2 caco-2 epithelial cells by campylobacter” -...
TRANSCRIPT
“Effect of growth phase and virulence factors on the adherence to and invasion of
Caco-2 epithelial cells by Campylobacter”
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M. Ganan1, R. Muñoz1, A.V. Carrascosa1, S. de Pascual-Teresa2, A. J. Martínez-
Rodríguez1 *
1- Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva, 3.
28006. Madrid, Spain.
2- Instituto del Frio (CSIC), Jose Antonio Novais 10. 28040. Madrid, Spain
Corresponding author:
Tel: (34)915622900, ext. 216
Fax: (34)915644853
e-mail: [email protected]
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Running title: Variables affecting Campylobacter adherence and invasion of Caco-2
cells
Keywords: Campylobacter, Caco-2 cells, adherence, invasion, virulence factors
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Abstract:
The effect of growth phase on the adherence to and invasion of Caco-2 epithelial cells
by five strains of Campylobacter was studied. No significant differences were observed
between the behaviors in the exponential or stationary phases for the most stress-
resistant strains (C. jejuni 118 and C. coli LP2), while the strains that produced a greater
reduction in the viability in the stationary phase owing to sensitivity to stress (C. jejuni
11351, C. jejuni 11168 and C. jejuni LP1), also presented reduced adherence to and
invasion of Caco-2 cells. The alleles presented in several loci (flaA, ciaB, cadF and
PldA), coding for putative virulence factors were identified in the studied strains. In
spite that C. jejuni 118 and C. jejuni 11168 strains showed a different adherence to and
invasion of Caco-2 cells behavior, they posses identical alleles for ciaB, cadF, and pldA
loci. From the virulence factors analyzed, only the flaA locus was different among both
strains. The results obtained showed that flagellin composition could be related to the
adherent or invasive behavior of Campylobacter against Caco-2 cells on stationary
phase.
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Campylobacter and related species are now recognised as the most common
causes of bacterial foodborne diarrhoeal disease throughout the world (19). Domestic
poultry are considered to be one of the most important reservoirs of human infection
since Campylobacter is a common inhabitant of the avian bowel (8). Although it can
colonize a variety of warm-blooded animals asymptomatically, this pathogen can also
cause diseases in humans ranging from self-limiting gastroenteritis to more serious
systemic infections, such as septicemia, meningitis, the hemolyticuremic syndrome,
pancreatitis and abortion (7). It has, also, been associated with other clinical conditions,
such as Guillain-Barré syndrome and reactive arthritis (15, 16).
The ability of Campylobacter to become established in the gastrointestinal tract
of chickens and/or humans is believed to involve the binding and colonization of
surface cells of the gastrointestinal tract (20). Previous studies of the ability of
Campylobacter to colonize and invade the gastrointestinal tract have used various
models (1, 14). Among them, Caco-2 epithelial cells are one of the most used, being
useful to mimic the behavior of Campylobacter in both chicken and human gut (2, 3).
Physiological studies have shown that the resistance of Campylobacter to stress
does not increase appreciably on entry to stationary phase (6). In fact, cells taken from
stationary phase cultures are more sensitive to stress than those in exponential phase,
which is the opposite of normal behaviour. Thus, genetic and physiological studies both
indicate that Campylobacter does not possess the normal stress response on entry to
stationary phase (9, 10). However, practically all the studies on the adherence to and
invasion of Caco-2 cells by Campylobacter have used cultures in the exponential phase
of growth, and the effect of stationary phase on this is unknown.
On the other hand, several virulence factors have been identified in previous
studies related with the ability to adhere and invade epithelial cells (17, 22). In the
present work, following the determination of in vitro adherence to and and invasion of
Caco-2 cells by different strains of Campylobacter, comparisons were made to analyse
the influence of growth phase and the presence and characteristics of some virulence
factors on the behaviour observed.
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Materials and methods
Bacterial strains, growth media, and culture conditions. Five strains of
Campylobacter were used in this work. C. jejuni NCTC 11351 and C. jejuni NCTC
11168 were bought from the National Collection of Type Cultures (NCTC, London,
UK). C. jejuni LP1 and C. coli LP2 were clinical isolates provided by Hospital La Paz
(Madrid, Spain). C. jejuni 118 was also a clinical isolate supplied by the Microbiology
Department of the Health Institute Carlos III (Madrid, Spain).
Cultures of Campylobacter were prepared as follows: a frozen bead was
inoculated into 50 ml of Brucella Broth (BB) (Bencton, Dickinson and Company, Le
Pont de Claix, France) contained in a 100 ml flask. This culture was incubated at 38º C
on a shaking platform at 150 rpm for 48 h under microaerobic conditions (85% (v/v)
nitrogen, 10% (v/v) carbon dioxide, 5% (v/v) oxygen) maintained using a Variable
Atmosphere Incubator (VAIN) (MACS-VA500, Don Whitley Scientific, Shipley, UK).
The cells were diluted 1:100 into 50 ml of fresh BB and grown for 18 h, until
exponential phase, or for 40 h until stationary phase, determined in previous studies of
growth behavior. Then, the culture was diluted 1:2 on BB and used as experimental
inoculums. Serial decimal dilutions were prepared in PBS (Lonza, Verviers, Belgium)
pH 7 and 20 µl volumes were spread onto fresh plates of Mueller Hinton Agar
supplemented with 5% defibrinated sheep blood (MHB) (Biomedics, Madrid, Spain).
The number of colony-forming units (CFU) was assessed after plates had been
incubated for 48 h.
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Epithelial cells. Caco-2 cells ( ECACC 86010202) were obtained from the
European Collection of Cell Cultures (ECACC, Salisbury, UK). The cells were grown
in Dulbecco’s Modified Eagle’s Medium (DMEM) (Cambrex BioScience, Wokingham,
UK), supplemented with 10 % fetal bovine serum (FBS) and 1 % non- essential amino
acids (Cambrex BioScience, Wokingham, UK) at 37 º in a 5 % CO2 humidified
atmosphere (Inco 2, Memmert, Germany). For the experimental assays, Caco-2 cells
were grown in 24 –well plastic plates (Sarstedt, Nümbrecht, Germany). The cells were
seeded at 2 x 104 cells per well and incubated for 7 days at 37º C in the conditions
described above until confluence. For washing cells, PBS without Calcium or
Magnesium was used (Cambrex BioScience, Wokingham, UK).
Adherence and invasion assay of Caco-2 cells. Bacterial inocula of 18 h or 40
h of growth, corresponding to exponential and stationary phases, respectively, were
centrifuged at 5000 rpm 10 min, washed and suspended with PBS-Ca+2/Mg+2 , and
cellular concentration adjusted to 108 UFC/ml and 0.5 ml of these suspensions was
inoculated into duplicate wells containing confluent monolayers of Caco-2 cells. The
real number of bacteria in the inoculum added to monolayers was determined
retrospectively by serial dilutions and plate counting.
Caco-2 cells were incubated for 1 h at 37 ºC and 5 % CO2 to allow bacterial
adherence and invasion. For determination of adherence the procedure was as follows:
Caco-2 cells were washed three times with PBS and the cell monolayer was lysed with
1 % Triton X-100 (Sigma Aldrich, St. Louis, Mo.) and total bacteria associated with the
cells (intracellular and extracellular bacteria) were counted by serial dilutions as
mentioned above. To determine bacterial invasion, Caco-2 cells were incubated for 1 h
at 37º C and 5 % CO2 and then washed twice with PBS and incubated in fresh PBS
containing 1 % FBS and 150 µg/ml gentamycin for 2 h to kill remaining viable
extracellular bacteria. Quantification of viable intracellular bacteria was performed by
washing the infected Caco-2 cells with PBS twice and then lysing with 1 % Triton X-
100. Following serial dilution in PBS, released intracellular bacteria were counted as
described for the adherence assay. Results were expressed as % bacteria adhered
relative to inoculum or % of invasive bacteria relative to inoculum. For each assay, the
mean value of adherent and invasive bacteria was determined and the standard error of
the mean from triplicate experiments was calculated. Statistical significance of
differences between the means was assessed by one-way ANOVA using Minitab
version 15.
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Study of the growth physiology of Campylobacter. Bacterial cultures were
prepared and incubated following the conditions described above. The number of CFU
was assessed after plates had been incubated for 48 h. Experiments were prepared in
triplicate and average values used to present the results obtained.
Detection of virulence genes. Chromosomal DNA of the different
Campylobacter strains was isolated from a 24 h culture prepared in the conditions
described above and following the protocol described by Sambrook et al. in 1989 (18).
PCR was used to detect the presence of four Campylobacter genes (flaA, ciaB, cadF,
and pldA). The PCR primers used for the specific amplification of these genes were
described in previous published studies (table 1). PCR was performed in a DNA
Thermal Cycler (Eppendorf, Hamburg, Germany) using the following cycling
parameters: 95º C during 10 min for initial denaturation followed by 35 cycles of
denaturation at 95º C for 1 min, annealing at 45º C for 1 min, primer extension step at
72º C for 2 min, and final extension step at 72 ºC for 10 min. The amplified PCR
fragments were resolved by electrophoresis on 0.7 % agarose gels. DNA bands were
stained with ethidium bromide and visualized using a UV transiluminator. The PCR
product of flaA gene was digested with DdeI and AluI (New England Biolabs, Ipswich,
MA) and analysed by electrophoresis on 2% agarose gels, ethidium bromide staining
and visualization. Gene sequences were determined and multiple sequence alignment
was done with CLUSTAL W (http://wwwebi.ac.uk).
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Results and Discussion
Effect of the growth phase on the adherence to and invasion of Caco-2
epithelial cells by Campylobacter. Figure 1 shows the percentage adherence to and
invasion of Caco-2 epithelial cells by different strains of Campylobacter in exponential
(a) and stationary phases (b). In the experiments carried out with exponential phase
cultures (18 h of growth), the levels of adherence to and invasion of Caco-2 cells were
significantly higher for C. jejuni 118 than for the other strains tested (2.56 % for
adherence and 1.88% for invasion, respectively). The other three strains of C. jejuni
(11351, LP1 and 11168) had lower levels of adherence to and invasion of Caco-2 cells,
ranging between 0.19 and 0.21 for adherence and 0.0037 and 0.0038 for invasion,
respectively. The strain with the lowest capacity to adhere to and invade Caco-2 cell
cultures was C. coli LP2, which had an adherence level of 0.02 and an invasion level of
0.00067, respectively.
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In the same study, but carried out in the stationary phase (40 h) (figure 1 b), C
jejuni 118 was found to hardly change its behavior at all, and the values of adherence to
and invasion of Caco-2 epithelial cells were not significantly different (p≤ 0.05) from
those obtained in the exponential phase; this strain had the greatest invasive and
adhesive capacity. Similarly, the behavior of LP2 was not significantly different either
to that observed in the exponential phase, and this strain had the least adhesive and
invasive capacity of all the strains studied. However, the other three strains (11351, LP1
and 11168) all behaved similarly and presented a significant decrease in both adhesive
and invasive capacity in the stationary phase.
Almost all previous studies of the adhesive and invasive capacities of
Campylobacter to Caco-2 cells have no analyzed the effect of the phase of growth on
the behavior obtained. This is perhaps surprising, since stationary phase condition is
especially relevant to Campylobacter survival in food and environment. Only one
previous study, Mihaljevic et al., in 2007 (11), found that a shortage of nutrients
affected the adhesion and invasion properties of Caco-2 cells by two strains of C. jejuni,
although these properties were not modified by oxidative stress. For most
microorganisms, when the stationary phase is reached, a series of genetically-regulated
responses are triggered, which are aimed at increasing the microorganism’s resistance to
conditions of stress. In E. coli, for example, the general stress response is controlled by
a sigma factor σs, a product of the rpoS gene, that controls expression of more than 50
genes. Orthologues of rpoS have been found in many other Gram-negative bacteria (4),
but Campylobacter is unusual in that it lacks σ
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s or any other sigma factor associated
with stationary phase adaptation (13). Nonetheless, it has been described that in the
absence of other mechanisms of adaptation to stress, the generation of new strains with
modified phenotypes and physiological changes could be an alternative way for
organisms in the stationary phase, with a limited capacity for genetic regulation and
adaptation to the environment, to survive stressful situations. (9, 10).
Studies developed as parts of the present work have revealed that both the
capacity to adhere to and invade Caco-2 epithelial cells mainly depend on the properties
of the strain used. These properties may or may not be modified in the stationary growth
phase depending on the strain’s capacity to overcome stressful conditions, independent
of its virulence in the exponential phase, as occurred in the case of strain C. jejuni 118
(more adherent and more invasive) and strain C. coli LP2 (less adherent and less
invasive).
Study of the physiology of Campylobacter growth. Figure 2 shows the results
obtained from the physiology study of the growth of each strain studied. We can
observe that, after 24 hours of growth, the culture enters the stationary phase and there
is a drastic reduction in the viable population for C. jejuni strains 11168, 11351 and LP1
(figure 2a). This reduction reached a mean value of 4.6 log, and all the strains presented
a similar behavior, without statistically significant differences among them (p≤ 0.05).
Similar profiles were obtained for strains 11168 and 11351 in previous studies
conducted in similar conditions (9, 10), showing the sensitivity of these strains to the
stationary phase. Likewise, the capacity of these three strains to adhere to and invade
Caco-2 cells was, also, significantly affected in the stationary phase, as described
previously. These results seem to indicate that these strains are more sensitive to
conditions of stress present in the stationary phase, and that this sensitivity affects their
capacity to adhere to and invade Caco-2 cells. In contrast, C. jejuni 118 and C. coli LP2
are more resistant ( figure 2b), producing a mean reduction in the viable population of 2
log between 24h and 48h. The adherence and invasion capacities of these strains was
not significantly affected either in the stationary phase (fig 1b), which indicates that
their resistance to stress could influence their capacities to adhere to and invade the
Caco-2 cells.
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Virulence factors and its relationship with the adherence to and invasion of
Caco-2 epithelial cells by Campylobacter. Several studies have revealed that both
Campylobacter adherence and invasion are multifactorial processes (1, 2, 3, 20).
Genomic sequencing data from several strains reveal that Campylobacter does not
contain homologues of classical bacterial enterotoxins, adhesins, invasions type III
protein secretion systems, or pathogenicity islands (5). However, several virulence
factors have been identified in previous studies (5). In this work, first we studied
whether all the strains carried the genes flaA, ciaB, cadF, and pldA, which have been
reported to encode proteins involved in the adherence and invasive capacities of
Campylobacter and can, therefore, be considered as possible virulence factors in this
species. The flaA gene, which encodes flagellin, is required for Campylobacter
adherence to and invasion of epithelial cell (21). The ciaB gene encodes a protein
involved in cell invasion (17), while cadF encodes a protein which interacts with the
host extracellular matrix of fibronectin, participating in colonization of the cellular
surface (12). The PldA gene has, also, been related to cellular invasion, and encodes a
protein involved in the synthesis of an outer membrane phospholipase (23). The results
obtained showed that the genes were present in all the strains studied, independently of
their origin and capacity of adherence and invasion to Caco-2 cells (data not shown).
Previously, Zheng et al. (2006), (22) in a study carried out on 43 strains of
Campylobacter, reported that the majority of strains had most of the virulence genes
studied. It is important to take into account that in this study, although 100% of the
strains had the genes cadF, pldA and flaA, only 91% of the strains analyzed had the
ciaB gene. On the other hand, it has been described that not only the presence or
absence of the virulence factor is important for adhesion or invasion, but that the same
virulence factor has, also, been described to have multiple alleles, each of which could
present different characteristics of adhesion and invasion.
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Since previous research has shown that the different alleles of flaA can be
distinguished by comparing the fragments obtained after digesting the PCR fragments
with the restriction enzymes AluI and DdeI, it was decided to compare this pattern in the
strains studied here. Results are showed in Figure 3. It can be observed that all the
strains analyzed present different alleles for the flaA gene. In the case of AluI digestion
(figure 3A), strains C. jejuni LP1 and C. jejuni 11351 present almost identical patterns,
although both alleles could be distinguished with the DdeI enzyme (figure 3B). Since
the existence of 1120 different flaA alleles, which encode for 287 different flagellins,
have been described to date, we decided to identify the flaA alleles present in the strains
studied here. First, the PCR-amplified fragment was purified and sequenced. Then, the
sequence of the flaA locus obtained for each of the Campylobacter strains analyzed was
compared with the sequences included in the database for the flaA locus
(http://pubmlst.org/perl/mlstdbnet/agdbnet.pl?file=flaA.xml). The comparison
confirmed the results obtained by digestion with the AluI and DdeI enzymes, in that the
strains studied presented different flaA alleles. Moreover, this comparison with the
sequences included in the databases permitted the flaA alleles of the strains studied to be
identified. Hence, the C. jejuni LP1 strain presented allele 233, the C. coli LP2 strain
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had allele 67, strain C. jejuni 118 allele 190, strain C. jejuni 1168 allele 285 and, finally,
strain C. jejuni 11351 allele 156 of the flaA locus.
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The great variability of flaA alleles in Campylobacter has been previously
described (see above), although the allele variability of the ciaB, cadF, and pldA loci in
this species is not yet known. Therefore, after demonstrating that all the strains present
the genes studied here, we decided to determine the variability of these loci in these
strains. The sequences obtained for each locus were compared by alignments made
using the ClustalW programme (http://www.ebi.ac.uk) (data not shown). Each of the
loci studied presents several different alleles. The ciaB and cadF loci present four
different alleles, each of which is shared by strains C. jejuni 118 and C. jejuni 11168. In
the case of the pldA locus, only three different alleles were found among the different
strains studied, since strains C. coli LP2, C. jejuni 118 and C. jejuni 11168 present the
same allele for this locus. From the alignments obtained, the percentage of identity
presented by different alleles from the same locus can be established (Table 2).
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The results obtained show that for the flaA locus, all the strains, except strain C.
jejuni 118, showed a high percent identity between them (96-98%). However, the flaA
locus of strain C. jejuni 118 only shared 84-85% identity with the other strains analyzed
here. A similar situation also occurs with strain C. coli LP2 for the cadF and ciaB locus.
The cadF and ciaB locus of strain C. coli LP2 present only 86-87% or 77% of identity,
respectively, while in these loci the other strains analyzed (strains C. jejuni 11168, C.
jejuni 11351, C. jejuni LP1 and C. jejuni 118) present a high identity (98-100%).
The results obtained can be related to the behavior observed in relation to
adherence to and invasion of the Caco-2 epithelial cells. Strains C. jejuni 11168, C.
jejuni 11351 and C. jejuni LP1 presented a similar pattern of adherence and invasion,
while strains C. jejuni 118 and C. coli LP2, behaved differently. Strain C. jejuni 118
was the most adherent and invasive of the strains studied, while strain C. coli LP2 was
the least adherent and invasive. Strains C. jejuni 118 and C. jejuni 11168 share the same
allele for the ciaB, cadF and pldA loci and the only difference among the genes studied
corresponded to the allele of the flaA locus. This seems to indicate that in the virulence
factors assayed, the different behavior observed between the strains can only be
explained by the possession of different alleles in the flaA locus. From the results
obtained, it could be inferred that the composition of flagellin from strain C. jejuni 118,
encoded by allele 190 of the flaA locus, could have a favorable influence on the
virulence of this strain, increasing the capacity to adhere to and invade the epithelial
cells, without this being significantly affected in the stationary growth phase. Similarly,
the results obtained rule out proteins encoded by the ciaB, cadF and pldA genes as
being involved in the different adherence and invasive capacity of strains C. jejuni 118
and C. jejuni 11168, although the allele 67 of the flaA locus could be involved in the
lower adherence and invasive capacity of the C. coli LP2 strain. The possibility that the
alleles present on the ciaB, cadF and pldA locus in the LP2 strain could be involved in
the lesser adherence and invasive capacity presented by this strain cannot be excluded
either. Moreover, other genes or virulence factors not studied here could also contribute
to the different behavior observed.
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In summary, this work shows that the capacity of Campylobacter to adhere to
and invade epithelial cells is closely related to the strain used. An increased resistance to
stress in the stationary growth phase could influence both the capacity to adhere to and
invade Caco-2 epithelial cells. Similarly, the flagellin composition could be
significantly related to the capacity of Campylobacter to adhere to and invade epithelial
cells. This multifactorial-conditioned behavior can be important in the modulation of
Campylobacter virulence in response to environmental stress factors in stationary phase,
which may have further implications in the pathogenesis of this organism.
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Figure legends:
Figure 1: Percentage of adherence to and invasion of Caco-2 epithelial cells by
different strains of Campylobacter in exponential (a) and stationary (b) phase of growth.
Figure 2: Physiology of growth during exponential and stationary phase for different
strains of Campylobacter. a) C. jejuni NCTC 11168, C. jejuni NCTC 11351 and C.
jejuni LP1, more sensitive strains to stationary phase stress; b) C. coli LP2 and C. jejuni
118, more resistant strains to stationary phase of growth.
Figure 3: Restriction analysis of flaA DNA fragments amplified by PCR from the
Campylobacter strains analyzed in this study. Restriction patterns of AluI (A) or B)
DdeI digest from C. jejuni LP1 (1), C. coli LP2 (2), C. jejuni 118 (3), C. jejuni NCTC
11168 (4) and C. jejuni NCTC 11351 (5). The 100 –pb ladder was included in the left of
the gels.
.
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Table 1. Primers used for the amplification of some Campylobacter virulence factors
Gen Primers Sequence 5’→3’ Reference
flaA flaA-F flaA-R
ATGGGATTTCGTATTAACAC CTGTAGTAATCTTAAAACATTTTG
Hänel et al., 2004
cadF cadFI-F2B cadFI-R1B
TTGAAGGTAATTTAGATATG CTAATACCTAAAGTTGAAAC
Zheng et al., 2006
ciaB ciaBI-652 ciaBI-1159
TGCGAGATTTTTCGAGAATG TGCCCGCCTTAGAACTTACA
Zheng et al., 2006
pldA pldA-361 pldA-726
AAGAGTGAGGCGAAATTCCA GCAAGATGGCAGGATTATCA
Zheng et al., 2006
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Table 2. Percentage of identity among sequences for the flaA, CadF, pldA and CiaB
loci in the Campylobacter analyzed strains.
Strains
Gen Strains
11168 11351 LP1 LP2
118 85 85 84 85 LP2 96 98 97 - LP1 97 97 - -
flaA
11351 96 - - - 118 100 98 99 87
LP2 87 87 86 - LP1 99 98 - -
cadF
11351 98 - - - 118 100 99 99 100
LP2 100 99 99 - LP1 99 99 - -
pldA
11351 99 - - - 118 100 98 99 77
LP2 77 77 77 - LP1 99 98 - -
ciaB
11351 98 - - -
Acknowledgments 392
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This work was founded through Projects CCG07-CSIC/AGR-2255, from the
CSIC and 25506 FUN C FOOD (CONSOLIDER-IMAGENIO 281 2010). M. Ganan is
the recipient of a predoctoral fellowship (I3P, CSIC) from the Spanish Ministry of
Science and Education (MEC). We thank Gema Campos Monfort for her technical
assistance.
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0.0001
0.001
0.01
0.1
1
10
11351 LP1 118 11168 LP2
% AdherenceInvasion
a
0.0001
0.001
0.01
0.1
1
10
11351 LP1 118 11168 LP2
% AdherenceInvasion
b
Figure 1
a
4
6
8
10
0 10 20 30 40 50
time (h)
log
cfu/
mL
1116811351LP1
4
6
8
10
0 10 20 30 40 50
time (h)
log
cfu/
mL
118
LP2
b
Figure 2
A B 1 2 3 4 5 1 2 3 4 5
Figure 3