| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Food and Environmental Safety, Veterinary Laboratories Agency, New Haw, Surrey, UK
Correspondence
Diane G. Newell
d.newell{at}vla.defra.gsi.gov.uk
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
whilst increasing secretion of interleukin (IL)-10. Preliminary studies using porcine in vitro organ culture (IVOC) tissue suggested that the flagella mutant was also attenuated in its ability to colonize intestinal tissue.
Present address: School of Biomedical and Natural Science, Nottingham Trent University, Clifton Lane, Nottingham, UK.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The organism is classified on the basis of serogrouping and biochemical characteristics into biotypes (BTs) and serotypes (Bottone, 1999). BT1b strains are considered highly pathogenic due to their lethality in a mouse infection model, and are most frequently associated with severe, often fatal, disease in humans (Bottone, 1999). BT2–5 isolates are mildly pathogenic in the mouse infection model, and account for the majority of human yersiniosis cases in northern Europe (Bottone, 1999). Finally, BT1A strains are avirulent in the mouse infection model and as a result have historically been classified as non-pathogenic.
The molecular pathogenesis of Y. enterocolitica has been extensively researched, with particular emphasis on BT1b. The recently sequenced Y. enterocolitica isolate 8081 is a BT1b O : 8 strain, which has been thoroughly characterized. Most research has been conducted on the pYV-encoded type III secretion system, responsible for injection of Yop effector proteins into host cells (Cornelis & Wolf-Watz, 1997). The Yop proteins are responsible for a large number of virulence traits, including the avoidance of macrophage ingestion (Grosdent et al., 2002), immunomodulation (Boland & Cornelis, 1998; Carlos et al., 2004; Denecker et al., 2002; Erfurth et al., 2004) and cytotoxicity (Andor et al., 2001; Zumbihl et al., 1999). The majority of the remaining research on pathogenesis of Y. enterocolitica has focused on invasin, and the adhesins Ail and YadA. Invasin has a major role in invasion of epithelial cells by Y. enterocolitica (Pepe & Miller, 1993) and also exhibits immunomodulatory properties (Kampik et al., 2000). YadA and Ail are adhesins involved in initial attachment to epithelial cells, as well as subversion of macrophages and immunomodulation (El Tahir et al., 2000; Grosdent et al., 2002; Schmid et al., 2004). All of these virulence determinants are present in BT1b and BT2–5 strains, but absent in BT1A strains (Bottone, 1999). In addition, several key publications have highlighted the key role of LPS and O-antigen variation in Y. enterocolitica pathogenesis (Bengoechea et al., 2003, 2004; Rebeil et al., 2004).
Despite the apparent lack of virulence attributes in BT1A Y. enterocolitica, there is growing evidence to suggest that this group of organisms are aetiological agents of human disease (Grant et al., 1998; Morris et al., 1991; Tennant et al., 2003). Recent work has shown that BT1A isolates are capable of invading and escaping from cultured epithelial cells in vitro, as well as persisting in cultured macrophages (Grant et al., 1999) and modulating secretion of cytokines by infected macrophages in vitro (McNally et al., 2006). In addition, previous work in our laboratory has highlighted the fact that BT1A strains are ubiquitous in the environment, accounting for 53 % of all Y. enterocolitica isolated from human disease cases in Great Britain during 1999–2000 (McNally et al., 2004).
The purpose of the present study was to further elucidate the interaction between BT1A Y. enterocolitica and epithelial and macrophage cells in vitro. Preliminary investigation of the ability of BT1A strains to invade HEp-2 cells uncovered a relationship between motility and invasion. The role of motility in Y. enterocolitica has been investigated earlier, suggesting that motility is required for invasion (Young et al., 2000), and implicating flagella in the secretion of effector proteins (Young et al., 1999a). In order to examine the role of flagella in BT1A isolates, a flagella mutant was constructed in a human faecal BT1A isolate and characterized in a number of in vitro pathogenesis assays.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). From this subset, strain 53/03 was chosen for further analysis, as it was a human isolate, and was motile and invasive. A list of strains, plasmids and primers used in this study is provided in Table 1. All Y. enterocolitica isolates were cultured aerobically for 16 h at 28 °C overnight on 5 % sheep blood agar plates, or in LB broth with shaking at 200 r.p.m., unless otherwise stated. Escherichia coli DH5 was employed for all cloning procedures and was grown in LB at 37 °C supplemented with ampicillin (Amp, 100 µg ml–1), kanamycin (Kan, 50 µg ml–1) or chloramphenicol (Cm, 25 µg ml–1). E. coli S17 
pir was employed for conjugative transfer of plasmids.
|
fliA knockout mutant.pir, and conjugations performed with 53/03 for 24 h at 28 °C on sterile filters placed on LB plates. The mating filter was resuspended in PBS and serial dilutions plated onto M9 minimal agar plates containing 6 % sucrose plus Kan. Colonies were replica-plated and checked by PCR for double recombination events. Potential mutants were confirmed by Southern blotting (using probes for npt and fliA), PCR, motility test, Western blotting and electron microscopy. The mutant AMC2 was complemented using pAM3 containing the cat gene excised from pUCCat, cloned into the KpnI site. The complemented strain was maintained under Amp, Kan and Cm selection to ensure plasmid stability.
Southern blotting.
Mutant and wild-type strains were grown overnight in 50 ml LB broth, and cultures harvested by centrifugation. Chromosomal DNA was prepared using the phenol/chloroform extraction method (Sambrook et al., 1989) and Phase Lock Gel tubes (Eppendorf). DNA was digested with XmnI, separated by agarose gel electrophoresis, and transferred to a nitrocellulose membrane (Amersham). Purified fliA and npt were labelled with the AlkPhos direct labelling kit, with hybridization and detection performed using the CPD-Star detection kit (Amersham).
Flagella secreted protein Western blots.
Bacteria were cultured for 16 h aerobically in L broth and then subcultured 1 : 50 into 50 ml fresh medium. Cultures were grown at 28 °C for 6 h, bacteria were pelleted by centrifugation, and the supernatant was collected and filtered through a 0.2 µm pore-size filter. Ice-cold TCA was added to a final volume of 10 % and the flagella secreted proteins (Fop) precipitated overnight at 4 °C. Precipitates were harvested by centrifugation at 4500 g for 30 min, the supernatants decanted, and the protein pellets suspended in a volume of Laemmli sample buffer (Sambrook et al., 1989) calculated according to the OD600 of the original bacterial culture. Proteins were separated on a 10 % SDS-polyacrylamide gel and stained by a commercially available silver stain kit (Amersham) or transferred to a nitrocellulose membrane using the Mini-Protean transfer system (Bio-Rad). The membrane was blocked in 0.1 M PBS+0.05 % Tween 20 (wash buffer) with 8 % (w/v) dried skimmed milk overnight at 4 °C, then washed in wash buffer. A 1 : 150 dilution was made of rabbit anti-flagella antiserum (Bleves et al., 2002) in wash buffer and this was hybridized to the membrane for 3 h at room temperature. The serum is a polyclonal serum raised against the BT1B O : 8 type strain ATCC 9610, as used in the Wauters typing scheme (Aleksic & Bockemuhl, 1984; Bleves et al., 2002), and contains antibodies against the somatic and flagellar antigens. Before use in flagella Western blots, serum was pre-absorbed with bacteria that had been grown at 37 °C to prevent flagella expression. The membrane was washed and then probed with a 1 : 1500 dilution of mouse anti-rabbit horseradish peroxidase (HRP)-conjugated antibody for 90 min. The membrane was washed and then developed using the classical 3-amino-9-ethylcarbazole (AEC)/H2O2 method (Sambrook et al., 1989).
LPS and total cell protein profiles.
Bacteria were grown on LB agar plates at 28 °C for 24 h, harvested and resuspended in 2 ml PBS to equal densities, as determined by OD600. LPS was extracted by adding 2 ml 90 % phenol, followed by incubation with constant agitation at 68 °C for 30 min. The supernatant was collected and 0.1 vols 20 % (v/v) sodium acetate plus 5 ml absolute alcohol were added. The sample was stored at 4 °C overnight and precipitated LPS harvested by centrifugation. The pellet was resuspended in 2 ml PBS and dialysed overnight in sterile distilled water. The LPS sample was diluted 1 : 1 with Laemmli sample buffer and separated by electrophoresis using standard SDS-PAGE techniques. The gel was stained with a commercially available silver stain (Amersham). Total cell protein extracts were prepared by resuspending the cell pellet in Laemmli sample buffer, followed by standard SDS-PAGE and Coomassie Blue staining.
Motility test.
Sixteen-hour, aerobic broth cultures were harvested by centrifugation and resuspended in 0.1 M PBS, pH 7.2. Ten microlitres of suspension was used to inoculate the centre of a 0.3 % tryptone soya agar plate, which was incubated for 16 h aerobically at 28 °C.
Transmission electron microscopy (TEM) of bacteria.
Bacteria were cultured for 16 h aerobically in L broth at 28 °C, harvested by centrifugation and resuspended in 0.1 M PBS. Formvar carbon-coated grids were placed onto a drop of the bacterial suspension for 15 min. The grid was removed, excess liquid drained by blotting paper, and the grid placed onto potassium phosphate tungsten-negative stain solution for 
10 s. Grids were dried and viewed using a Philips CM 10 transmission electron microscope.
Cell cultures.
HEp-2 human epithelial cells were cultured in Minimal Essential Medium (MEM, Life Technologies) supplemented with 10 % heat-inactivated fetal calf serum (Life technologies), 1 % L-glutamine and 1 % non-essential amino acids (Sigma). Cells were grown at 37 °C in a humidified 5 % CO2 atmosphere, and subcultured every 4 days. Two days prior to infection, cells were seeded into a 24-well tissue-culture plate (Nunc) at approximately 2x105 cells ml–1. U937 human monocytic cells were maintained in RPMI medium (Sigma) supplemented as above. Seventy-two hours prior to infection, cells were seeded into a 24-well tissue-culture plate (Nunc) at approximately 2x105 cells ml–1 and treated with 10 nmol phorbol-myristate-acetate (PMA, Sigma) to trigger differentiation into active macrophages.
Association and invasion assays.
Association with and invasion of bacteria into cultured HEp-2 cells was assayed as described elsewhere (Finlay & Falkow, 1988; Miller & Falkow, 1988). Bacteria were cultured for 16 h aerobically in LB broth with shaking, harvested by centrifugation, and resuspended in supplemented tissue-culture medium to 2x107 c.f.u. ml–1. One millilitre of bacterial suspension was added in triplicate to wells of a pre-seeded 24-well plate at an m.o.i. of 100. The infected cells were incubated for 3 h at 37 °C. Cells were washed three times with 0.1 M PBS, and fresh supplemented tissue-culture medium containing 100 µg gentamicin ml–1 (Sigma) was added and the cells further incubated for 2 h. Cells were then washed twice in PBS and lysed with a 1 % Triton X-100 (Sigma) solution for 5 min. Dilutions of the lysed infected cells were plated onto LB agar in triplicate and incubated overnight, and colonies were counted to give the total number of invasive bacteria per millilitre. In a parallel series of wells, the cells were lysed after the initial 3 h infection period to ascertain the total number of associated organisms per millilitre.
Persistence within U937 human macrophage cell line.
Bacteria were grown, harvested and resuspended as for the HEp-2 invasion assay. Differentiated U937 cells were infected at the same m.o.i. as for the HEp-2 invasion assay and incubated for 1 h at 37 °C. Cells were washed three times with 0.1 M PBS, and fresh medium containing 100 µg gentamicin ml–1 was added with a further incubation for 5 h. Cells were washed twice in PBS and lysed by addition of a 1 % Triton X-100 solution for 10 min. Dilutions of the lysed cell infection were plated onto LB agar in triplicate and incubated at 28 °C aerobically for 16 h, and colonies were counted to give the total number of invasive bacteria per millilitre. In a parallel series of wells the cells were lysed 15 min after the initial addition of gentamicin in order to ascertain the number of organisms internalized by the cells after the 1 h infection.
Secretion of cytokines by infected U937 cells.
U937 human macrophages were infected as above for a fixed time point of 3 h. The supernatant was removed and centrifuged at 13 000 g for 5 min. Two hundred microlitres of the supernatant was stored at –20 °C until required. The levels of secretion for interleukin (IL)-6, IL-10 and tumour necrosis factor (TNF)- were ascertained by using the Quantikine sandwich ELISA kit (R&D Systems). For assays involving crude flagellin samples, approximately 10 ng protein was added to cells, followed by incubation for 3 h, as for bacterial infections.
Preparation of crude flagellin sample.
Bacteria were cultured as described above for the preparation of Fop proteins. Precipitated protein was resuspended in 1.5 M Tris/HCl, pH 8.8, and then subjected to size-exclusion filtration on a YM 30 kDa centrifugation device (Centricon). The filtrate was collected and then centrifuged again using a YM 10 kDa kit (Centricon). The retentate was then stored at –20 °C. For use in assays, the protein concentration was determined using the Bio-Rad protein assay kit and a BSA standard dilution series (Bio-Rad). Confirmation of the presence of flagellin in the preparation was performed by Western blotting using unabsorbed serum, confirming the presence of flagellin and the absence of detectable levels of LPS.
TEM of Y. enterocolitica-infected U937 cells.
U937 cells were infected as above for 1 or 6 h with strains 53/03 or AMC2. Cells were then treated with 3 % glutaraldehyde in PBS for 5 min and removed from the tissue-culture well. Cells were pelleted by centrifugation at 1500 r.p.m. for 5 min, washed in 0.1 M PBS and post-fixed in 1 % (w/v) osmium tetroxide, dehydrated in ethanol, and placed in hexamethyldisizone for 5 min. Specimens were subjected to critical-point drying with liquid CO2, then fixed to aluminium stubs with silver conductive paint and sputter-coated with gold. Specimens were then examined using a Cambridge Stereoscan S250 Mark III transmission electron microscope at 10–20 kV.
Construction of a GFP fusion containing, Y. enterocolitica.
The gfp+ gene was amplified from pMN402 (Scholz et al., 2000) using primers GFP5 and GFP3. Plasmid pAM4 was constructed by digesting pACYC184 with ClaI, blunt-ended by treatment with the large Klenow fragment of DNA polymerase I, and religated using T4 DNA ligase. Plasmid pAM4 was digested with EcoRV, phosphatase-treated, and ligated to the amplified gfp+ gene to create pAM6. Clones were selected by loss of tetracycline resistance. The gyrA promoter was amplified from Y. enterocolitica 8081 using primers Gyr5 and Gyr3. The amplified product and pAM6 were digested with EcoRV and ClaI, the linearized plasmid was phosphatase-treated, and the fragments were ligated to create pAM19. Electrocompetent Y. enterocolitica strains were electroporated with 1 µl pAM19 at 1.5 kV, 2.5 µF, 200 
.
Porcine in vitro organ culture (IVOC).
Two commercial cross-bred, three-month-old male pigs were euthanized on separate days. Immediately after euthanasia the terminal ileum and spiral colon sections were harvested. The contents of the tissues were emptied and 2 cm2 sections prepared, washed in pre-warmed 0.1 M PBS, pH 7.2, and placed on sterile gauze. The tissue sections were placed individually in a square Petri dish and partially submerged in pre-warmed DMEM containing 1 % L-glutamine and 1 % non-essential amino acids. Overnight cultures of bacteria containing the GFP fusion plasmid were harvested by centrifugation and resuspended in PBS. One hundred microlitres of culture was pipetted directly onto the apical surface of the tissue and incubated at 37 °C in 5 % CO2 for 6 h, with media changed after 3 h. Tissues were washed vigorously in PBS and fixed in ice-cold 4 % paraformaldehyde for 24 h.
Tissue preparation and confocal fluorescence microscopy.
Tissue samples were fixed in 4 % paraformaldehyde and snap-frozen in isopentane pre-cooled in liquid nitrogen to between –40 and –60 °C. Tissue sections were then cut using a cryostat (–20 °C) to a thickness of 4–6 µm and collected directly onto slides. The slides were mounted with Vectashield with 4',6-diamidino-2-phenylindole (DAPI) mounting medium and stored in the dark at room temperature. Confocal fluorescence microscopy was performed on a Leica microscope with an excitation wavelength of 488 nm and an emission filter of 507 nm.
Statistics.
P values for differences between sets of data for the wild-type, mutant and complemented mutant were performed using Student's t test.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). However, three of the isolates were completely non-invasive in HEp-2 cells; two were ovine isolates, and one was isolated from an asymptomatic human who had been in contact with a confirmed human yersiniosis case, as part of a national intestinal infectious disease survey. All three isolates were serotype O : 5, non-motile, and failed to express flagellin (data not shown).
|
fliA mutant in a clinical BT1A isolate) and by Southern blotting to confirm the insertion of the Kan cassette into fliA by hybridization, with both a fliA (Fig. 2B) and an npt (Fig. 2C) probe. The mutant was aflagellate (Fig. 2D), non-motile (Fig. 2E) and failed to express flagellin detectable by SDS-PAGE (Fig. 2F) or by Western blotting of secreted proteins (Fig. 2G). After complementation of the mutant with pAM3, motility was restored (Fig. 2E), and flagellin secretion was restored as indicated by Western blotting (Fig. 2G). The flagellin detected in BT1A isolates migrated to a different extent to that of strain 8081 and BT3 and BT4 isolates, but was confirmed as flagellin by 2D gel electrophoresis and MS analysis. Any potential regulatory effects as a result of the mutation in fliA were investigated by examining whole-cell protein extracts (Fig. 2H) and LPS profiles (Fig. 2I) of the mutant and wild-type strain. The results suggested no obvious changes in protein expression or LPS production between the mutant and wild-type strain.
|
fliA mutant was characterized in the HEp-2 cell association and invasion assay. The mutant demonstrated an almost complete loss of invasiveness in vitro when compared with the wild-type and complemented strains (102.5) (P=0.000). Complementation of the mutant resulted in partial restoration of the parental invasive phenotype compared with the wild-type (P=0.001), with differences possibly due to overexpression of fliA from the plasmid. Numbers of associated bacteria were similar for the wild-type (105), mutant (105) and complemented strains (104), although the complemented strain was slightly reduced in its ability to adhere (P=0.045), indicating that the loss of invasiveness was not due to the inability of the mutant to actively swim towards or adhere to the cell layer. Additionally, pre-growth of the organism at 37 °C (which is inhibitory to flagella expression) prior to cell infection completely inhibited cell invasion, but had no effect on association with cells (data not shown).
An aflagellate BT1A Y. enterocolitica isolate is unable to persist within macrophages in vitro
Previous studies have shown BT1A strains to be capable of surviving within cultured macrophages for extended periods of time (Grant et al., 1999). To assess the effect of flagella on this persistence, human U937 macrophage cell lines were infected with mutant, wild-type and complemented strains, and the number of intracellular organisms after 6 h was compared with the total number of associated organisms recovered after an initial 1 h infection period. The aflagellate strain was completely cleared by the macrophages by 6 h, whilst the mutant and complemented strains persisted (P=0.000) (Fig. 3A). A time-course experiment showed that the mutant was ingested by the macrophages to the same extent as the wild-type, but was completely killed by the macrophages between 3 and 4 h post-infection (Fig. 3B). TEM of infected macrophages showed that the mutant strain was contained within a large phagosomal vacuole, whilst the wild-type was present inside the cells in tight vacuoles with no free surrounding space (Fig. 3C, D, E). Additionally, pre-growth of the organism at 37 °C (which is inhibitory to flagella expression) prior to cell infection completely inhibited the ability of the organism to survive within macrophages, with no detectable bacteria at 1 h post-infection (data not shown).
|
. There was no difference between the levels of IL-6 secreted by cells infected with the mutant and those of cells infected with the wild-type (data not shown). Whilst the wild-type strain induced high levels of secretion of the anti-inflammatory cytokine IL-10, U937 infected with the mutant secreted lower levels (P=0.0014) (Fig. 4B). Complementation of the mutant partially restored the IL-10 secretion phenotype (P=0.021). Similarly, U937 infected with the mutant strain secreted approximately twofold greater amounts of the pro-inflammatory cytokine TNF- compared with the wild-type (P=0.0031) and the complemented strain (P=0.0045) (Fig. 4A). These results suggested that flagellin induces some cytokine responses in vitro. To investigate this hypothesis, the effect of isolated flagellin on the cytokine response of host cells was tested. Unfortunately, attempts to amplify the fleABC operon from BT1A isolates using primers designed against the published sequence (Kapatral & Minnich, 1995), in order to generate recombinant flagellin, proved unsuccessful. Instead, crude flagellin samples (53/03 Flg or AMC2 Flg) were prepared by size-exclusion filtration and used to treat cultured U937 cells. The crude flagellin samples were subjected to Western blotting with serum containing antibodies against the flagellar and somatic O antigens, with no detectable endotoxin present in the sample. The addition of this crude flagellin material had no obvious cytotoxic effect on the cells, as determined microscopically (data not shown). The cells treated with 53/03Flg or AMC2Flg did not secrete IL-10 (Fig. 4B). However, cells secreted approximately twofold lower levels of TNF- when exposed to flagellin from 53/03 compared with flagellin from AMC2 (P=0.041) (Fig. 4A).
|
), but semi-quantitative evaluation indicated that the number of wild-type bacteria associated was approximately 50-fold higher than that observed for the aflagellate mutant bacteria. Similarly, although there was less colonization of the spiral colon by wild-type bacteria, it was still approximately 10-fold greater than with the aflagellate mutant bacteria.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 1999; McNally et al., 2004; Morris et al., 1991; Tennant et al., 2003). Nevertheless, little is known about the interaction of this group of organisms with the infected host, or the potential virulence mechanisms involved. During a preliminary investigation of the phenotypic properties of BT1A strains from a variety of sources (McNally et al., 2006), three isolates were identified as both non-invasive and non-motile. Consequently, the role of flagella as a potential virulence factor in BT1A Y. enterocolitica was further investigated by comparison of the characteristics of an aflagellate mutant with those of the wild-type parent.
Although the natural target for mutagenesis would have been the fleABC operon, which encodes the structural flagellin genes, attempts at amplifying the fleABC operon from BT1A isolates were unsuccessful, using primers based on the previously published operon sequence (Kapatral & Minnich, 1995) or on the recently completed genome sequence of Y. enterocolitica 8081 BT1b. Attempts at amplifying the entire fleABC operon with primers upstream and downstream, generating a 3.9 kb product, were also unsuccessful, as were attempts at amplifying individual genes or even promoter regions from the area. As there are high levels of homology between fleA, B and C, the failure to amplify individual genes may not be surprising; however, our results seem to confirm that the flagellin region in BT1A isolates is variable (Tennant et al., 2003). Interestingly, the three flagellin genes successfully hybridized with BT1A strains in a comparative microarray study (Howard et al., 2006), suggesting that the sequence variation may be occurring at a low level. The flagellin-coding region of BT1A strains is currently the focus of further investigation.
An alternative strategy adopted was to target the fliA gene for mutagenesis. The fliA gene is an appropriate target, as the gene product is essential for expression of flagella in a BT1b strain, but its mutation has no effect on expression of other key virulence genes (Iriarte et al., 1995; Young et al., 1999b, 2000). The fliA gene was insertionally inactivated with a Kan cassette using the pKNG101 suicide vector. The resulting mutant (AMC2) was confirmed by PCR and Southern blotting, electron microscopy, Western blotting and loss of motility.
Investigations using the fliA mutant (AMC2) suggested that flagella play a role in invasion of epithelial cells in vitro. The aflagellate mutant was almost completely attenuated in its ability to invade HEp-2 cells, with the phenotype partially restored by complementation with the entire fliA gene under the control of its native promoter. Similar observations have been made using a fliA mutant constructed in a BT1b strain (Young et al., 2000), and in our study, growth of the wild-type strain at 37 °C, which is inhibitory to flagella expression, prior to cell infection completely inhibited cell invasion. One explanation would be that the lack of motility precludes interaction with the cell monolayer. However, our results indicated that the number of associated bacteria was the same for both the wild-type and the aflagellate mutant after an initial 3 h infection, as well as for the bacteria pre-grown at 37 °C. Thus, it appears that the flagella of BT1A strains play an active role in the invasion of host cells. The active involvement of flagella in invasion by other enteric pathogens, including Salmonella (La Ragione et al., 2003), as well as in adherence by enteropathogenic E. coli (Giron et al., 2002), has been previously reported.
Comparison of the wild-type and mutant strains also suggests that flagella play a role in survival of BT1A Y. enterocolitica within human macrophages in vitro. Similar findings have been observed in both naturally occurring and experimentally constructed flagella mutants of Salmonella Typhimurium (Schmitt et al., 1994, 2001). However, whether flagella are expressed within macrophages by Y. enterocolitica BT1A has yet to be ascertained. The mechanism of this intracellular persistence is unclear. TEM of infected macrophages showed that the wild-type was contained within tight vacuoles, with little free space, whilst the mutant was contained within a large spacious vacuole. Earlier work using murine macrophages has also demonstrated that BT1A bacteria are contained within vacuoles which appear to fuse with each other (Grant et al., 1999). Thus, the vacuolar space surrounding the bacteria may be relevant to intracellular persistence.
As flagella from a wide range of bacterial pathogens have been shown to have immunomodulatory effects on host cells, primarily through interaction with Toll-like receptor (TLR)5 (Donnelly & Steiner, 2002; Wyant et al., 1999; Zhou et al., 2003), the cytokine responses to challenge of U937 cells with the wild-type and aflagellate mutant were compared. Incubation with the aflagellate mutant AMC2 resulted in elevated secretion of the pro-inflammatory cytokine TNF-, and decreased secretion of the anti-inflammatory cytokine IL-10, compared with the wild-type strain. This suggests that the flagella may play a role in depression of the host inflammatory immune response to infection with Y. enterocolitica. Support for this hypothesis was obtained using a crude preparation of secreted flagellin, which induced a twofold reduction in TNF- secretion compared with the equivalent protein sample prepared from the aflagellate strain.
As the aflagellate mutant was constructed by mutation of a regulatory gene, the possibility exists that other factors which contribute to BT1A pathogenesis were affected, such as outer-membrane protein profiles, or perhaps LPS composition. The effect of a fliA mutation on global gene expression and LPS composition in BT1A strains was partially characterized by examining whole-cell protein extracts and LPS profiles of the mutant and wild-type strains, and there were no observable differences between the two. Whilst fliA mutants have been shown to have no effect on expression of virulence genes in pathogenic Y. enterocolitica, previous work has shown that there is cross-talk between the flagella operon and other type III secretion systems (Bleves et al., 2002), and that flagella actively export virulence factors (Young et al., 1999a). Whilst we have failed to find any secreted proteins in BT1A strains by SDS-PAGE and silver staining (data not shown), it cannot be discounted that abolition of flagella could result in the absence of an as-yet-unidentified virulence factor.
The aflagellate mutant was also attenuated in its ability to colonize porcine intestinal tissue in vitro. This attenuation was more evident with terminal ileal material, which is the area of the porcine gastrointestinal tract that is dense in follicle-associated epithelium, than with spiral colon tissue. This is in disagreement with the association observed with HEp-2 cells in vitro, and also with previous work describing the expression of flagella in Y. enterocolitica at 28 but not at 37 °C. Clearly, there is a high degree of complexity involved in flagellin production, and the expression of flagella by BT1A Y. enterocolitica in vitro and in vivo is currently under further investigation.
In contrast to our results, earlier studies using flagella mutants of pathogenic biotypes of Y. enterocolitica have indicated that flagella have no detectable effect in the mouse model (Young et al., 2000). Given the well-recognized similarity between the pig and human intestinal tracts, our results suggest that such virulence properties are host specific for Y. enterocolitica and indicate that the ability of the mouse model to reflect the disease potential of BT1A strains may need to be reconsidered, although a mouse model of colonization has been described (Tennant et al., 2003).
In conclusion, this study reports the successful mutagenesis of a potential virulence factor, flagellin, in a BT1A Y. enterocolitica isolate. The aflagellate mutant was attenuated in its ability to invade cultured epithelial cells and colonize porcine intestinal tissue in vitro, and was unable to survive within cultured human macrophages over 3 h. The flagellin proteins were also shown to influence secretion of TNF- by infected macrophages, and potentially induce production of the anti-inflammatory cytokine IL-10. Further work is required to begin to fully understand the potentially pathogenic BT1A Y. enterocolitica, but this is hampered by a lack of knowledge of the genome sequence. It is well recognized that at the genetic level BT1A isolates may possess as little as 41 % similarity to BT2–5 isolates (Fearnley et al., 2005). Such differences in sequence similarity have hampered our ability to further confirm our findings by genetic-based methods. Investigation of BT1A isolates as a separate group of pathogenic bacteria will facilitate understanding of this zoonotic organism.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: P. W. O'Toole
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Andor, A., Trulzsch, K., Essler, M., Roggenkamp, A., Wiedemann, A., Heesemann, J. & Aepfelbacher, M. (2001). YopE of Yersinia, a GAP for Rho GTPases, selectively modulates Rac-dependent actin structures in endothelial cells. Cell Microbiol 3, 301–310.[CrossRef][Medline]
Bengoechea, J. A., Brandenburg, K., Arraiza, M. D., Seydel, U., Skurnik, M. & Moriyon, I. (2003). Pathogenic Yersinia enterocolitica strains increase the outer membrane permeability in response to environmental stimuli by modulating lipopolysaccharide fluidity and lipid A structure. Infect Immun 71, 2014–2021.
Bengoechea, J. A., Najdenski, H. & Skurnik, M. (2004). Lipopolysaccharide O antigen status of Yersinia enterocolitica O : 8 is essential for virulence and absence of O antigen affects the expression of other Yersinia virulence factors. Mol Microbiol 52, 451–469.[CrossRef][Medline]
Bleves, S., Marenne, M. N., Detry, G. & Cornelis, G. R. (2002). Up-regulation of the Yersinia enterocolitica yop regulon by deletion of the flagellum master operon flhDC. J Bacteriol 184, 3214–3223.
Boland, A. & Cornelis, G. R. (1998). Role of YopP in suppression of tumor necrosis factor alpha release by macrophages during Yersinia infection. Infect Immun 66, 1878–1884.
Bottone, E. J. (1999). Yersinia enterocolitica: overview and epidemiologic correlates. Microbes Infect 1, 323–333.[CrossRef][Medline]
Carlos, I. Z., Monnazzi, L. G. S., Falcao, D. P. & Machado de Medeiros, B. M. (2004). TNF-, H2O2 and NO response of peritoneal macrophages to Yersinia enterocolitica O : 3 derivatives. Microbes Infect 6, 207–212.[CrossRef][Medline]
Chang, A. C. & Cohen, S. N. (1978). Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134, 1141–1156.
Cornelis, G. R. & Wolf-Watz, H. (1997). The Yersinia Yop virulon: a bacterial system for subverting eukaryotic cells. Mol Microbiol 23, 861–867.[CrossRef][Medline]
Denecker, G., Totemeyer, S., Mota, L. J., Troisfontaines, P., Lambermont, I., Youta, C., Stainier, I., Ackermann, M. & Cornelis, G. R. (2002). Effect of low- and high-virulence Yersinia enterocolitica strains on the inflammatory response of human umbilical vein endothelial cells. Infect Immun 70, 3510–3520.
Donnelly, M. A. & Steiner, T. S. (2002). Two nonadjacent regions in enteroaggregative Escherichia coli flagellin are required for activation of toll-like receptor 5. J Biol Chem 277, 40456–40461.
El Tahir, Y., Kuusela, P. & Skurnik, M. (2000). Functional mapping of the Yersinia enterocolitica adhesin YadA. Identification of eight NSVAIG-S motifs in the amino-terminal half of the protein involved in collagen binding. Mol Microbiol 37, 192–206.[CrossRef][Medline]
Erfurth, S. E., Grobner, S., Kramer, U., Gunst, D. S., Soldanova, I., Schaller, M., Autenrieth, I. B. & Borgmann, S. (2004). Yersinia enterocolitica induces apoptosis and inhibits surface molecule expression and cytokine production in murine dendritic cells. Infect Immun 72, 7045–7054.
Fearnley, C., On, S. L., Kokotovich, B., Manning, G., Cheasty, T. & Newell, D. G. (2005). Application of fluorescent amplified fragment length polymorphism to the comparison of human and animal Yersinia enterocolitica. Appl Environ Microbiol 71, 4951–4959.
Finlay, B. B. & Falkow, S. (1988). Comparison of the invasion strategies used by Salmonella cholerae Suis, Shigella flexneri and Yersinia enterocolitica to enter cultured animal cells: endosome acidification is not required for bacterial invasion or intracellular replication. Biochimie 70, 1089–1099.[Medline]
Giron, J. A., Torres, A. G., Freer, E. & Kaper, J. B. (2002). The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells. Mol Microbiol 44, 361–379.[CrossRef][Medline]
Grant, T., Bennett-Wood, V. & Robins-Browne, R. M. (1998). Identification of virulence-associated characteristics in clinical isolates of Yersinia enterocolitica lacking classical virulence markers. Infect Immun 66, 1113–1120.
Grant, T., Bennett-Wood, V. & Robins-Browne, R. M. (1999). Characterization of the interaction between Yersinia enterocolitica biotype 1A and phagocytes and epithelial cells in vitro. Infect Immun 67, 4367–4375.
Grosdent, N., Maridonneau-Parini, I., Sory, M. P. & Cornelis, G. R. (2002). Role of Yops and adhesins in resistance of Yersinia enterocolitica to phagocytosis. Infect Immun 70, 4165–4176.
Howard, S. L., Gaunt, M. W., Hinds, J., Witney, A. A., Stabler, R. & Wren, B. W. (2006). Application of comparative phylogenomics to study the evolution of Yersinia enterocolitica and to identify genetic differences relating to pathogenicity. J Bacteriol 188, 3645–3653.
Iriarte, M., Stainier, I., Mikulskis, A. V. & Cornelis, G. R. (1995). The flia gene encoding Sigma(28) in Yersinia enterocolitica. J Bacteriol 177, 2299–2304.
Kampik, D., Schulte, R. & Autenrieth, I. B. (2000). Yersinia enterocolitica invasin protein triggers differential production of interleukin-1, monocyte chemoattractant protein 1, granulocyte-macrophage colony-stimulating factor, and tumour necrosis factor alpha in epithelial cells: implications for understanding the early cytokine network in Yersinia infections. Infect Immun 68, 2484–2492.
Kaniga, K., Delor, I. & Cornelis, G. R. (1991). A wide-host-range suicide vector for improving reverse genetics in Gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene 109, 137–141.[CrossRef][Medline]
Kapatral, V. & Minnich, S. A. (1995). Co-ordinate, temperature-sensitive regulation of the three Yersinia enterocolitica flagellin genes. Mol Microbiol 17, 49–56.[Medline]
La Ragione, R. M., Cooley, W. A., Velge, P., Jepson, M. A. & Woodward, M. J. (2003). Membrane ruffling and invasion of human and avian cell lines is reduced for aflagellate mutants of Salmonella enterica serotype Enteriditis. Int J Med Microbiol 293, 261–272.[CrossRef][Medline]
McNally, A., Cheasty, T., Fearnley, C., Dalziel, R. W., Paiba, G., Manning, G. & Newell, D. G. (2004). Comparison of the biotypes of Yersinia enterocolitica isolated from pigs, cattle and sheep at slaughter and from humans with yersiniosis in Great Britain during 1999–2000. Lett Appl Microbiol 39, 103–108.[CrossRef][Medline]
McNally, A., Dalton, T., La Ragione, R. M., Stapleton, K., Manning, G. & Newell, D. G. (2006). Yersinia enterocolitica isolates of differing biotypes from humans and animals are adherent, invasive and persist in macrophages, but differ in cytokine secretion profiles in vitro. J Med Microbiol 55, 1725–1734.
Miller, V. L. & Falkow, S. (1988). Evidence for two genetic loci in Yersinia enterocolitica that can promote invasion of epithelial cells. Infect Immun 56, 1242–1248.
Morris, J. G., Prado, V., Ferreccio, C., Robins-Browne, R. M., Bordun, A., Cayazzo, M., Kay, B. A. & Levine, M. M. (1991). Yersinia enterocolitica isolated from two cohorts of young children in Santiago, Chile: incidence of and lack of correlation between illness and propsed virulence factors. J Clin Microbiol 29, 2784–2788.
Parke, D. (1990). Construction of mobilizable vectors derived from plasmids RP4, pUC18 and pUC19. Gene 93, 135–137.[CrossRef][Medline]
Pepe, J. C. & Miller, V. L. (1993). Yersinia enterocolitica invasin: a primary role in the initiation of infection. Proc Natl Acad Sci U S A 90, 6473–6477.
Portnoy, D. A., Moseley, S. L. & Falkow, S. (1981). Characterization of plasmids and plasmid-associated determinants of Yersinia enterocolitica pathogenesis. Infect Immun 31, 775–782.
Rebeil, R., Ernst, R. K., Gowen, B. B., Miller, S. I. & Hinnebusch, B. J. (2004). Variation in lipid A structure in the pathogenic yersiniae. Mol Microbiol 52, 1363–1373.[CrossRef][Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schmid, Y., Grassl, G. A., Buhler, O. T., Skurnik, M., Autenrieth, I. B. & Bohn, E. (2004). Yersinia enterocolitica adhesin A induces production of interleukin-8 in epithelial cells. Infect Immun 72, 6780–6789.
Schmitt, C. K., Darnell, S. C., Tesh, V. L., Stocker, B. A. & O'Brien, A. D. (1994). Mutation of flgM attenuates virulence of Salmonella Typhimurium, and mutation of fliA represses the attenuated phenotype. J Bacteriol 176, 368–377.
Schmitt, C. K., Ikeda, J. S., Darnell, S. C., Watson, P. R., Bispham, J., Wallis, T. S., Weinstein, D. L., Metcalf, E. S. & O'Brien, A. D. (2001). Absence of all components of the flagellar export and synthesis machinery differentially alters virulence of Salmonella enterica serovar Typhimurium in models of typhoid fever, survival in macrophages, tissue culture invasiveness, and calf enterocolitis. Infect Immun 69, 5619–5625.
Scholz, O., Thiel, A., Hillen, W. & Niederweis, M. (2000). Quantitative analysis of gene expression with an improved green fluorescent protein. Eur J Biochem 267, 1565–1570.[Medline]
Tennant, S. M., Grant, T. H. & Robins-Browne, R. M. (2003). Pathogenicity of Yersinia enterocolitica biotype 1A. FEMS Immunol Med Microbiol 38, 127–137.[CrossRef][Medline]
Woodcock, D. M., Crowther, P. J., Doherty, J., Jefferson, S., DeCruz, E., Noyer-Weidner, M., Smith, S. S., Michael, M. Z. & Graham, M. W. (1989). Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res 17, 3469–3478.
Wyant, T. L., Tanner, M. K. & Sztein, M. B. (1999). Salmonella typhi flagella are potent inducers of proinflammatory cytokine secretion by human monocytes. Infect Immun 67, 3619–3624.
Young, G. M., Schmiel, D. H. & Miller, V. L. (1999a). A new pathway for the secretion of virulence factors by bacteria: the flagellar export apparatus functions as a protein-secretion system. Proc Natl Acad Sci U S A 96, 6456–6461.
Young, G. M., Smith, M. J., Minnich, S. A. & Miller, V. L. (1999b). The Yersinia enterocolitica motility master regulatory operon, flhDC, is required for flagellin production, swimming motility, and swarming motility. J Bacteriol 181, 2823–2833.
Young, G. M., Badger, J. L. & Miller, V. L. (2000). Motility is required to initiate host cell invasion by Yersinia enterocolitica. Infect Immun 68, 4323–4326.
Zhou, X., Giron, J. A., Torres, A. G., Crawford, J. A., Negrete, E., Vogel, S. N. & Kaper, J. B. (2003). Flagellin of enteropathogenic Escherichia coli stimulates interleukin-8 production in T84 cells. Infect Immun 71, 2120–2129.
Zumbihl, R., Aepfelbacher, M., Andor, A., Jacobi, C. A., Ruckdeschel, H., Rouot, B. & Heesemann, J. (1999). The cytotoxin YopT of Yersinia enterocolitica induces modification and cellular redistribution of the small GTP-binding protein RhoA. J Biol Chem 274, 29289–29293.
Received 21 August 2006;
revised 18 December 2006;
accepted 16 January 2007.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
