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J Med Microbiol 55 (2006), 1725-1734; DOI: 10.1099/jmm.0.46726-0
© 2006 Society for General Microbiology
ISSN 1473-5644

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

Alan McNally1,{dagger}, Tracey Dalton2, Roberto M. La Ragione1, Kenneth Stapleton1, Georgina Manning1,{dagger} and Diane G. Newell1

1 Veterinary Laboratories Agency, Woodham Lane, New Haw, Surrey KT15 3NB, UK

2 Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E, UK

Correspondence
Diane G. Newell
d.newell{at}vla.defra.gsi.gov.uk

Received 16 May 2006
Accepted 12 August 2006

Previous epidemiological studies have demonstrated a potential link between the serotypes of Yersinia enterocolitica recovered from cattle, sheep and pigs and those isolated from human disease cases. Further studies utilizing amplified fragment length polymorphisms have shown a relationship at the genetic level between strains of biotypes 3 and 4 from humans and livestock, and also suggested that some biotype 1A isolates, classically defined as non-pathogenic, are closely related to biotype 3 and 4 isolates. This study sought to understand further the pathogenic potential of Y. enterocolitica isolates from livestock in Great Britain. A range of surrogate in vitro models, such as invasion of epithelial tissue cultures, survival in cultured macrophages and cytokine secretion response, was employed to assess the pathogenicity of 88 strains. The results suggested that all isolates examined were capable of adhering to and invading epithelial cells and of surviving within macrophages. However, the inflammatory response of the infected macrophages differed with the infecting Y. enterocolitica subtype, with the response to pathogenic biotype 3 and 4 isolates different to that observed with biotype 1A isolates, and with the biotype 3 O : 5,27 isolates recovered exclusively from animals. Infections of porcine tissue also suggested the possibility of host-tissue tropism within Y. enterocolitica subtypes.

Abbreviations: AFLP, amplified fragment length polymorphism; GFP, green fluorescent protein; IL, interleukin; IVOC, in vitro organ culture; TNF, tumour necrosis factor.

{dagger}Present address: School of Biomedical and Natural Science, Nottingham Trent University, Clifton Lane, Nottingham, UK. Back


   INTRODUCTION
TOP
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Yersinia enterocolitica is a human pathogen that can cause a range of diseases from mild diarrhoea to the severe complication of mesenteric lymphadenitis (Bottone, 1999). Disease is generally self-limiting, although severe disease can occur, especially in immunocompromised individuals. Y. enterocolitica infection involves invasion of the gastrointestinal epithelium, primarily at the follicle-associated epithelium (Grassl et al., 2003; Hamzaoui et al., 2004). The organisms then escape from the host cell via the basolateral membrane, allowing the bacteria to propagate in the lamina propria, where the production of a variety of virulence factors aids subversion of the host immune response (Cornelis & Wolf-Watz, 1997).

The virulence factors and mechanisms of pathogenesis of Y. enterocolitica are well documented, primarily in several well-studied biotype 1b O : 8 strains. The organism has been shown to adhere to and invade a wide selection of eukaryotic cells in vitro (Finlay & Falkow, 1988; Miller & Falkow, 1988), with the genes encoding the adhesins and invasin protein identified and characterized (Grassl et al., 2003; Pepe & Miller, 1993). The interaction of Y. enterocolitica with macrophages in vitro has also been studied in detail. However, most of the emphasis to date has been on how the organism avoids uptake by macrophages (Grosdent et al., 2002), rather than how it survives within macrophages (Grant et al., 1999; Yamamoto et al., 1996). The genetic basis of resistance to macrophages has been well characterized, especially the role of the pYV virulence plasmid and the encoded Yop proteins (Grosdent et al., 2002; Neyt & Cornelis, 1999; Tardy et al., 1999; Trülzsch et al., 2004). In particular, the ability of Y. enterocolitica to modulate the host immune response has been highlighted, with various Yop proteins and other virulence factors shown to play a role in down-regulating the secretion of various pro-inflammatory cytokines, such as tumour necrosis factor (TNF)-{alpha} (Boland & Cornelis, 1998; Carlos et al., 2004; Ruckdeschel et al., 1997), interleukin (IL)-6 (Denecker et al., 2002; Dube et al., 2004), and IL-8 (Schulte & Autenrieth, 1998). Increased synthesis of the anti-inflammatory cytokine IL-10 has also been demonstrated in vitro (Sing et al., 2002a).

Previous studies have highlighted Y. enterocolitica as ubiquitous in livestock. The organism has been isolated from 26.1 % of pigs, 10.7 % of sheep and 6.3 % of cattle at slaughter in Great Britain (McNally et al., 2004). Of those isolates recovered, 58 % belonged to biotype 1A, which is classically defined as non-pathogenic due to its lack of lethality in the mouse infection model (Tennant et al., 2003). Biotype 1A isolates also lack the majority of the defined virulence determinants of Y. enterocolitica found in the presumptively pathogenic biotypes 1b and 2–5 (Cornelis & Wolf-Watz, 1997; Tennant et al., 2003). Despite this, recent investigations using in vitro and in vivo models suggest that biotype 1A isolates may still be capable of causing disease in humans (Grant et al., 1998, 1999; Morris et al., 1991; Tennant et al., 2003). Such a hypothesis is consistent with the evidence that 53 % of Y. enterocolitica strains isolated from humans in Great Britain between 1999 and 2000 were biotype 1A (McNally et al., 2004).

Amplified fragment length polymorphisms (AFLPs) have been used to genotype selected Y. enterocolitica isolates from humans and animals (Fearnley et al., 2005). Two main clusters have been identified, one comprising solely biotype 1A isolates and the other comprising primarily, but not exclusively, biotype 2–5 isolates. Within the two main clusters, isolates were further subdivided according to serotype. There was no obvious relationship with source, with the exception of biotype 4 O : 3 human strains, which formed a distinct group from biotype 4 O : 3 isolates from pigs.

This study has elaborated on the relationship between human and animal Y. enterocolitica strains by means of phenotypic comparisons. A variety of virulence mechanisms was examined in selected strains of human and animal origin, using defined in vitro assays. These included association with and invasion of eukaryotic cells (Miller & Falkow, 1988), resistance to intra-macrophage killing (Yamamoto et al., 1996, 1997) and modulation of host-cell cytokine secretion (Denecker et al., 2002; Dube et al., 2004; Meyer-Bahlburg et al., 2004; Schulte & Autenrieth, 1998). In addition, some preliminary work was undertaken on the interaction with animal intestinal tissue through in vitro organ culture (IVOC) (Fitzhenry et al., 2002a, b; Phillips et al., 2000).


   METHODS
TOP
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial strains and growth conditions. The sources and bioserotyping of strains has been described previously (McNally et al., 2004). Strains were selected to represent a cross-section of biotypes and serotypes present in humans and animals in Great Britain (Table 1Go), with all biotype 3 and 4 strains confirmed as pYV-positive by growth on CMO agar and microarray comparison in a linked study (Howard et al., 2006). The Y. enterocolitica sequenced strain 8081 (biotype 1B) and Escherichia coli DH5-{alpha} were used as controls in all phenotypic assays. All Y. enterocolitica strains were grown routinely at 28 °C for 18 h on 10 % (v/v) sheep blood agar or in Luria–Bertani (LB) broth with shaking, unless otherwise stated. E. coli was grown at 37 °C for 18 h on 10 % sheep blood agar or in LB broth with shaking, unless otherwise stated.


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  		Table 1. Representation of the biotype, serotype and source of the 88 strains selected for characterization by pathogenesis potential phenotypes

 
Cell cultures. HEp-2 human epithelial cells were cultured in minimal essential medium (MEM; Life Technologies) supplemented with 10 % (v/v) heat-inactivated fetal calf serum (Life Technologies), 1 % (v/v) L-glutamine and 1 % (v/v) 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 a concentration of approximately 2x105 cells ml–1 (as determined by cell cytometer counts). U937 human monocytic cells were maintained in RPMI 1640 (Sigma) supplemented as above. Seventy-two hours prior to infection, cells were seeded into a 24-well tissue culture plate (Nunc) at a concentration of approximately 2x105 cells ml–1 and treated with 10 nM phorbol myristate acetate (Sigma) upon seeding to trigger differentiation into active macrophages (Song et al., 2003).

Association and invasion of epithelial cells by Y. enterocolitica isolates. Bacterial association with and invasion of HEp-2 cells was assayed as described previously (Dibb-Fuller et al., 1999; Finlay & Falkow, 1988; Miller & Falkow, 1988). Bacteria were grown overnight in LB broth, harvested by centrifugation and resuspended in MEM plus 1 % amino acids and 1 % L-glutamine, to give approximately 2x107 c.f.u. ml–1 (as determined by plate counts). Bacterial suspension (1 ml) was added in triplicate to wells of a pre-seeded 24-well plate (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, followed by the addition of MEM plus 1 % amino acids, 1 % L-glutamine and 100 µg gentamicin ml–1. The cells were further incubated for 2 h. Cells were then washed twice in PBS and lysed with a 1 % Triton X-100 solution for 5 min. Dilutions of the lysed cells were plated onto LB agar in triplicate and incubated overnight. Colonies were counted to give the total number of invasive bacteria per millilitre. In a parallel series of wells, cells were lysed after the initial 3 h infection period to ascertain the total number of associated bacteria per millilitre.

Persistence of Y. enterocolitica isolates within U937 cells. 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, before a further incubation for 5 h. Cells were then washed twice in PBS and lysed by addition of a 1 % Triton X-100 solution for 10 min. Dilutions of the lysed cells were plated onto LBG agar in triplicate and incubated at 28 °C overnight. 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 at 1 h post-challenge.

Trypan blue exclusion assay. The integrity of infected U937 cells was examined by their ability to exclude trypan blue dye. Cells were exposed to bacteria as above and then overlaid with 0.5 % trypan blue for 5 min. At least 1000 cells were examined per well and the proportion of cells with dye uptake was recorded. Experiments were repeated in triplicate.

Cytokine production in infected U937 cells. To examine the effect of different Y. enterocolitica isolates on cytokine secretion, differentiated U937 cells were challenged as described above and incubated for 3 h. Supernatants were collected and centrifuged at 13 000 r.p.m. for 5 min in a bench-top microcentrifuge, and 200 µl of the supernatant was collected and stored at –20 °C until analysed. IL-6, IL-8, IL-10 and TNF-{alpha} levels were assessed using Quantikine sandwich ELISA kits (R&D systems) according to the manufacturer's instructions, with quantities presented as levels of cytokine normalized against levels present in cells unstimulated by bacteria.

Construction of a green fluorescent protein (GFP) : : gyrA fusion in Y. enterocolitica. In order to visualize the interaction of Y. enterocolitica strains with intestinal tissue, fusions constitutively expressing GFP were constructed in seven Y. enterocolitica isolates, which were selected to represent each biotype from each host. The list of strains, plasmids and primers used is shown in Table 2Go. Y. enterocolitica isolates were made electrocompetent by repeated washing of a mid-exponential broth culture in ice-cold 10 % glycerol solution. Cells were then stored at –80 °C until use (Sambrook et al., 1989). All molecular biology techniques were performed according to standard protocols (Sambrook et al., 1989). 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, treated with calf intestinal phosphatase 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 treated with phosphatase, 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 {Omega}.


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  		Table 2. List of specific strains used in this study for IVOC, and the plasmids and primers used in molecular biology techniques

 
IVOC. Two 3-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 removed and 2 cm2 tissue 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. Bacterial overnight cultures were harvested by centrifugation and resuspended in PBS to give approximately 108 c.f.u. ml–1. 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 fluorescent microscopy. Fixed tissue samples were snap frozen in isopentane pre-cooled in liquid nitrogen to between –40 and –60 °C. Tissue sections were 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 DAPI mounting medium and stored in the dark at room temperature. Confocal fluorescent microscopy was performed on a Leica microscope with an excitation wavelength of 488 nm and an emission filter of 507 nm.

Statistical analysis. For each of the four sets of cytokine data, an analysis of variance was done to compare the mean cytokine responses to the strain types, with the results averaged into groups of biotype 3 O : 5,27, biotype 1A, biotype 4 O : 3 and biotype 3 O : 9. The control results were all zero due to normalization and hence omitted, as were all other zero or missing results. As the variance of the data tended to increase with the mean, the data were transformed to their log10 values for the analysis. When the overall F test for the effects of strains was significant (P<0.05), all pairs of strain were compared using Tukey's HSD test based on the Studentized range.


   RESULTS
TOP
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Adherence to and invasion of epithelial cells in vitro by Y. enterocolitica of various biotypes and sources

Association and invasion assays on cultured HEp-2 cells were performed on all 88 isolates to ascertain the ability of Y. enterocolitica of different biotypes and from various hosts to adhere to and invade epithelial cells in vitro. HEp-2 cells were chosen over intestinal cell lines as Y. enterocolitica has previously been shown to invade these at higher levels than INT-407 and Caco-2 cells (Finlay & Falkow, 1988). The arithmetic mean association and invasion of each source/biotype combination is presented in Fig. 1Go. All of the Y. enterocolitica biotypes were capable of adhering to epithelial cells in high numbers, with approximately 10 % of the organisms added associating with the HEp-2 cells (~106 c.f.u. ml–1 from 107 c.f.u. ml–1 added for infection). The data confirmed that isolates from biotypes 2–4 are more invasive than biotype 1A strains (Grant et al., 1999), with the pathogenic biotype isolates invading at 104–105 c.f.u. ml–1, compared with 102–103 c.f.u. ml–1 for biotype 1A strains. Nevertheless, it was clear that biotype 1A isolates were invasive. Averaged association with and invasion of epithelial cells of biotypes 3 O : 5,27, 3 O : 9 and 4 O : 3 showed that all bio/serotype strains invaded at statistically similar levels. Invasiveness appeared to be independent of the source of the isolate, although anomalies were observed; in particular, the sheep biotype 4 O : 3 isolates displayed reduced invasion compared with other biotype 3 and 4 strain types.


Figure 1
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  		Fig. 1. Mean levels of association and invasion of cultured HEp-2 epithelial cells by biotype 1A (all serotypes), biotype 4 O : 3, biotype 3 O : 9, biotype 3 O : 5,27 and ‘other’ bio/serotype (BT1b; BT2; BT3 O : 5, O : 15, O : ?; BT4 O : 5,27) isolates of human, porcine, ovine and bovine origin. Results are displayed as c.f.u. bacteria ml–1 recovered from an infected well and are means±SEM of individual duplicate experiments performed in triplicate. White portions of bars, mean association; black portions of bars, mean invasion.

 
Persistence of Y. enterocolitica isolates within U937 macrophages

The ability of isolates of different biotypes and sources to survive within macrophages was assessed in differentiated U937 cells. In order to determine the optimal times for the experiment, a time-course assay was performed using a single biotype 1A isolate and the sequenced 8081 pathogenic strain (Fig. 2aGo). The growth kinetics mirrored those previously reported for intra-macrophage survival of various Y. enterocolitica strains (Grant et al., 1999; Yamamoto et al., 1996, 1997) and those for other bio/serotypes tested (data not shown), suggesting that these phenotypes are representative of the phenotypes likely to be observed for test strains. The results suggested that, at 5 h post-challenge, the bacterial numbers began to increase. This was selected as the time point for examination rather than, for example, the time at which Yop expression was maximal, as it allowed a fairer comparison between pYV+ and pYV– strains based solely on growth kinetics within the macrophages. The results obtained for the persistence of Y. enterocolitica isolates within U937 cells (Fig. 2bGo) suggested that Y. enterocolitica is capable of survival within macrophages irrespective of biotype or source. The mean number of organisms recovered after 5 h was similar to the number recovered after the initial 1 h infection period among biotypes 1A, 3 and 4; however, there was some variation within individual strain types, with sheep and pig biotype 1A isolates persisting to higher levels than the other strain types tested. The number of biotype 4 O : 3 isolates recovered after 5 h was slightly lower than that for the other strain types; however, trypan blue exclusion assays indicated that this was due to a concomitant increase in the number of non-viable macrophages in infections carried out with biotype 4 O : 3 isolates.


Figure 2
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  		Fig. 2. Persistence of YE 53/03 (BT1A isolate) and Y. enterocolitica 8081 within cultured U937 human macrophages over a 24 h time course (a), and the mean levels of persistence of biotype 1A, 4 O : 3, 3 O : 9 and 3 O : 5,27 isolates of human, porcine, ovine and bovine origin (b). Results are displayed as c.f.u. bacteria ml–1 recovered from an infected well after 5 h (T5) compared with bacteria recovered after an initial 1 h infection (T0) and are means±SEM of duplicative experiments performed in triplicate.

 
U937 responses to Y. enterocolitica

The pYV virulence plasmid, encoding the Yop proteins known to modulate cytokine secretion during Y. enterocolitica infection, is absent from biotype 1A strains. In order to assess the secretion of cytokines following infection with different strain types, supernatants from infected U937 cells were harvested and assayed using commercial ELISA kits (Fig. 3Go). Infections were carried out with four selected isolates from each pathotype/host combination, namely: YE111/02, YE149/02, YE203/02 and YE238/02 (animal BT3 O : 5,27); YE201/02, YE204/02, YE220/02 and YE227/02 (pig BT4 O : 3); YE13/03, YE35/03, YE38/03 and YE53/03 (human BT1A); YE214/02, YE218/02, YE236/02 and YE237/02 (pig BT3 O : 9); YE03/03, YE07/03, YE08/03 and YE12/03 (human BT4 O : 3); YE02/02, YE13/02, YE208/02 and YE221/02 (animal BT1A); and YE39/03, YE56/03, YE58/03 and YE74/03 (human BT3 O : 9).


Figure 3
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  		Fig. 3. Mean levels of secretion of IL-6 (a), IL-8 (b), IL-10 (c) and TNF-{alpha} (d) by U937 human macrophages after infection with biotype 1A, 4 O : 3, 3 O : 9 and 3 O : 5,27 isolates of human, porcine, ovine and bovine origin. Data are presented as fold difference in the level of cytokine production relative to the unstimulated control. Cells were infected with four randomly selected isolates from each biotype/source combination. Duplicative experiments were performed in duplicate and results are shown as means±SEM.

 
Modulation of IL-6 secretion. Secretion of IL-6 was increased with biotype 3 O : 5,27 isolates compared with the other strain types tested (P=0.061), with levels of IL-6 secretion slightly elevated for biotype 1A strains (P=0.2) (Fig. 3aGo). The biotype 4 O : 3 and 3 O : 9 strains exhibited decreased levels of IL-6 secretion, particularly the pig biotype 4 O : 3 and human biotype 3 O : 9.

Modulation of IL-8 secretion. Secretion of IL-8 by infected U937 cells was also slightly, although significantly, increased with biotype 3 O : 5,27 isolates compared with the other strain types (P=0.045) (Fig. 3bGo). IL-8 secretion with biotype 1A strains of both human and animal origin was also elevated compared with biotype 3 O : 9 and 4 O : 3 strains (P=0.05), although secretion levels were slightly higher in the environmental biotype 1A isolates than in the human strains.

Modulation of IL-10 secretion. Levels of secreted IL-10 were similar for all of the isolates tested and did not differ significantly from the levels secreted by uninfected cells, although they were greatly reduced compared with an LPS control (Fig. 3cGo).

Modulation of TNF-{alpha} secretion. Infected U937 cells secreted large amounts of TNF-{alpha} upon infection with human or animal biotype 1A strains. Secretion induced by animal biotype 1A strains was on average 3.9-fold greater than that observed upon infection with pathogenic strains (P=0.022). Secretion induced by human biotype 1A isolates was approximately a further twofold greater than that observed after infection with animal biotype 1A strains (P=0.061).

Colonization of pig terminal ileum by IVOC

The seven isolates selected for IVOC analysis were successfully transformed with the plasmid containing the constitutive gfp : : gyrA fusion. Fluorescence from the fusion was confirmed using a confocal fluorescent microscope and compared with an untransformed strain as a control (data not shown). All seven isolates successfully colonized porcine terminal ileum and spiral colon tissues during IVOC (Fig. 4Go). There was little evidence of bacteria closely adhered to intestinal tissue or within tissues, with the majority of organisms appearing to be more closely associated with mucus and cellular debris. The strains of animal origin tested associated with the porcine tissue in greater numbers than the strains of human origin (Table 3Go) in both the terminal ileum and spiral colon. All of the strains tested displayed a preference towards colonization of the follicle-associated, epithelium-rich spiral colon tissue compared with the terminal ileum.


Figure 4
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  		Fig. 4. Colonization of porcine intestinal tissue by GFP-tagged strains of Y. enterocolitica in IVOC experiments. The panels show spiral colon tissue colonized by GFP-tagged strains YE149/02 (a), YE201/02 (b), YE208/02 (c), YE237/02 (d), YE12/03 (e), YE53/03 (f) and YE56/03 (g). Images were captured using a Leica confocal fluorescent microscope and a x63 objective and were then magnified a further threefold using the Leica confocal microscope capture software. Large arrows indicate cell nuclei counterstained with DAPI; small arrows indicate GFP-tagged bacteria.

 

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  		Table 3. Enumeration of bacteria colonizing IVOC porcine intestinal tissue

The results represent the mean number of bacteria enumerated from five randomly selected fields of view. Strains were scored according to the number of organisms per field (+, 0–20; ++, 20–100; +++, 100–500; ++++, >500).

 

   DISCUSSION
TOP
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The aim of this study was to investigate the human pathogenic potential of Y. enterocolitica isolates of different biotypes and from various veterinary sources by comparing their phenotypes with isolates from human disease cases. A total of 88 isolates was selected for characterization to represent the various biotypes and serotypes present in pigs, cattle, sheep and humans collected contemporaneously within England and Wales (McNally et al., 2004). No statistical differences between biotypes could be detected in the ability of isolates to adhere to and invade cultured epithelial cells or to persist in macrophages other than those already reported between biotype 1A strains and pathogenic biotypes. However, differences were observed in the response of cultured human macrophages to infection with some isolates. Biotype 3 O : 5,27 infection resulted in slightly increased levels of IL-6 (P=0.061) and significantly increased (P=0.045) IL-8 secretion when compared with other biotypes. Infection with biotype 1A isolates resulted in greatly increased levels of TNF-{alpha} secretion (P=0.022). These results suggested that, whilst all Y. enterocolitica biotypes may possess pathogenic potential, the duration and severity of infection or disease pathology may differ due to differences in host response to some biotypes.

The levels of invasion observed for the pathogenic biotype isolates were higher than those observed for the biotype 1A strains, which is consistent with previous data (Grant et al., 1998, 1999). There was no significant difference in the ability of strain types of bovine, porcine, ovine or human origin to invade HEp-2 cells.

All isolates were capable of persisting in macrophages. Previous research has focused on the ability of Y. enterocolitica to avoid host macrophage ingestion via the Yop type III secreted effector proteins (Cornelis & Wolf-Watz, 1997). Increasingly, the importance of studying Y. enterocolitica inside macrophages has been raised (Pujol & Bliska, 2005) and in this study it was decided to examine bacteria actively persisting within intact macrophages. Using U937 macrophage-like cells, no statistical differences in levels of intra-macrophage survival were found between biotypes or source. The levels of bacteria recovered were, on average, slightly lower for the pathogenic biotype 4 O : 3 and 3 O : 9 strains tested than for the biotype 1A isolates, but this phenomenon was explained by the increased number of non-viable macrophages, as determined by trypan blue staining, which was almost certainly due to the production of Yop effector proteins upon contact with the U937 cells (Cornelis & Wolf-Watz, 1997). The levels of persistence for the biotype 1A isolates were surprising and differed slightly from those published previously (Grant et al., 1999). However, our study utilized human macrophage-like cell lines, whilst previous work has focused on the mouse RAW macrophage cell line. The relevance of data with RAW cells is questionable, as biotype 1A isolates are non-pathogenic in a mouse infection model (Bottone, 1997).

IVOC has been developed as a powerful tool for investigating host–pathogen relationships (Fitzhenry et al., 2002a, b; Phillips et al., 2000). In order to investigate differences in association with whole intestinal tissue, one isolate from each biotype/source combination was selected and transformed with a plasmid containing a constitutive gyrA : : gfp+ fusion. These strains were then used to conduct IVOC studies on porcine tissue. The GFP+ derivative was chosen, as it has been shown to be a more stable version of GFP and has been used successfully in vivo (Hautefort et al., 2003; Scholz et al., 2000). The porcine IVOC study showed that all of the strains were capable of colonizing pig intestinal tissue in vitro. The animal strains appeared to colonize porcine tissues at higher numbers than the human isolates, suggesting that there may be differences in tissue tropism between strains. This was in contradiction to the association observed in the in vitro cell line-based assays; however, there were numerous other factors present in the IVOC assay, including the presence of a mucous layer, that may influence the way that the organism interacts with the surface. The biotype 1A strains were equally as capable of colonizing the IVOC tissue as the other biotypes tested, although there was a slight reduction in the numbers of biotype 1A strains present in intestinal tissues compared with the pathogenic biotypes. The possibility of tissue tropism in Y. enterocolitica isolates requires further work, in particular the interaction of Y. enterocolitica biotypes with human intestinal tissue.

The host-cell response to infection with different Y. enterocolitica biotypes was investigated. This was undertaken using commercial sandwich ELISA kits to measure the levels of secretion of several cytokines by infected U937 cells. This method has been used extensively in previous Y. enterocolitica cytokine studies (Denecker et al., 2002; Dube et al., 2004; Kampik et al., 2000). The pro-inflammatory cytokine TNF-{alpha} was selected for analysis, as modulation of its secretion by Y. enterocolitica Yop effectors has been reported (Boland & Cornelis, 1998; Carlos et al., 2004; Ruckdeschel et al., 1997). Similarly, secretion of IL-6 (Denecker et al., 2002; Dube et al., 2004) and IL-8 (Denecker et al., 2002; Kampik et al., 2000; Schmid et al., 2004; Schulte & Autenrieth, 1998; Schulte et al., 1996, 2000) was examined, as both have been shown to play a role in Y. enterocolitica infection. Lastly, the anti-inflammatory cytokine IL-10 was studied, as Y. entercolitica has been shown to increase production of IL-10 to avoid the immune response (Erfurth et al., 2004; Sing et al., 2002a, b, 2003).

The data showed that infection with biotype 3 O : 5,27 isolates, which are ubiquitous in animals in Great Britain but not in humans (McNally et al., 2004), resulted in increased secretion when compared with other biotypes of IL-6 and IL-8, two cytokines that play key roles in clearing Y. enterocolitica infection. It is therefore tempting to speculate that the absence of biotype 3 O : 5,27 strains in humans is a consequence of an elevated IL-6/IL-8-mediated immune response. Future work, including responses to infection in a variety of host tissue types, is currently being pursued to address this. The biotype 1A strains tested displayed IL-6 and IL-8 secretion to levels equivalent to those observed in biotype 4 O : 3 and 3 O : 9 strains and also displayed IL-10 secretion to similar levels. However, the biotype 1A isolates displayed large-scale secretion of TNF-{alpha} by human macrophages, with greatly elevated levels observed for both human and animal isolates. Indeed, infection with human biotype 1A isolates induced an approximately twofold increase in TNF-{alpha} secretion compared with animal-source biotype 1A isolates. It may therefore be hypothesized that infection of humans with biotype 1A strains leads to a different pathology than infection with biotype 3 O : 9 or 4 O : 3, supported by statistically similar levels of IL-6 and IL-8 secretion but massively increased TNF-{alpha} secretion. This would be consistent with macrophages being a preferred environment for biotype 1A organisms, with TNF-{alpha} release leading to migration of more macrophages to the site of infection. The relevance of these findings requires more in-depth analysis, including analysis of the active role played by the bacteria in the differences in host response.

In conclusion, this study has highlighted that all of the Y. enterocolitica isolates tested in this study from livestock had the ability to adhere to and invade cultured epithelial cells, as well as to persist in cultured human macrophages. However, Y. enterocolitica biotype 3 O : 5,27, which was isolated from animals and not humans, led to elevated IL-6 and IL-8 release by infected macrophages compared with other biotype 3 and 4 isolates. In contrast, Y. enterocolitica biotype 1A isolates showed no significant differences in IL-6 and IL-8 secretion compared with biotype 3 and 4 isolates, but infection of human macrophages led to a significant release of TNF-{alpha}. These findings indicate that the differences in biotype–disease association in human infection are a reflection of host responses to the various biotypes. The data suggest that the differences may reside in the ability of the organisms to interact with the host intestinal surface or to control the host immune response.


   ACKNOWLEDGEMENTS
 
This work was funded by the Department for Environment, Food and Rural Affairs (DEFRA), grant OZ0405. We would like to thank William Cooley for assistance with confocal fluorescent microscopy, Professor Michael Niederweis, University of Nurnberg, for kindly supplying pMN402 and Drs Alan Phillips and Stephanie Schuller for guidance with IVOC. Statistical analysis was performed by Robin Sayers.


   REFERENCES
TOP
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.[Abstract/Free Full Text]

Bottone, E. J. (1997). Yersinia enterocolitica: the charisma continues. Clin Microbiol Rev 10, 257–276.[Abstract]

Bottone, E. J. (1999). Yersinia enterocolitica: overview and epidemiologic correlates. Microbes Infect 1, 323–333.[CrossRef][Medline]

Carlos, I. Z., Monnazzi, L. G. S., Falcão, D. P. & Machado de Medeiros, B. M. (2004). TNF-{alpha}, H2O2 and NO response of peritoneal macrophages to Yersinia enterocolitica O : 3 derivatives. Microbes Infect 6, 207–212.[CrossRef][Medline]

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., Tötemeyer, 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.[Abstract/Free Full Text]

Dibb-Fuller, M. P., Allen-Vercoe, E., Thorns, C. J. & Woodward, M. J. (1999). Fimbriae and flagella-mediated association with and invasion of cultured epithelial cells by Salmonella Enteriditis. Microbiology 145, 1023–1031.[Abstract/Free Full Text]

Dube, P. H., Handley, S. A., Lewis, J. & Miller, V. L. (2004). Protective role of interleukin-6 during Yersinia enterocolitica infection is mediated through the modulation of inflammatory cytokines. Infect Immun 72, 3561–3570.[Abstract/Free Full Text]

Erfurth, S. E., Gröbner, S., Kramer, U., Gunst, D. S. J., 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.[Abstract/Free Full Text]

Fearnley, C., On, S. L. W., 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, 4960–4965.[Abstract/Free Full Text]

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]

Fitzhenry, R. J., Pickard, D. J., Hartland, E. L., Reece, S., Dougan, G., Phillips, A. D. & Frankel, G. (2002a). Intimin type influences the site of human intestinal mucosal colonisation by enterohaemorrhagic Escherichia coli O157 : H7. Gut 50, 180–185.[Abstract/Free Full Text]

Fitzhenry, R. J., Reece, S., Trabulsi, L. R., Heuschkel, R., Murch, S., Thomson, M., Frankel, G. & Phillips, A. D. (2002b). Tissue tropism of enteropathogenic Escherichia coli strains belonging to the O55 serogroup. Infect Immun 70, 4362–4368.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

Grassl, G. A., Bohn, E., Müller, Y., Bühler, O. T. & Autenrieth, I. B. (2003). Interaction of Yersinia enterocolitica with epithelial cells: invasin beyond invasion. Int J Med Microbiol 293, 41–54.[CrossRef][Medline]

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.[Abstract/Free Full Text]

Hamzaoui, N., Kernéis, S., Caliot, E. & Pringault, E. (2004). Expression and distribution of ß1 integrins in in vitro-induced M cells: implications for Yersinia adhesion to Peyer's patch epithelium. Cell Microbiol 6, 817–828.[CrossRef][Medline]

Hautefort, I., Proença, M. J. & Hinton, J. C. D. (2003). Single-Copy Green Fluorescent Protein gene fusions allow accurate measurement of Salmonella gene expression in vitro and during infection of mammalian cells. Appl Environ Microbiol 69, 7480–7491.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

Kampik, D., Schulte, R. & Autenrieth, I. B. (2000). Yersinia enterocolitica invasin protein triggers differential production of interleukin-1, interleukin-8, monocyte chemoattractant protein 1, granulocyte–macrophage colony-stimulating factor, and tumor necrosis factor alpha in epithelial cells: implications for understanding the early cytokine network in Yersinia infections. Infect Immun 68, 2484–2492.[Abstract/Free Full Text]

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]

Meyer-Bahlburg, A., Greil, S., Kruse, N., Marienfeld, R., Serfling, E. & Huppertz, H.-I. (2004). Yersinia enterocolitica leads to transient induction of TNF-{alpha} and activates NF-{kappa}B in synovial fibroblasts. Clin Exp Rheumatol 22, 278–284.[Medline]

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.[Abstract/Free Full Text]

Morris, J. G., Jr, Prado, V., Ferreccio, C., Robins-Browne, R. M., Bordun, A.-M., 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 proposed virulence factors. J Clin Microbiol 29, 2784–2788.[Abstract/Free Full Text]

Neyt, C. & Cornelis, G. R. (1999). Insertion of a Yop translocation pore into the macrophage plasma membrane by Yersinia enterocolitica: requirement for translocators YopB and YopD, but not LcrG. Mol Microbiol 33, 971–981.[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.[Abstract/Free Full Text]

Phillips, A. D., Navabpour, S., Hicks, S., Dougan, G., Wallis, T. & Frankel, G. (2000). Enterohaemorrhagic Escherichia coli O157 : H7 target Peyer's patches in humans and cause attaching/effacing lesions in both human and bovine intestine. Gut 47, 377–381.[Abstract/Free Full Text]

Pujol, C. & Bliska, J. B. (2005). Turning Yersinia pathogenesis outside in: subversion of macrophage function by intracellular yersiniae. Clin Immunol 114, 216–226.[CrossRef][Medline]

Ruckdeschel, K., Machold, J., Roggenkamp, A., Schubert, S., Pierre, J., Zumbihl, R., Liautard, J.-P., Heesemann, J. & Rouot, B. (1997). Yersinia enterocolitica promotes deactivation of macrophage mitogen-activated protein kinases extracellular signal-regulated kinase-1/2, p38, and c-Jun NH2-terminal kinase. Correlation with its inhibitory effect on tumor necrosis factor-{alpha} production. J Biol Chem 272, 15920–15927.[Abstract/Free Full Text]

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., Bühler, 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.[Abstract/Free Full Text]

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]

Schulte, R. & Autenrieth, I. B. (1998). Yersinia enterocolitica-induced interleukin-8 secretion by human intestinal epithelial cells depends on cell differentiation. Infect Immun 66, 1216–1224.[Abstract/Free Full Text]

Schulte, R., Wattiau, P., Hartland, E. L., Robins-Browne, R. M. & Cornelis, G. R. (1996). Differential secretion of interleukin-8 by human epithelial cell lines upon entry of virulent or nonvirulent Yersinia enterocolitica. Infect Immun 64, 2106–2113.[Abstract]

Schulte, R., Grassl, G. A., Preger, S., Fessele, S., Jacobi, C. A., Schaller, M., Nelson, P. J. & Autenrieth, I. B. (2000). Yersinia enterocolitica invasin protein triggers IL-8 production in epithelial cells via activation of Rel p65-p65 homodimers. FASEB J 14, 1471–1484.[Abstract/Free Full Text]

Sing, A., Roggenkamp, A., Geiger, A. M. & Heesemann, J. (2002a). Yersinia enterocolitica evasion of the host innate immune response by V antigen-induced IL-10 production of macrophages is abrogated in IL-10-deficient mice. J Immunol 168, 1315–1321.[Abstract/Free Full Text]

Sing, A., Rost, D., Tvardovskaia, N., Roggenkamp, A., Wiedemann, A., Kirschning, C. J., Aepfelbacher, M. & Heesemann, J. (2002b). Yersinia V-antigen exploits toll-like receptor 2 and CD14 for interleukin 10-mediated immunosuppression. J Exp Med 196, 1017–1024.[Abstract/Free Full Text]

Sing, A., Rost, D., Tvardovskaia, N., Roggenkamp, A., Geiger, A. M., Kirschning, C. J., Wiedemann, A., Aepfelbacher, M. & Heesemann, J. (2003). Mechanisms of Yersinia enterocolitica evasion of the host innate immune response by V antigen. Adv Exp Med Biol 529, 165–167.[Medline]

Song, C.-H., Lee, J.-S., Kim, H.-J., Park, J.-K., Paik, T.-H. & Jo, E.-K. (2003). Interleukin-8 is differentially expressed by human-derived monocytic cell line U937 infected with Mycobacterium tuberculosis H37Rv and Mycobacterium marinum. Infect Immun 71, 5480–5487.[Abstract/Free Full Text]

Tardy, F., Homblé, F., Neyt, C., Wattiez, R., Cornelis, G. R., Ruysschaert, J. M. & Cabiaux, V. (1999). Yersinia enterocolitica type III secretion-translocation system: channel formation by secreted Yops. EMBO J 18, 6793–6799.[CrossRef][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]

Trülzsch, K., Sporleder, T., Igwe, E. I., Rüssmann, H. & Heesemann, J. (2004). Contribution of the major secreted Yops of Yersinia enterocolitica O : 8 to pathogenicity in the mouse infection model. Infect Immun 72, 5227–5234.[Abstract/Free Full Text]

Yamamoto, T., Hanawa, T., Ogata, S. & Kamiya, S. (1996). Identification and characterization of the Yersinia enterocolitica gsrA gene, which protectively responds to intracellular stress induced by macrophage phagocytosis and to extracellular environmental stress. Infect Immun 64, 2980–2987.[Abstract]

Yamamoto, T., Hanawa, T., Ogata, S. & Kamiya, S. (1997). The Yersinia enterocolitica GsrA stress protein, involved in intracellular survival, is induced by macrophage phagocytosis. Infect Immun 65, 2190–2196.[Abstract]




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A. McNally, R. M. La Ragione, A. Best, G. Manning, and D. G. Newell
An aflagellate mutant Yersinia enterocolitica biotype 1A strain displays altered invasion of epithelial cells, persistence in macrophages, and cytokine secretion profiles in vitro
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