominant secreted
protein in the ECPs of EPEC and EHEC.
EspB
a EspB
b
78
0
2
4
6
8
10
12
1.5 3 4.5 6 7.5 9 10.5 12
Culture time
ECP
conc
entra
tion
(µg/
ml)
Fig. IV.2. Protein secretion of EPEC strains in DMEM at 37oC with 5% (v/v) CO2. Open bar, ECP production of EPEC E2348/69; grey bar, ECP production of EPEC ∆espB; black bar, ECP production of ∆espB / pQEespB. Results are expressed as means ± SEM from three independent experiments.
79
Fig. IV.3. Comparative proteomics analysis of ECP from EPEC E2348/69 wild type strain and the ∆espB mutant. Proteins were separated by 2-D SDS PAGE using IPG of pH 3-10NL. Proteins highlighted with circles were secreted proteins identified by MALDI-TOF-MS. The EspB protein was missing from the ECP of ∆espB mutant.
Fig. IV.4. Western blot analysis of Triton X-100 insoluble and soluble fractions of HeLa cells after infected with the EPEC wild type E2348/69 and the ∆espB mutant and detected with anti-Tir antibody.
Tir
EspB EspD
EspA
EspC
pH 3 pH 10 E2348/69
49
20
35
28
MW (kDa) 124 80
pH 3 pH 10
EspA
EspD
Tir EspC ∆espB
80
IV.3.3. Mutation of espB mutant abolished the translocation of effectors
Although the ∆espB mutant did not affect the secretion of other proteins, EspB was
shown to be inserted into the target cell membrane which facilitates the translocation of
effectors (Ide et al., 2001). To further characterize the translocator property of EspB,
HeLa cells were infected with the EPEC wild type and the ∆espB mutant. After the
infection for 5 h, HeLa cells were fractionated by 1% Triton X-100. Western blot by
using anti-Tir antibody showed that Tir can be detected in the TritonX-100 soluble
fraction of the HeLa cells infected with the EPEC wild type but not in the sample infected
with the ∆espB mutant (Fig. IV.4.). This result indicated that EspB was important for the
translocation of TTSS effectors.
IV.3.4. EspB is translocated into infected HeLa cells
Although EspB is proposed to have some characteristics of an effector and can be
detected in cytoplasm of infected cells (Tailor et al., 1998; Wolff et al., 1998), the
translocation of EspB was not verified. To elucidate the character of EspB as an effector,
the TEM β-lactamase system was used to visualize the translocation of EspB into the host
cells. Full length of EspB was fused to β-lactamase to create EspB-TEM fusion protein
using plasmid pCX341 (Charpentier & Oswald. 2004). The plasmid expressing a full
length EspB-TEM fusion protein (pCXespB) was transformed into the EPEC wild type
strain E2348/69 and the escN::TnphoA mutant and assayed for its translocation by
loading infected HeLa cells with fluorescent β-lactamase substrate CCF2/AM. Under the
fluorescence microscope, HeLa cells infected with EPEC / TEM (negative control, empty
vector) remained green, indicating the absence of TEM-1 activity in the cell cytosol (Fig.
81
IV.5a). In contrast, cells infected with EPEC strains expressing EspB-TEM appeared blue,
indicating that TEM-1 was translocated into the host cells (Fig. IV. 5b). However, only
green fluorescent signal was detected within living HeLa cells infected with the EPEC
escN::TnphoA mutant (Fig. IV. 5d), which indicated that EspB-TEM fusion protein is not
translocated into infected cells by the EPEC escN::TnphoA mutants.
It was reported that the first 20 codons of certain TTSS effectors mediated secretion and
translocation of LEE encoded effectors (Charpentier and Oswald 2004). To assess if
EspB similarly possesses the effector characteristic secretion and translocation signal at
its N-terminus, the first 20 residues of EspB was fused to TEM-1 (pCXespB-20) and the
translocation property was assayed in EPEC wild type and escN::TnphoA mutant
backgroud. It was found that blue fluorescent signal was detected in HeLa cells infected
with EPEC wild type but not escN::TnphoA strain expressing EspB20-TEM (Fig. IV.5c
and 5e). These results showed that EspB was an effector protein which can be
translocated into host cells. The translocation signal is in the first 20 amino acids of EspB
and the translocation of EspB was TTSS dependent.
82
Fig. IV.5. Demonstration of the translocation of EPEC EspB proteins into live HeLa cells by using TEM-1 fusions and fluorescence microscopy. HeLa cells were infected with EPEC wild type strains E2348/69 and escN::TnphoA mutant expressing different TEM-1 fusion proteins. β-lactamase activity in HeLa cells is revealed by the blue fluorescence emitted by the cleaved CCF2/AM product, whereas uncleaved CCF2 emits a green fluorescence.
83
IV.3.5. EspB is involved in the autoaggregation
During the analysis of ECP production, EPEC wild type strain E2348/69 was found to
aggregate in the culture tube and settle down on the bottom after 6 h of culturing in
DMEM medium at 37oC without shaking. On the other hand, the ∆espB mutant did not
display the autoaggregation phenotype as the wild type. However the complemented
strain E2348/69 ∆espB / pQEespB could restore autoaggregation and form precipitate in
the culture tube (Fig. IV.6). Further more, the EPEC ler mutant and ihf mutant which
could not secrete any EspB protein were found to diffuse in the broth culture without
forming any aggregation (data not shown). These observations triggered the speculation
that EspB may contribute to EPEC aggregation phenotype. This is an interesting
phenotype since the autoaggregation associates with the micro-colony formation and will
contribute to the virulence of EPEC. Further experiments were carried out to identify the
role of EspB which involves in the aggregation property.
IV.3.6. EspB mutant does not form the autoaggregation
To further verify that the autoaggregation of the EPEC bacteria was due to the
interactions of the bacteria cells, Phase-contrast microscopy was used to show the
aggregation formation of EPEC. After culturing the EPEC wild type, ∆espB mutant and
∆espB / pQEespB strains in DMEM for 6 h, bacteria culture were stained with crystal
violet and transfer to glass slides and observed under phase-constrast microscopy.
Microscopic observation showed that many bacterial cells were clumped together in the
EPEC wild type, but no clumping of cells was observed in the ∆espB (Fig. IV.7.).
84
Individual bacteria of espB mutants were found in the culture medium. The clumping of
bacteria was observed also in the complemented ∆espB with pQEespB. This clumping
phenotype suggested that the EPEC wild type strain can form autoaggregation when
cultured in minimal medium DMEM and EspB may be involved in the aggregation
phenotype and this phenotype can be restored in the ∆espB mutant by suppling EspB in
tran.
Fig. IV.6. Autoaggregation of EPEC in DEM culture is conferred by EspB. After 6 h of culturing, the EPEC wild type and ∆espB /pQEespB strains formed aggregation at the bottom of the culture tubes and the supernatant were clear, while ∆espB mutant failed to aggregate and a distributed throughout the DMEM culture.
E2348/69 espB espB/pQEespB
85
Fig. IV.7. Microscopic observation of EPEC demonstration of EPEC E2348/69 (wild type), ΔespB and ΔespB / pQEespB strains cultured in DMEM for 6 h. E2348/69 and ΔespB / pQEespB strains showed clumping pattern while ΔespB strain grew in a diffusive pattern with single cells in the medium. Arrows indicate the clumping of bacterial cells.
E2348/69 espB espB / pQEespB
86
Fig. IV.8. Transmission electron microscopy showed the extracellular filamentous appendages of the EPEC wild type E2349/69 (a) and ∆espB (b) mutant. The arrows are highlighting the filamentous structures on the extracellular surface of EPEC wild type but on the ∆espB mutant.
IV.3.7. ∆espB mutant showed less extracellular filamentous appendages
To further access what structures may contribute to the aggregation phenotype of EPEC,
transmission electron microscopy was used to examine the surface feature of the EPEC
wild type and the ∆espB mutant. Electron micrographs of the wild type EPEC cells and
the ∆espB mutant grown in DMEM showed that the EPEC cells either do not possess
flagella or rarely. The EPEC wild type had many extracellular filamentous appendages on
the surface but not on the surface of the ∆espB mutant (Fig. IV. 8). These results
indicated that EspB may interact or affect the expression of some surface exposed
ba
87
structures of EPEC results in the aggregation phenotype. And this surface exposed
structure might be the extracellular filamentous TTSS appendages.
IV.3.8. ∆espB mutant has lower invasion ability
Although EPEC is minimally internalized into non-intestinal cell lines in vitro, this
phenotype has been very useful in studying the molecular genetics of EPEC because
many of the genes involved in invasion are also involved in forming the A/E lesion
(Donnenberg et al., 1989; Donnenberg et al., 1990; Jepson et al., 2003).
In order to further characterize the role of EspB in the cellular invasion by EPEC, the
invasion of HeLa cells by the EPEC wild type and the ∆espB mutant were compared. A
gentamicin HeLa cell assay was used and the recovery of the EPEC wild type and the
∆espB mutant were measured. A three fold less invasion ratio of the ∆espB mutant
compared to the wild type EPEC was observed (Fig. IV. 9). And the complemented strain
∆espB/pQEespB partially restored the invasion phenotype in ∆espB (Fig. IV. 9)
indicating that EspB did involve in the invasion process.
88
0
0.2
0.4
0.6
0.8
1
1.2
E2348/69 ∆espB ∆espB/pQEespB
EPEC strains
% S
ervi
val r
ate
of b
acte
ria w
ithin
Hel
a ce
lls (a
gain
st E
PEC
wild
type
)
Fig. IV.9. EPEC espB mutant showed reduced ability to invade HeLa cells. HeLa cells were infected with the EPEC wild type E2348/69, ΔespB and ΔespB / pQEespB mutants for 6 h. HeLa cells were washed, harvested in PBS and lysed with PBS containing 1% Triton X-100. The cell lysates were diluted serially and plated on LB plates. After overnight incubation, colony forming unit (CFU) were scored and presented as a percentage of those observed with the wild type.
89
IV. 4. Discussion
Many key virulence factors shared by the A/E pathogens are found at the site of
enterocyte effacement (Frankel et al., 1998). EspB is one of the secreted proteins which
is identified in the culture medium and on the membrane of the infected cells. Due to the
multiple roles of EspB, different forms of EspB may exist after modifications to perform
different functions. By the proteomics approach, two dominant EspB spots were found in
both EHEC EDL933 and EPEC E2348/69 extracellular proteomes (Figs. III. 3 & 4). N-
terminal sequencing of these two EspB proteins and alkaline phosphatase analysis did not
show any detectable modification. The two EspB spots in the 2-D gel may not be due to
the improper separation since different pH range gel strips were used to separate EPEC
and EHEC ECPs while the two forms of EspB always appeared together. Further
characterization is needed to determine the possible modification and function of these
two forms of EspB proteins.
EspB is a dominant secreted protein in EPEC and EHEC when cultured in minimal
medium. Some other translocators and effectors have also been identified in the ECPs of
EPEC and EHEC when cultured in DMEM. It is possibile that bacteria will express
different amount of secretion molecules to favorite certain environmental conditions and
there may be competition between translocators and effectors. Thus, it was speculated
that a ∆espB mutant may increase the secretion of certain effectors. However, the ECP of
a ∆espB mutant in EPEC did not show increased secretion of effectors. This result was
similar to a triple deletion mutant (∆espABD) in CR (Deng et al., 2005) which did not
increase the effectors secretion. This phenomenon indicated that the secretion of
90
translocators and effectors may under different regulation network and some other
internal or external signals may trigger the expression of the translocators and effectors.
Although EspB was found to have no effect on the secretion of effectors, EspB was
shown to be needed for the translocation of other effectors. The ∆espB mutant abolished
the translocation of Tir indicating that EspB, which secreted by the TTSS, is needed for
efficient delivery of effectors to the host cell. These results further supported the role of
EspB as a critical translocator component which is indispensable for TTSS effector
translocation.
Interestingly, besides acting as a translocator, EspB was identified to have the character
of an effector. There are reports showing identification of EspB in the cytoplasm of the
infected host cells (Taylor et al., 1998; Wollf et al., 1998). However, some researchers
argued that the limitation of fractionation method may introduce contamination in
between different cell fractions (Blocker et al., 2000). Using β-lactamase in combination
with fluorescent substrate CCF2/AM in this study, EspB was shown to be translocated
into the cytoplasm of infected HeLa cells. The identification of the secretion and
translocation signal in the N-terminal of EspB further showed that EspB can be a TTSS
secreted effector. Collectively, EspB was demonstrated to possess duel characters as a
TTSS translocator and a TTSS translocated effector.
During the analysis of ECP production, the autoaggregation phenotype of EPEC was
observed when cultured in DMEM. In the present study, the wild type EPEC showed
91
autoaggregation after 6 h of culture in DMEM, while the ∆espB did not aggregate. And
the complemented strain of ∆espB can form aggregation similar to the wild type EPEC.
The incapable formation of autoaggregation in the ∆espB mutant indicated that there may
be a significant change in the cell surface properties of the ∆espB mutant. There are
various surface components implicated in the autoaggregation of E. coli including antigen
43 (Ag43) (Hasman et al., 1999), curli (Prigent-Combaret et al., 2001) and fimbriae
(Schembri et al., 2001). To further characterize the possible surface change in the ∆espB
mutant, transmission electron microscope was used to visualize the surface structure of
the ∆espB mutant. Transmission electron microscopy pictures showed that the surface of
the ∆espB mutant was smoother than the EPEC wild type strain which showed some
filamentous appendages on the extracellular surface of bacteria cells. Based on the length,
the protrusion structure on the surface of EPEC wild type could be the filamentous TTSS
needle complex. It is reported that A/E pathogens use EspA, EspD and EspB together to
form the filamentous needle structure to contact with host closly. It is possible that the
∆espB mutant abolished the filamentous needle structure which resulted in the smooth
surface without filamentous protrusion. To further verify this hypothesis, immuno-
staining using antibody against translocators are needed to verify the component of the
protrusion structure. The results here suggest that the autoaggregation may be related to
the TTSS proteins such as EspB and the TTSS needle complex may serve as surface
signal conductor which is involved in the EPEC cell communication and aggregation.
Unlike true intracellular pathogens such as Shigella spp., EPEC strains do not appear to
be specifically adapted for intracellular survival. However, the invasion phenotype has
92
been very useful in studying the molecular genetics of EPEC because many of the genes
involved in invasion are also involved in forming the A/E lesion. Donnenberg et al. (155)
have used TnphoA and the gentamicin protection assay to isolate mutants deficient in cell
entry and found certain invasion mutant with the defect genes within TTSS cluster. In
this study, EspB was found to be important for invasion process which support the
finding that TTSS genes are not only responsible for A/E lesion but also are involved in
the invasion process. EspB may be involved in the invasion process as a component of
the TTSS needle complex, and may act as a signal molecule interacting with host cells,
which implied by the effector property of EspB.
IV. 5. Conclusion
In this study, two forms of EspB were identified from 2-D SDS PAGE of ECPs from
EHEC and EPEC, which indicated the modification and multiple functions for EspB. It
was found that the N-terminals of the two forms of EspB were not cleaved. No
detectable phosphorylation of these two forms of EspB was identified either. Further
experiments are needed to find out the possible modification of these two forms of EspB.
In this study, EspB was shown to be a multi-functional molecule which served as a
translocator and an effector. As a translocator, EspB was demonstrated to be necessary
for effector translocation. For the first time, EspB was visualized to be a TTSS
translocated effector by TEM-1 reporter system. At the same time, EspB was found to be
involved in the autoaggregation and invasion process of EPEC. These data further
indicated that EspB is a virulence factor of dual functions and involved in many
pathogenesis processes.
93
Chapter V. Identification and Characterization of NleI, a New Non-
LEE-encoded Effector of Enteropathogenic Escherichia coli (EPEC)
Part of this chapter has been submitted for publication:
Li, M., I. Rosenshine, H.B. Yu, C. Nadler, E. Mills, C.L. Hew, and K.Y. Leung.
Identification and characterization of NleI, a new non-LEE-encoded effector of
enteropathogenic Escherichia coli (EPEC). (Submitted)
94
Abstract
EPEC, a major gastro-intestinal pathogen and a cause of infantile diarrhea in many
developing countries, is a prototype for a group of pathogens causing characteristic A/E
lesions on intestinal epithelia. A/E pathogens utilize a LEE-encoded TTSS as a molecular
apparatus to deliver effector proteins into host cells. These effectors have been identified
as potential virulence factors in A/E pathogens including EPEC, EHEC, rabbit EPEC
(REPEC) and Citrobacter rodentium (CR). We used a proteomics approach to identify a
new non-LEE-encoded effector, NleI, from the EPEC sepL and sepD mutants. The nleI
gene is located in a prophage-associated island and next to the gene cluster of nleBCD.
Southern blot and bioinformatics analyses showed that nleI was present in other A/E
pathogens including EHEC and CR but not in E. coli K12. We demonstrated that NleI is
secreted and translocated into HeLa cells in a TTSS-dependent manner and that the first
20 amino acids of NleI contain the secretion and translocation signal. CesT was found to
be involved in the translocation but not the stabilization of NleI. At the transcription level,
the expression of NleI is found to be under the control of SepD but not Ler and SepL. We
showed that NleI did not affect filopodia and pedestal formation of HeLa cells during
infection. After translocation, NleI can be detected in the cytoplasmic and membrane
fractions of HeLa cells. Accumulated NleI can also be visualized throughout the HeLa
cells by using fluorescence microscopy.
95
V.1. Introduction
EPEC is an important enteric pathogen for humans, causing severe diarrhea in infants.
Infection with EPEC induces A/E lesions in enterocytes, characterized by the localized
destruction of brush border microvilli, intimate adhesion and formation of actin pedestals
beneath the attached bacteria (Frankel et al., 1998). A/E pathogens include EHEC, which
can lead to hemorrhagic colitis and hemolytic uremic syndrome in humans (Nataro and
Kapper, 1998), and several other animal pathogens including Citrobacter rodentium (CR)
and rabbit enteropathogenic E. coli (REPEC) (Agin et al., 1996; Vallance et al., 2002).
One of the emerging themes in the pathogenesis of EPEC and other A/E organisms is that
they employ a conserved protein secretion machinery, TTSS, to cause diseases in their
hosts (Cornelis and Gijsegem, 2000). The TTSS of A/E pathogens is located within a
chromosomal PAI designated the LEE which is necessary for the formation of A/E
lesions. This LEE-encoded TTSS is evolutionarily related to the flagellar apparatus and
consists of more than 20 proteins that form a molecular complex spanning both inner and
outer membranes of the bacteria. It is postulated that the TTSS apparatus acts as a
molecular syringe, secreting and injecting bacterial proteins into the culture medium and
the host cell cytoplasm (Cornelis, 2002). Some of these secreted proteins have been
shown to subvert the host cell functions, and to facilitate bacterial proliferation and
disease development.
TTSS dependent secretion proteins can be characterized into two categories, translocators
and effectors (Galan, 2001; Collmer et al., 2002). Translocators such as EspA, EspB and
EspD are TTSS secreted proteins that are required for the translocation of the effector
96
proteins into the host cytoplasm (Frankel et al., 1998). EspA, EspB and EspD are
essential for the pathogenesis of EPEC and are demonstrated as virulence factors in
human volunteer studies (Abe et al., 1998; Ide et al., 2001; Shaw et al., 2001; Tacket et
al., 2000; Wachter et al., 1999). Effector proteins are translocated from the bacterial
cytoplasm directly into the host cell cytoplasm by the TTSS, whereby causing
modulation of signal transduction pathways and cellular processes (Hueck, 1998). Genes
encoding translocated effectors have been found both within and outside of the LEE
pathogenicity island. To date, seven EPEC LEE-encoded effectors have been identified:
EspB, Tir (EspE), EspF, EspG, EspH, Map and SepZ (EspZ) (Elliott et al., 2001; Kanack
et al., 2005; Kenny et al., 1997; Kenny and Jepson 2000; McNamara et al., 2001; Tu et
al., 2003; Wolff et al., 1998). In addition, A/E pathogens secrete a number of non-LEE-
encoded effectors including Cif, NleA/EspI, TccP/EspFu, EspJ and EspG2 (Marches et
al., 2003; Gruenheid et al., 2004; Mundy et al., 2004; Garmendia et al., 2004;
Campellone et al., 2004; Dahan et al., 2005; Matsuzawa et al., 2005; Shaw et al., 2005).
Recently, seven new non-LEE-encoded effectors of NleB, NleC, NleD, NleE, NleF,
NleG, and NleH have been identified from CR and EPEC by systematic mutagenesis and
proteomics approaches (Deng et al., 2004; Thomas et. al., 2005). These Nles have been
shown to be secreted by CR in a TTSS-dependent manner, and NleC and NleD from
EPEC and EHEC are reported to be translocated into HeLa cells via the LEE-encoded
TTSS (Marches et al., 2005). However, the exact functions of these non-LEE-encoded
effectors have yet to be further characterized.
97
sepL is the first gene in the LEE4 operon of EPEC which contains espA, espD and espB.
SepL, a soluble cytoplasmic protein associating with the bacteria membrane fraction, is
required for the A/E effect (Kresse et al., 2000; O’Connell et al., 2004). sepD, also
known as rorf6, is located within the LEE2 operon of EPEC. It is reported that SepD is an
important component of the EPEC TTSS and interacts with SepL to form a membrane-
bound complex (Creasey et al., 2003a; O’Connell et al., 2004). Both SepL and SepD are
indicated to be essential for the secretion of translocators (Deng et al., 2005). An
interesting feature from the sepL and sepD mutants is that both of them exhibit enhanced
secretion of effectors without secreting any translocators (Deng et al., 2004). It is
postulated that SepL and SepD form a molecular switch that controls the secretion of
translocators and effectors as a conserved feature in all A/E pathogens, including CR,
EHEC and EPEC (Deng, et al., 2005).
Here, we used a proteomics approach to identify a new non-LEE-encoded effector, NleI,
from the EPEC sepL and sepD mutants. We demonstrated that NleI is secreted into the
culture medium and translocated into infected cells by EPEC through the LEE-encoded
TTSS. CesT was found to be involved in the translocation but not stabilization of NleI.
At the transcriptional level, the GFP reporter assay showed that NleI is regulated by
SepD but not by Ler and SepL. After infection, NleI can be identified by Western blot
analysis and by confocol fluorescence microscopy inside the HeLa cell cytoplasm and
cell membrane. Thus, our data indicates that NleI is a new non-LEE-encoded effector of
EPEC.
98
V.2. Material and methods
V.2.1. Bacteria strains and culture conditions
The bacterial strains and plasmids used for this study are listed in Table V.1. E. coli
strains were routinely cultured at 37oC in Luria-Bertani broth or in Dulbecco modified
Eagle medium (DMEM) without shaking. When required, the medium was supplemented
with ampicillin (100 μg/ml), chloramphenicol (30 μg/ml), kanamycin (50 μg/ml), and
streptomycin (50 µg/ml). Cultivation of bacteria in DMEM was carried out in 37oC, 5%
(v/v) CO2 atmosphere.
V.2.2. Tissue culture conditions
HeLa cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at
37oC in a humidified atmosphere of 5% CO2. Before infection, HeLa cells were washed
three times with DMEM to remove FBS and antibiotics. Infections were carried out in 10
mm tissue culture dishes, except for infections carried out in 24-well plates in which
HeLa cells were grown on round coverslips.
V.2.3. Construction of deletion mutants and plasmids
The primers used for the construction of deletion mutants and recombinant plasmids are
listed in Table V.2. Nonpolar deletion mutants were constructed according to the method
for suicide plasmid pRE112 (Edwards et al., 1998) or overlap extension PCR (Ho et al.,
1989). Primer sets dnleIF1, dnleR1, dnleF2, and dnleR2 were used to generate in-frame
deletion mutant of ΔnleI, and dsepDF1, dsepDR1, dsepDF2, and dsepDR2 were used to
99
generate ΔsepD from either the EPEC wild type E2348/69 chromosome or the EPEC
sepL::Tn10 chromosome. For the construction of ΔcesT, the cesT gene was replaced by a
kanamycin cassette using the lambda Red system (Datsenko and Wanner, 2000) with
primers dcesTF and dcesTR. For the construction of pACYCnleI, the complete nleI gene
was obtained by PCR with EPEC E2348/69 genomic DNA as the template and cloned
into pACYC184. Plasmids pSAnleI, pSAsepD, pSAnleI-6His and pSAnleI-Flag were
constructed by amplifying the corresponding DNA fragments with the primers listed in
Table V.2. and cloning into pSA10. For the construction of a GFP reporter system, a
promoterless complete gfp gene derived from pVIK105 (Kalogeraki and Winans, 1997)
was cloned into plasmid pDN19lacΩ to create plasmid pDNgfp. An nleI putative
promoter region fused to gfp was cloned into pDN19lacΩ to create plasmid pDNnleI-gfp.
To construct plasmids expressing TEM fusions, nleI, map and tir were amplified from
EPEC genomic DNA using specific primers (Table 2) and cloned into the pCX341
plasmid (Charpentier and Oswald, 2004) to create effector-TEM fusions.
V.2.4. Flow cytometric analysis
The EPEC wild type E2348/69 and mutant strains carrying the appropriate plasmids were
cultured in LB overnight, subcultured at 1:100 dilution into DMEM and incubated at
37oC in 5% (v/v) CO2 incubator without shaking. In each experiment, the EPEC wild
type E2348/69 was used as a negative control for non-fluorescence sample. For each
sample, 50,000 bacterial-sized particles were analyzed in a FACS Calibur cytometer
(Becton Dickison). The GFP was detected at 525 nm in the FL1 channel. Data were
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analyzed by using software WinMDI (Version 2.8). The geometric mean of the
fluorescence of each strain in three independent experiments was calculated.
V.2.5. 2-D SDS-PAGE and proteomics
Extracellular proteins of EPEC strains were prepared as described previously (Li et al.,
2004). Protein spots of interested were excised manually and in-gel digestion of proteins
was performed using a trypsin digestion protocol. Peptides were analyzed with Applied
Biosystems 4700 Proteomics Analyzer MALDI-TOF/TOF (Applied Biosystems) Mass
Spectrometer. Spectra were searched against the NCBI database with MASCOT (Matrix
Science) or the on-going EPEC genome from Sanger Institute
(http://www.sanger.ac.uk/projects/Escherichia_Shigella).
V.2.6. Fractionation of infected HeLa cells
Infection of HeLa cells with EPEC and fractionation of EPEC infected HeLa cells were
prepared as described previously (Tu et al., 2003; Gruenheid et al., 2004). Briefly, HeLa
cells (5 x 106 cells/100 mm dish) were infected with 100 µl of overnight bacterial culture
expressing NleI-6His tags. After 3 h infection, IPTG (1mM) was added and infection was
allowed to proceed for an additional 2 h. After 5 h of infection, HeLa cells were washed 3
times and lyzed by 1% Triton X-100. Centrifugation was applied to precipitate all
insoluble components including cell membrane, nuclei, large cytoskeleton complexes,
and attached bacteria. Cytosolic proteins in the supernatant were recovered as the soluble
fraction. For sub-cellular fractionation, after 5 h infection, HeLa cells were washed three
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times with ice-cold PBS and resuspended in homogenization buffer (3 mM imidazole,
250 mM sucrose, 0.5 mM EDTA, pH 7.4) supplemented with COMPLETE protease
inhibitor cocktail (Roche) and mechanically disrupted by passage through a 21.5-gauge
needle. The homogenate was centrifuged gently (3000 xg) for 15 min at 4oC to pellet
unbroken cells, bacteria, nuclei and cytoskeletal components. The supernatant was
subjected to high speed ultracentrifugation at 100000 xg for 30 min at 4oC in a TL100
centrifuge (Beckman) to separate host cell membranes (pellet) from cytoplasm
(supernatant). The pellets were resuspended in 1x Laemmli buffer to make up the equal
volume as supernatant. Equal volume of both fractions was dissolved in SDS-PAGE
sample buffer. After boiling for 5 minutes, samples were separated by SDS-PAGE and
subjected to Western blot analysis.
V.2.7. Expression and Immunoblot analysis
Overnight bacterial cultures grown without shaking at 37ºC, were diluted 1:50 in DMEM,
and grown without shaking, in 5% CO2, at 37ºC, for 3.5 h. The pellet containing total
proteins were recovered and lysed as well. Samples for Western blot analysis were
resolved by SDS-PAGE (10 to12 % polyacrylamide). Proteins were transferred to PVDF
membrane, and immunoblots were blocked in 5% non-fat dried milk in TBS, pH 7.4,
containing 0.1% Tween 20 (TBST) for 1 h at room temperature or overnight at 4oC and
then incubated with primary antibody in TBST for 1 h at room temperature. Membranes
were washed 3 times in TBST and then incubated with a 1:2000 dilution of goat anti-
mouse antibody (Santa Cruz) for 1 h at room temperature. Membranes were washed as
described above. Antigen-antibody complexes were visualized with an enhanced
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Chemiluminescence Detection Kit (Pierce), followed by exposure on to Kodak film
(Kodak). The following primary antibodies were used: anti-β-lactamase (QED
Biosciences), anti-6His (Qiagen), anti-calnexin (Stressgen) and anti-tubulin (Sigma). For
TEM assay, the different bacteria cultures was adjusted according to the cell density
(OD600 measurements), mounted on SDS-PAGE gel and then transferred to a
nitrocellulose membrane. Binding of secondary anti-mouse IgG conjugated to alkaline
phosphatase (Sigma) was detected using BCIP/NBT (Promega).
V.2.8. Translocation assay
Translocation assay was performed as described previously (Charpentier and Oswald,
2004) with following modifications. Bacterial strains were diluted by a factor of 5
immediately before infection to achieve multiplicity of infection of about 40:1 (bacteria
to HeLa cell). Infection was allowed to proceed for only 60, 90 or 120 min and no IPTG
was added (as pCX341 and its derivatives have a significant level of basal expression).
Fluorescence was quantified on a SPECTRAFluor Plus microplate reader (TECAN) with
excitation at 405 nm, and emission detected via 465-nm and 535-nm filters. Translocation
was expressed as the emission ratio at 465/535 nm to normalize the ß-lactamse activity to
cell loading and number of cells present in each well, corrected for the background
fluorescence in both channels.
V.2.9. Fluorescence microscopy for observation of translocation, transfection and
actin condensation
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Translocation of NleI and the first 20 amino acids of NleI were assayed with the β-
lactamase reporter system as described by Charpentier and Oswald (2004). Briefly,
overnight LB cultures of the EPEC wild type and the mutants of escN::TnphoA carrying
pCX341, pCXtir, pCXNleI and pCXNleI20 were diluted 1:50 in DMEM and incubated at
37oC in a 5% CO2 atmosphere for 2 h. The preactivated bacteria at an MOI of 100:1 were
added to HeLa cells grown on cover slips in 24-well culture plates. Infections were
allowed to proceed for 1h and then induced with 1 mM IPTG for an additional 1h.
Monolayers were then washed three times with Hanks’ balanced salt solution (HBSS)
(Invitrogen), loaded for 1 h with 1 μM CCF2/AM (Invitrogen) and washed twice with
HBSS. The cover slips were mounted to slides and were observed on a fluorescence
microscope (Olympus, BX60) with a 4’,6’-diamidino-2-phenylindole (DAPI) filter set
(340- to 380-nm excitation and 425-nm long-pass emission). Pictures were taken under a
true color camera. To analyze the NleI location, HeLa cells were transfected for 48 h, and
fixed for 30 min in 4% formaldehyde. Actin stress fibers and nuclei were labeled with
phalloidin (Alexa 633, Sigma) and Hoechst, respectively and observe on a fluorescence
microscope (Olympus, BX60) with a FITC filter set or a RFP filter set.
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Table V.1. Bacteria strains and plasmids used in this study Strain or plasmid Description Reference or source
Strains
DBS100 Wild type C. Rodentium ATCC 51459 Schauer and Falkow, 1993
EDL933 Wild type EHEC O157:H7, Slt1, Slt2-negative derivative J. Leong
E2348/69 Prototypic EPEC strain, O127:H6, Smr Donnenberg et al. (1990)
MG1655 Wild type E. coli K12
∆cesT::kan (EM2018) E2348/69 cesT::kan This study
∆nleI E2348/69 in-frame deletion of nleI This study
∆sepD E2348/69 in-frame deletion of sepD This study
ler::kan (DF2) E2348/69 ler::kan Friedberg et al. (1999)
escN::TnphoA (27-3-2(1)) E2348/69 escN::TnphoA Donnenberg et al. (1990)
sepL::Tn10 (SN23) E2348/69 sepL::Tn10 This study
sepL::Tn10 ∆ nleI E2348/69 sepL::Tn10; in-frame deletion of NleI This study
SM10 (λpir ) thi thr leu tonA lacY supE recA-RP4-2-Tc-Mu Kmr pir Rubirés et al. (1997)
Plasmids
pACYC184 Tetr, Cmr Amersham
pACYCnleI pACYC with nleI fragment This study
pCX341 pBR322 derivative, cloning vector used to fuse EPEC effectors to the mature form of TEM-1 β-lactamase Charpentier et al.(2004)
pCXmap (pCX394) Residues 1-204 of Map plus Map regulatory region fused to TEM-1 Charpentier, unpublished data
pCXtir (pCX392) Residues 1-551 of Tir plus Tir regulatory region fused to TEM-1 This study
pCXnleI Residues 1-193 of NleI fused to TEM-1 This study
pCXnleI20 Residues 1-21 of NleI fused to TEM-1 This study
pDN19lacΩ Promoterless lacZ oriV oriT Tetr Strr Ω fragment Totten et al. (1990)
pDN-gfp pDN19 fused to GFP This study
pDNnleI-gfp pDN19 with nleI putative promoter region fused to GFP This study
pEGFP-N1 Kanr BD Biosciences Clontech
pEGFPnleI pEGFP-N1 with nleI fragment This study
pGEMT-Easy Ampr Promega
pRE112 pGP704 suicide plasmid; pir dependent; Cmr; oriT oriV sacB Edwards et al. (1998)
pRE∆nleI pRE112 with nleI fragment deleted 477 base pair This study
pSA10 pKK177-3 derivative, lacI, Ampr Schlosser-Silverman et al. 2000
pSAnleI pSA10 with nleI fragment This study
pSAnleI-6His pSA10 with nleI fragment and 6his tag at C terminal This study
pSAsepD pSA10 with sepD fragment This study
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Table V.2. Oligonucleotides used in this study
Name Sequence (5’ to 3’) dnleI-F1 GGGAGCTCAAGGGAGATAAAGGAGAG dnleI-R1 GGGAGCTCGTGCACCTGAATATCTCT dnleI-F2 ATGGATCCTGATTGTAAGTCCAGAAC dnleI-R2 ATGGATCCGAGGATGATTGAATACCT dsepD-F1 GCTATTTGTTTGACAGTG dsepD-R1 CCTCTTTATCAAGACTTT dsepD-F2 GAACAATAATAATGGCATAGCATAGGACGAATTGTGTAA dsepD-R2 GCTATGCCATTATTATTGTTC dcesTF ATGTCATCAAGATCTGAACTTTTATTAGATAGGTTTGCGGAAAAAATTGGGTGTA
GGCTGGAGCTGCTTC dcesTR TTATCTTCCGGCGTAATAATGTTTATTATCGCTTGAGCTAATTTCCTCTATCATAT
GAATATCCTCCTTAG pACYC-nleIF GGGAATTCGGGTTCACAAAAAAACCG pACYC-nleIR GGAGTACTGTTAACATATAGCTTGGG pCX-mapF GGTTGGGTACCGTATGTGCAAGATCTTTGCAAAATTG pCX-mapR GCGAATTCCCCAGCCGAGTATCCTGCACATTG pCX-nleIF CGGCATATGCCATCATTAGTTTCAGG pCX-nleIR CGGGAATTCTTATCCTTTATGACAAAGTT pCX-nleI20R CGGGAATTCCGAAGAACCTGCATTCCGGT pCX-tirF GGTTGGGTACCCAAAAAGGTCTCTATAGACGTTTAAA pCX-tirR GCGAATTCCCAACGAAACGTACTGGTCCCGG pEGFP-nleIF GGGAATTCATGCCATCATTAGTTTCAGG pEGFP-nleIR GGGGATCCTTATCCTTTATGACAAAG pSA-nleI-F GGGAATTCATGCCATCATTAGTTTCAGG pSA-nleI-R GGGTCGACTCACTTATCCTTTATGACAAAGT pSA-nleI-hisR GGGTCGACTCAGTGGTGGTGGTGGTGGTGCTTATCCTTTATGACAAAGT pSA-nleI-flagR CGGGTCGACTCACTTGTCGTCATCGTCTTTGTAGTCCTTATCCTTTATGACAAAGTpSA-sepDF CGGAATGAATTCATGAACAATAATAATGGC pSA-sepDR CGGAATGTCGACTTACACAATTCGTCCTAT pDNFuz-gfp-F GCGAGGAATTCTCGAAATTCGGAGGAAACAAAGATGAGTA pDNFuz-gfp-R GCAGTGGATCCCTATTTGTATAGTTCATCCATGCCA pDNPro-nleI-F CGGGAATTCCCATGGCAGGATGTTAAG pDNPro-nleI-R CGGGGATCCGCTCTGCGTATTAACCGA
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V.3. Results
V.3.1. Identification of NleI from EPEC sepL and sepD mutants
SepL was reported to be an important component of TTSS and to control the secretion of
TTSS proteins (O’Connell et al., 2004). To have an integrated view of the secretion
profile of the EPEC sepL mutant, we used a proteomics approach to compare the
extracellular proteomes of the EPEC wild type E2348/69 and the EPEC sepL::Tn10
mutant (Figs. V.1.A and B). The two-dimensional (2-D) SDS PAGE profile of the ECP
of the sepL::Tn10 mutant showed that majority of the secreted proteins are those of TTSS
effectors, namely Tir, EspF, EspG, EspH, Map and NleA (Fig. V.1.B). No translocators,
such as EspA, EspB, and EspD, were identified in the ECP of the EPEC sepL::Tn10
mutants but they were present in the ECP of the wild type E2348/69 (Fig. V.1.A; Li et al.,
2004). There is a report showing that SepL and SepD control the TTSS hierarchy of
translocators and effectors in EPEC, EHEC and CR (Deng et al., 2005). A double mutant
of sepL and sepD was found to exhibit the same secretion phenotype as their single
mutants (Deng et al., 2004). To further characterize whether the sepD mutant will also
have similar proteomics profile in EPEC, a sepD non-polar deletion mutant was created
and the ECP of ∆sepD was generated. Similar extracellular proteomic profile from the
∆sepD mutant and sepL::Tn10 mutant could be observed (Fig. V.1.C). These results
confirmed the earlier observation that SepL and SepD positively regulated the expression
and secretion of translocators and that their respective mutants altered the secretion
profile of CR (Deng et al., 2005). Among these secreted proteins, we identified one new
protein with molecular weight of approximately 20 kDa. This protein is homologous to
Z2075 from the EHEC EDL933 which is located in a prophage island CP933O. Since the
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EPEC sepL::Tn10 and the ∆sepD mutants preferentially secrete effectors, this newly
identified protein may be an effector protein from EPEC. We named this protein NleI
since it is located outside the LEE locus in EPEC E2348/69.
V.3.2. NleI is located within a prophage-associated island in EPEC
NleI is located in a region where nleBCD and many other phage-related genes are present
(Fig. V.2.). Comparing the genome of EPEC E2348/69 with that of E. coli K12 MG1655
showed that this region is absent from the genome of the non-pathogenic E. coli K-12
MG1655 strain (Fig. V.2.) (coli-BASE website, http://colibase.bham.ac.uk). The region is
inserted between the conserved backbone genes (torT and torS) of E. coli K12 MG1655.
It has many characteristics of a prophage-associated island, such as the presence of phage
structural proteins, putative transposase fragments, phage tail proteins and other
prophage-associated open reading frames (ORFs). Thus, NleI may be encoded within a
pathogenicity island (PAI) containing horizontally transferred genes. However, the
overall G + C content (50%) of this region did not differ significantly to that of the EPEC
E2348/69, indicating that it might have been acquired from species with G + C content
similar to that of EPEC, or that the base composition of this region has gradually adapted
to the genome of EPEC.
Many virulence factors are phage encoded, including cholera toxin and Shiga toxin
(Waldor and Mekalanos, 1996; Huang et al., 1987). Newly identified non-LEE-encoded
effectors, NleB, NleC and NleD, are encoded in the same prophage-associated regions as
NleI in EPEC. Secretion and tranlocation of NleC and NleD have been demonstrated in
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EHEC, EPEC and CR although their exact roles in pathogenesis remain to be elucidated
(Deng et al., 2004; Marches et al., 2005). Considering the presence of nleI in a prophage-
associated island and its location close to nleBCD, we speculate that nleI may be
horizontally transferred with nleBCD and play a yet undefined role in the pathogenicity
of EPEC.
To further investigate the number of copies and distribution of nleI gene in EPEC, EHEC
and CR, an nleI probe was prepared and a Southern blot was carried out against the
EcoRV and HindIII digested genomic DNA from EPEC E2348/69, EHEC EDL933, CR
DBS100, and E. coli K12 MG1655. The Southern blot showed that there was only one
full copy of nleI in EPEC E2348/69 while there were several copies in EHEC EDL933
and CR DBS100 (Fig. V.3.) No signal was detected from the E. coli K12 MG1655
genomic DNA (Fig. V.3.), indicating that nleI may be absent from the E. coli K12
MG1655 strain. These results are consistent with our bioinformatics analysis. Although
NleI has only one full copy encoded in the EPEC E2348/69 genome, it has many
homologues in the EHEC EDL933, such as Z2075 encoded by a prophage CP-933O,
Z6025 encoded by a cryptic prophage CP-933P, Z2339 encoded by a prophage CP-933R
and Z2149, a phage or prophage encoded protein. This information suggests that the
region where nleI resides in EPEC is possibly acquired through horizontal gene transfer,
and EHEC and CR may contain more than one insertion from the same phage.
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Fig. V.1. Comparative analysis of the extracellular proteomes (ECPs) from the EPEC wild type E2348/69 (A), the sepL::Tn10 mutant (B), and the ∆sepD mutant (C). The proteins were separated by 2-D SDS PAGE using IPG at pH 3 to 10. The circled proteins were identified by MALDI-TOF/TOF MS. The new non-LEE-encoded effector is labeled as NleI.
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Fig. V.2. Graphic representation of the putative prophage-associated island that contains NleI by comparing the EPEC E2348/69 and the E. coli K12 MG1655 genomes. The transcriptional direction of each orf is indicated by an arrowhead (coli-BASE website, http://colibase.bham.ac.uk, 6). Annotations of orfs are based on the homologs from the published EHEC EDL933 genome.
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Fig. V.3. Southern blot analysis of genomic DNA from EPEC E2348/69 (lanes 1 and 5), Citrobacter rodentium DBS100 (CR) (lanes 2 and 6), EHEC EDL933 (lanes 3 and 7), and E. coli K12 MG1655 (lanes 4 and 8). The genomic DNA samples were digested with EcoRV (lanes 1 to 4) or HindIII (lanes 5 to 8), and labeled with nleI DNA as a probe.
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Fig. V.4. Comparative proteome analysis of the ECPs from EPEC sepL::Tn10 mutant (A), sepL::miniTn10Kan ∆nleI mutant (B) and sepL::miniTn10Kan ∆nleI / pACAYCnleI mutant (C). The proteins were separated by 2-D SDS PAGE using IPG at pH 3 to 10. The circled proteins indicated the new non-LEE-encoded effector, NleI and were identified by MALDI-TOF-MS-MS.
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V.3.3. NleI is a secreted protein and the secretion of NleI is TTSS-dependent
The non-LEE-encoded effectors of EPEC, EHEC and CR were reported to be secreted by
LEE-encoded TTSSs (Marches et al., 2003; Gruenheid et al., 2004). To confirm the
expression and secretion of NleI, we created a nleI deletion mutant in the EPEC
sepL::Tn10 mutant background. The analysis of ECPs of EPEC sepL::Tn10, sepL::Tn10
∆nleI, and the complementary mutant sepL::Tn10 ∆nleI /pACYCnleI verified the
expression and secretion of NleI (Fig. V.4. A, B and C). To confirm the secretion of
NleX was TTSS dependent, a plasmid expressing NleI with 6xHis-tag at its C-terminal
was constructed (pSAnleI-6His) and transformed into the EPEC wild-type E2348/69 and
a TTSS defective mutant escN::TnphoA. Total bacteria-associated proteins and secreted
proteins were obtained from these strains and detected by anti-6His antibody. Western
blot analysis of the bacterial pellets and culture supernatant revealed that the NleI-6His
fusion protein was produced at a similar level in both bacterial pellets (Fig. V.5).
However, NleI-6His could be detected only in the extracellular proteins of EPEC wild-
type E2348/69 but not in the TTSS defective mutant escN::TnphoA (Fig. V.5). These
results indicate that NleI is a secreted protein and it is secreted via the LEE-encoded
functional TTSS.
To further verify the secretion property of NleI, we constructed a NleI-TEM fusion
protein using plasmid pCX341 (Charpentier and Oswald. 2004). The plasmid expressing
NleI-TEM fusion protein (pCXnleI) was transformed into the EPEC wild type E2348/69
and the escN::TnphoA mutant. Western blot analysis of the bacterial pellets and culture
supernatant revealed that the NleI-TEM fusion protein was produced at similar levels in
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the wild type E2348/69 and the escN::TnphoA mutant bacterial pellets (Fig. V.6.A).
However, NleI-TEM could be detected only in the culture supernatant from the EPEC
wild type E2348/69 but not from the TTSS defective mutant escN::TnphoA (Fig. V.6.B).
These results indicate that NleI is secreted via the LEE-encoded functional TTSS.
Fig. V.5. Western blot analysis of secreted proteins and total proteins of EPEC E2348/69 wild type and the TTSS secretion mutant escN::TnPhoA expressing pSAnleI-6His with anti-6His antibody.
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Fig. V.6. NleI is secreted into the culture supernatant and translocated into live HeLa cells in a TTSS-dependent manner and the secretion and translocation signal is at the first 20 amino acids. Western blot analyses of TEM-1 fusion proteins of bacterial pellets (A) and culture supernatants (B) from different EPEC strains and probed with anti-β-lactamase antibody . Demonstration of the translocated NleI into live HeLa cells by using TEM-1 fusion proteins from different EPEC strains. The presence of TEM fusions in HeLa cells is revealed by a blue fluorescence due to cleaving of CCF2/AM by the β-lactamase activity of TEM, whereas uncleaved CCF2/AM emits a green fluorescence (B).
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V.3.4. NleI is translocated into the host cells
To verify the translocation property of NleI, we used the EPEC wild type and various
mutants expressing NleI-TEM fusion protein to infect HeLa cells and assayed its
translocation probability by loading infected HeLa cells with fluorescent β-lactamase
substrate CCF2/AM. Under the fluorescence microscope, HeLa cells infected with
EPEC/pCX341 (negative control, vector encoding unfused TEM) remained green,
indicating the absence of TEM-1 activity in HeLa cell cytosol (Fig. V.7.A). In contrast,
cells infected with EPEC strains expressing NleI-TEM and Tir-TEM (positive control)
appeared blue, indicating that TEM-1 was translocated into the host cells (Fig. V.7.B and
D). However, only green fluorescent signal was detected within living HeLa cells when
infected with the EPEC escN::TnphoA mutant expressing NleI-TEM and Tir-TEM,
indicating that TEM fusion proteins were not translocated into infected cells in the TTSS
defective mutant background (Fig. V.7.E and G).
We also compared the translocation efficiency of NleI-TEM into HeLa cells with Map-
TEM and unfused TEM in EPEC. After infecting HeLa cells for 60, 90 or 120 min,
EPEC strains were then washed off and the HeLa cells were incubated with the ß-
lactamase substrate, CCF2/AM. The blue to green fluorescence ratio, indicative of the
amount of TEM-fused protein within the HeLa cells, was determined (Fig. V.8.). HeLa
cells infected with EPEC expressing NleI-TEM had a significantly higher blue to green
fluorescence ratio than HeLa cells infected with EPEC expressing unfused TEM
(background), showing that the NleI-TEM fusion was translocated into HeLa cells.
However, the NleI-TEM fusion was translocated less efficiently than Map-TEM as the
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latter induced a higher blue to green fluorescence ratio in HeLa cells compared with the
former, albeit similar protein expression levels (Fig. V.8. lanes 1 and 2).
It was reported that the first 20 codons of Tir, EspF, Map and Cif mediated secretion and
translocation of these LEE and Non-LEE encoded effectors (Charpentier and Oswald
2004). To assess if NleI similarly possesses secretion and translocation signals at its N-
terminus, we constructed fusions of the first 20 residues of NleI to TEM (pCXnleI-20).
This plasmid was expressed in the EPEC wild type E2348/69 and the escN::TnphoA
mutant to observe the translocation efficiency using anti-β-lactamase antibody and
fluorescence microscopy. As expected, blue fluorescent signal was detected only in the
wild type but not in the escN::TnphoA background, indicating the first 20 amino acids
was sufficient for the secretion and translocation of NleI (Fig. V.7.C and F). These results
show that NleI is an effector protein which can be translocated into host cells. The
translocation signal is in the first 20 amino acids of NleI and the translocation is TTSS
dependent.
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Fig. V.7. Demonstration of the translocation of EPEC effector proteins into live HeLa cells by using TEM-1 fusion proteins. β-lactamase activity in HeLa cells is revealed by the blue fluorescence emitted by the cleaved CCF2/AM product, whereas uncleaved CCF2/AM emits a green fluorescence.
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V.3.5. CesT is involved in the translocation but not the stabilization of NleI
CesT is a LEE-encoded chaperone shown to affect the translocation efficiency of a large
number of EPEC effectors (Abe et. al., 1999; Creasey et. al., 2003b; Thomas et. al.,
2005). In order to determine whether NleI translocation was CesT dependent, the NleI-
TEM fusion was introduced into a ∆cesT background. While the NleI-TEM expression
was not affected (Fig. V.8. lanes 2 and 3), its translocation was reduced to background
level in the ∆cesT background (Fig. V.8.). The NleI translocation efficiency is therefore
dependent on CesT. CesT was shown to stabilize several effectors such as Tir and NleA
(Thomas et al., 2005). However, similar amount of NleI-TEM and two fusion protein
bands were observed in both the EPEC wild type (Fig. V.8. lane 2) and in the ∆cesT
mutant (Fig. V.8. lane 3), indicating that CesT is not required for NleI stabilization. In
contrast, less Tir and Map were observed in the ∆cesT mutant, which indicated that CesT
is required for the stabilization of Tir and Map (data not shown; Elliott et. al., 1999;
Creasey et al., 2003b).
V.3.6. NleI is localized in the host cytoplasm and membrane
HeLa cells were infected with the EPEC wild type E2348/69 or with the EPEC
escN::TnphoA mutant, both harboring pSANleIpSAnleI-6His. After 5 h of infection,
Triton X-100 soluble and insoluble factions were analyzed by 1-DE and Western blotting
using the anti-6His antibody. NleI was found in the Triton X-100 soluble fraction
prepared from the wild type EPEC infected cells but not from the escN::TnphoA mutant
infected cells (Fig. V.9.A). The presence of NleI in the Triton X-100 soluble fraction
suggests that NleI may interact with some cytosolic molecules. The NleI from the
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insoluble fraction was mainly from the attached bacteria, but might also be from NleI
associated with host cell components insoluble in Triton X-100. To further localize NleI,
sub-fractionation of the infected HeLa cells was conducted. Proteins from different sub-
cellular fractions were subjected to Western blotting using anti-6His, anti-α-tubulin and
anti-calnexin antibodies. α-tubulin and calnexin are sub-cellular markers for cytosolic and
membrane proteins in HeLa cells, respectively. Data showed that NleI-6His is localized
in both host cell cytoplasmic and membrane fractions (Fig. V.9.B). To visualize the
location of NleI, a plasmid pEGFP-nleI was constructed and transfected into HeLa cells.
After 24 h of transfection, HeLa cells were fixed and stained with Hoechst and Pholloidin
and analyzed by confocal microscopy. The GFP signal could be observed throughout the
HeLa cells including the nuclei, similar to HeLa cells transfected with the empty vector
pEGFP-N1 (Fig. V.10).
.
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Fig. V.8. NleI is translocated by the EPEC wild type E2348/69 into HeLa cells in a CesT-dependent manner. Translocation into HeLa cells by EPEC/pCXmap (filled circles), EPEC/pCXnleI (filled squares), EPEC/pCX341 (filled triangles) or ΔcesT/pCXNleI (open squares). The results shown are the average of duplicate wells in three independent experiments. The insets shows the whole cell lysates of the EPEC wild type E2348/69 carrying pCXmap (lane 1, Map-TEM), pCX341 (lane 4, TEM) or pCXnleI (lanes 2, NleI-TEM) as well as a lysate of a ΔcesT mutant carrying pCXnleI (lane 3, NleI-TEM) were subjected to Western blot analysis and probed with anti-β-lactamase antibody. TEM-1 fused or unfused proteins are marked on the right. NleI-TEM fusion proteins were expressed as two protein bands. Molecular mass markers, in kilo Daltons, are indicated on the left.
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Fig. V.9. Western blot analysis of Triton X-100 soluble and insoluble fractions of HeLa cells after infection of the EPEC E2348/69 wild type and the TTSS secretion mutant escN::TnphoA expressing pSAnleI-6His probed with anti-6His antibody (A). Western blot analysis of infected HeLa cell fractions (B). HeLa cells were infected with the EPEC wild type E2348/69 or with the escN::TnPhoA mutant expressing 6His-tagged NleI and subjected to subcellular fractionation by differential centrifugation. Fractions were analyzed by Western blot using anti-6His, anti-tubulin and anti-calnexin antibodies.
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Fig. V.10. Confocal microscopy demonstrates that NleI is localized in the cytoplasm and the nuclei of HeLa cells. HeLa cells were transfected with pEGFPnleI for 24 h and fixed with 4% para-formaldehyde.
124
V.3.7. NleI is regulated by SepD but not regulated by Ler and SepL at the
transcriptional level
It is reported that the LEE-encoded auto-regulator, Ler, can positively regulate effectors
such as Tir and non-LEE-encoded protein TagA (Elliott et al., 2000; Haack et al., 2003).
Our proteomics results (Fig. V.1.A, B and C) also showed that the secretion levels of
NleI and other effectors is much higher in the EPEC sepL::Tn10 and ∆sepD mutants than
the EPEC wild type E2348/69. To determine if the expression of NleI is regulated by Ler,
SepL and SepD at the transcriptional level, a plasmid which contained the nleI promoter
region fused with a promoterless gfp gene (pDNnleI-gfp) was introduced into the EPEC
wild type E2348/69, ler::kan, sepL::Tn10 and ∆sepD mutants. The green fluorescence
signals were determined by flow cytometry and were compared among all these EPEC
strains (Fig. V.11). The wild type EPEC containing pDNnleI-gfp had a significant higher
gfp expression than the wild type containing pDNgfp (vector without putative promoter
region), indicating that a promoter is present in front of the nleI gene. The GFP intensity
of the EPEC wild type, ler::kan and sepL::Tn10 mutants containing pDNnleI-gfp were
found to be comparable, which suggested that NleI is not under the control of Ler and
SepL at the transcriptional level. However, the GFP intensity of the ∆sepD/pDNnleI-gfp
strain had a significant higher level compared to the other strains (Fig. V.11). And the
complementary strain ∆sepD/pDNnleI-gfp /pSAsepD displayed weaker GFP intensity
compared to ∆sepD/pDNnleI-gfp strain, which was similar to the GFP intensity of wild
type harboring pDNnleI-gfp. These results suggest that SepD negatively regulates NleI
while Ler and SepL may not regulate NleI at the transcriptional level.
125
V.3.8. NleI is not involved in the filopodia and pedestal formation
EPEC uses the TTSS to translocate effector proteins to interact with the eukaryotic
cytoskeleton and cause actin condensation and cytoskeleton rearrangement (Frankel et al.,
1998). Tir is one of the effector proteins delivered by EPEC into host cells, becoming an
integral plasma membrane protein and a receptor for the EPEC outer membrane adhesin,
intimin (Jerse et al., 1990; Kelly et al., 1990). The interaction between Tir and intimin
leads to intimate attachment of EPEC to the host cell membrane and formation of an
actin-rich pedestal beneath the adherent bacterium (Moon et al., 1983; Knutton et al.,
1989). EspH is also shown to be a modulator of the host actin cytoskeleton, down-
regulating filopodia formation and enhancing pedestal formation (Tu et al., 2003). To
investigate if NleI is also involved in the process of the cell cytoskeleton re-arrangement,
actin staining was carried out to examine the actin condensation underneath the EPEC
attaching sites. HeLa cells were infected with the EPEC wild type, the ∆nleI mutant, and
the overexpressor of NleI (E2348/69/pSAnleI) for 15, 45 and 90 min and then fixed and
stained with phalloidin-rhodamine. Fluorescence microscopy was used to observe the
formation of filopodia and pedestals. Actin staining results suggested that the intensity of
actin condensation beneath the EPEC wild type, ∆nleI mutant and E2348/69/pSAnleI was
at comparable levels (Fig. V.12). These results indicated that NleI was not involved in the
filopodia and pedestal formation in infected HeLa cells.
126
Fig. V.11. Flow cytometric analysis of the GFP intensity of the EPEC wild type E2348/69, ler::Kan (DF2), sepL::Tn10, and ∆sepD mutants, and a complementary strain ∆sepD/pSAsepD carrying pDN-gfp or pDNnleI-gfp. In each individual experiment 50,000 bacterial-sized particles were analyzed. The fluorescence intensity of each particle is reported on the x axis and the number of bacterial-sized particles is shown on the y axis (A). The geometric mean of the fluorescence detected by the flow cytometry for EPEC strains are analyzed (B). The values represent the means ± the standard deviations (SD) from three independent experiments.
B
127
Fig.V.12. NleI is not affecting actin condensation underneath the EPEC infection sites. Transfected cells were infected with EPEC wild type, ∆nleI and ∆nleI/pSAnleI for 15min, fixed and stained with phalloidin-rhodamine. The arrows highlighted the actin condensation underneath the attached EPEC strains.
E2348/69
E2348/69 ∆nleI
E2348/69 ∆nleI / pSAnleI
RFP Phase-contrast
128
V.4. Discussion
To date, six LEE-encoded effectors and 12 non-LEE-encoded effectors have been
identified by various research groups. After comparing the ECPs of the EPEC wild type,
the sepL::Tn10 and the ΔsepD mutants, we have identified a new non-LEE-encoded
effector, NleI. By using TEM-1 reporter system, NleI was shown to contain secretion and
translocation signals at its N-terminal. Two protein bands were found from the NleI-TEM
fusion proteins in both the EPEC wild type and the escN::TnphoA mutant background
(Fig. V.6.A1). This might be due to the cleavage of NleI inside the bacterial cytoplasm. It
is speculated that effector proteins will quickly undergo degradation if they are not
translocated. This may explain why NleI is not identified in the ECP of the EPEC wild
type when grown in DMEM medium (Fig. V.1.). Interestingly, the first 20 residues of
NleI-TEM fusion showed only one band (Fig. V.6.A1), which indicate that the cleavage
site is located at the N-terminal probably after the first 20 amino acids. The TTSS
chaperone CesT was shown to maintain the steady-state of Tir and Map in bacterial
cytosol (Abe et al., 1999; Elliott et al., 1999; Creasey et al., 2003b) and also to facilitate
the secretion of at least 7 effectors, including 3 non-LEE encoded effectors NleA, NleF
and NleH (Thomas et al., 2005). We found that CesT is also actively involved in the
secretion and translocation of NleI while it does not seem to be required for the stability
of NleI in the EPEC cytoplasm. This result extends the roles of CesT in facilitating
efficient secretion and translocation of non-LEE-encoded effectors.
Using sub-cellular fractionation and GFP tagged transfection, we showed that NleI is
localized throughout the HeLa cells (Fig. V.9 and 10). Although the GFP signal was
129
detected in the HeLa cell nuclei, it is possible that the GFP could have diffused into the
nuclei since the GPF signal from the vector was also observed in the nuclei (Fig. V.10).
Further experiments are needed to verify that whether the GFP signal in nuclei is due to
NleI or just due to diffusion. We have also used immuno-staining to localize NleI-Flag in
EPEC infected HeLa cells. The signal was localized throughout the cells and no specific
localization was identified (data not shown). Some EPEC effectors, such as EspH, were
shown to be toxic to host cells (Tu et al. 2003). However, no significant cytotoxic effect
on HeLa cells was observed when comparing HeLa cells transfected with the pEGFP-nleI
and the pEGFP empty vector. We also overexpressed NleI in yeast cells using the pYES2
plasmid to test for its cytotoxicity. Yeast cells harboring the NleI overexpression vector
grew similarly to yeast cells harboring the empty pYES2 vector under β-galactose
induction (data not shown). Our data indicate that NleI is localized in eukaryotic cells in a
diffused pattern and there is no obvious cytotoxic effect to eukaryotic cells.
In this study, a proteomics approach was used to show that sepL::Tn10 and ΔsepD
mutants display a higher secretion level of effectors than the wild type EPEC.
Our data confirm previous studies (Deng et al., 2005) that the sepL and sepD mutants
exhibited an increased secretion of effectors but a reduced secretion of translocators.
Deng et al. (2005) also reported that SepL and SepD did not regulate the translocators
and effectors that are encoded within the LEE regions at the transcriptional level.
However, by measuring the expression of GFP in EPEC strains containing pDNnleI-gfp,
we found that SepD but not SepL negatively regulated the expression of NleI at the
transcriptional level (Fig. V.11.). The expression of EPEC LEE-encoded effectors and
130
LEE-encoded TTSS components including SepD are regulated by Ler (Elliott et al., 2000;
Deng et al., 2004). It is therefore intriguing that nleI expression was increased in the
sepD mutant but not in the ler mutant. In addition, we have found that the basal level of
SepD from in tran plasmid pSAsepD in the ΔsepD background can restore the secretion
of translocators and depress the secretion of some effectors to the similar level of the
EPEC wild type (data not shown). It is possible that very low levels of SepD are
sufficient for nleI repression. Whether this regulatory cascade is unique to NleI or
common to other Nles requires further investigation.
Garmendia and Frankel (2005) reported that the expression of effectors EspJ and
TccP/EspFu is not regulated by Ler. Our data here add one more non-LEE-encoded
effector, NleI, which, while not regulated by Ler, is dependent on TTSS for secretion and
translocation. It is possible that some other regulatory molecules control the expression of
the non-LEE encoded effectors. Besides genetic regulations on effectors, environmental
factors including host contact may also influence the secretion and translocation of
effector molecules (Galan and Collmer, 1999).
V.5. Conclusion
One new non-LEE encoded effector, NleI, was identified by comparative proteomics.
NleI was shown to be secreted and translocated into HeLa cells. The expression of NleI
was shown to be regulated by SepD at transcriptional level. It was demonstrated that NleI
possesses secretion and translocation signal at its N-terminal and the translocation of NleI
is facilitated by CesT. NleI was found to localize in host cells while the exact function
131
remains elusive. These results add a new member to the growing pool of effectors from
the A/E pathogens. Further analysis of the interactions of NleI and host may illuminate
how NleI is involved in the cellular signal transduction and provide more insights into the
EPEC pathogenesis.
132
Chapter VI. General conclusions and future directions
VI.1. General conclusions
EPEC and EHEC strains are well recognized enteric pathogenic E. coli and still remain as
public threat worldwide. Though many research groups have devoted much effort to
studying the virulence of EPEC and EHEC, there are still many other virulence factors
from EPEC and EHEC to be discovered and understood. Efforts aimed at identification
and characterization of virulence factors and elucidation of the functions of the identified
virulence factors are the major focus of this study.
Since most virulence factors are secreted or are located on the bacterial cell surface
(Finlay, 1996), an attempt was made to compare the ECP profiles of EPEC and EHEC
and their various mutants to identify virulence factors from EPEC and EHEC. Proteomics
approach using 2-D SDS PAGE in combination with MALDI-TOF MS followed by a
protein database search were used in the first step to analyze the extracellular protein
profiles of EHEC EDL933 and EPEC E2348/69, and their various mutants (Li et al.,
2004). This study resulted in the identification of many virulence determinants such as
EspA, EspB, EspD, Tir, EspC, EspP and StcE (TagA). Several potential virulence factors
were also identified in this study, including some phage related proteins and hypothetical
proteins. The proteomics profiles of EHEC and EPEC have demonstrated both the
similarity and divergence of these two pathogens in an integrative view. The results
obtained will help in understanding the pathogenesis of EPEC and EHEC and highlight
some candidate proteins for further study of EPEC and EHEC pathogenesis.
133
Since EspB is one of the prominent secreted protein identified from EPEC and EHEC by
proteomic approach, it was further characterized. Results showed that the two dominant
forms of EspB from EPEC and EHEC were not due to the N-terminal cleavage or
phosphorylation. EspB was identified as an effector which possesses the secretion and
translocation signal at its N-terminus. The dependence of the secretion and translocation
of EspB on TTSS substantially supported the notion that EspB was a TTSS dependent
effector protein. Interestingly, EspB was found to be involved in the autoaggregation of
EPEC and the property of EspB involved in this process was further characterized. It was
indicated that EspB may affect the TTSS assembly which resulted in the defect of TTSS
filamentous structure and further affected the autoaggregation and invasion of EPEC.
This study suggests that one of the TTSS translocator component, EspB, may be involved
in the EPEC self interaction as well as interaction with host cells.
Since SepL and SepD can control the secretion of translocators and effectors,
comparative proteomics was applied to identify effector proteins from EPEC sepL and
sepD mutants. This led to the identification of a new effector protein, NleI, which was
present in the ECP profiles of both EPEC sepL and sepD mutants but absent from that of
the wild type. Construction of deletion mutants and reporter fusions confirmed the
expression and translocation property of NleI. CesT was demonstrated to be involved in
translocation but not stabilization of NleI. Cellular experiments showed that NleI was not
involved in the actin condensation and pedestal formation. No specific location of NleI
can be observed in host cells and no cytotoxicity effect of NleI to the host cell was
134
detected. More intriguingly, SepD was first shown to regulate the expression of NleI at
the transcriptional level, which is a new regulation network for Nles. Collectively, these
results added in a new effector into the effector pools of A/E pathogens and provided
clues for the roles of non-LEE encoded effectors in the pathogenesis of EHEC and EPEC.
The experimental data in these studies may reveal features common to other enteric
pathogens as well as strategies unique to A/E lesion-causing bacteria. New discovery
here may accelerate the processes of diagnostics and curative approaches to diseases
caused by EPEC and EHEC and may pave the way towards the development of new
therapies for diarrheas.
VI.2. Future directions
Using proteomics approach, many new proteins were identified from the ECPs of EPEC
and EHEC (Table III 1 and 2). Some of them displayed certain virulence properties
similar to known proteins from E. coli or other species, such as putative adhesin, putative
receptor. Two phage related proteins were also identified from the ECPs of EPEC and
EHEC. To characterize the secretion property of these secreted proteins and the
localization of the adhesins would be the pursuit in the next step. The involvements of
these novel proteins in the cellular process need to be further verified by in vitro
experiments, such as adhesion and invasion assay, FAS assay, cell cytotoxicity assay.
And functional studies of these novel proteins revealed by this proteomic data may lead
to the discovery of novel virulence determinants from EPEC and EHEC.
135
The mechanism of EspB as a TTSS translocator is well characterized. However, it may
still be a new concept for EspB to act as a TTSS effector. From the effector aspect, EspB
may be actively involved in the interplay of bacteria and host cells. The contribution of
EspB in the EPEC autoaggregaton and invasion showed some clues for EspB as to play
dual roles in bacteria and host interface. Immuno-staining using anti-EspB antibody to
display the participation of EspB in the extracellular filamentous structure in EPEC is
needed to verify the involvement of TTSS in EPEC autoaggregation. Structural biology
provides an extensive tool for the understanding the obscure mechanism of certain
molecules and gives chances to solve interesting biological problems. EspB can be
overexpressed, purified and optimized for crystallography study to solve its molecular
structure. Identification of the biochemical and structural properties of EspB in addition
to functional studies may provide important implications for the multiple roles of EspB
and the embedded mechanism of EspB in pathogenesis possess.
NleI is a novel non-LEE encoded effector from EPEC identified in this study. The
secretion and translocation property of NleI have been verified in this study. However,
the cellular function of NleI remains elusive. As for the functional assay for NleI, many
approaches have been employed, such as the adhesion and invasion assay, the inducible
nitric oxide synthase (iNOS) assay, the fluorescence actin staining (FAS) assay, and the
cell apoptosis assay (data not shown). Unfortunately, all tests failed to come out with
concrete phenotype for NleI. Pull down assay can be done by overexpressing NleI in
mammalian cells to search for the possible binding partner of NleI. Limited phenotypic
assays and non-availability of suitable animal models for EPEC add to the difficulty to
136
assign functions for effectors in EPEC. NleI showed many homologues in CR and
southern blot also verified the multi-copies of NleI in CR. nleIs can be knocked out in CR
and the infection property of CR wild type and nleI mutants in mice can be compared.
These experiments will be better ways to characterize the virulence characteristics of NleI
in animal model. The additional experiments, such as the transepithelia electrical
resistance (TER) assay by using polarized MDCK cells, the anti-phagocytosis activity
against macrophage, can also be used to characterize NleI as a new effector from the
cellular level.
137
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