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The Journal of Immunology, 1999, 162: 2875-2883.
Copyright © 1999 by The American Association of Immunologists

Coordinate Involvement of Invasin and Yop Proteins in a Yersinia pseudotuberculosis-Specific Class I-Restricted Cytotoxic T Cell-Mediated Response1

Géraldine Falgarone*, Hervé S. Blanchard*, François Virecoulon*, Michel Simonet{ddagger} and Maxime Breban2,*,{dagger}

* Institut National de la Santé et de la Recherche Médicale, Unit 477, and {dagger} Institut de Rhumatologie, Hôpital Cochin, Assistance Publique-Hopitaux de Paris, Université René Descartes, Paris, France; and {ddagger} Laboratoire de Bactériologie, Faculté de Médecine Henri Warembourg, Lille, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Yersinia pseudotuberculosis is a pathogenic enteric bacteria that evades host cellular immune response and resides extracellularly in vivo. Nevertheless, an important contribution of T cells to defense against Yersinia has been previously established. In this study we demonstrate that Lewis rats infected with virulent strains of Y. pseudotuberculosis, mount a Yersinia-specific, RT1-A-restricted, CD8+ T cell-mediated, cytotoxic response. Sensitization of lymphoblast target cells for cytolysis by Yersinia-specific CTLs required their incubation with live Yersinia and was independent of endocytosis. Although fully virulent Yersinia did not invade those cells, they attached to their surface. In contrast, invasin-deficient strain failed to bind to blast targets or to sensitize them for cytolysis. Furthermore, an intact virulence plasmid was an absolute requirement for Yersinia to sensitize blast targets for cytolysis. Using a series of Y. pseudotuberculosis mutants selectively deficient in virulence plasmid-encoded proteins, we found no evidence for a specific role played by YadA, YopH, YpkA, or YopJ in the sensitization process of blast targets. In contrast, mutations suppressing YopB, YopD, or YopE expression abolished the capacity of Yersinia to sensitize blast targets. These results are consistent with a model in which extracellular Yersinia bound to lymphoblast targets via invasin translocate inside eukaryotic cytosol YopE, which is presented in a class I-restricted fashion to CD8+ cytotoxic T cells. This system could represent a more general mechanism by which bacteria harboring a host cell contact-dependent or type III secretion apparatus trigger a class I-restricted CD8+ T cell response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Three species of the Gram-negative bacteria genus Yersinia are pathogenic for humans and rodents. The most harmful, Y. pestis, is the causative agent of plague, whereas the closely related Y. pseudotuberculosis and the more distant Y. enterocolitica are responsible for gastrointestinal infections, manifested by acute ileitis and mesenteric lymphadenitis 1 . Infection with the two latter species is sometimes complicated with reactive sterile arthritis, especially in patients bearing the HLA-B27 class I MHC allele 2 . Such manifestation is thought to be mediated by the immune system and may involve a pathogenic HLA-B27-restricted Yersinia-specific CD8+ T cell 3 .

Enteric infections with Y. pseudotuberculosis and Y. enterocolitica share similar characteristics. After crossing the acidic environment of the stomach, bacteria reach the intestine and invade the lamina propria of the terminal ileum through M cells. The bacteria multiply in Peyer’s patches and drain into the mesenteric lymph nodes (LN),3 causing acute mesenteric lymphadenitis. The chromosomal outer membrane protein invasin, which promotes binding to the ß1 chain of integrins and internalization into eukaryotic cells in vitro 4 , is necessary for efficient translocation of bacteria across the intestinal epithelium 5, 6 . All pathogenic strains also harbor a 70- to 75-kb plasmid pYV that encodes several factors essential for bacterial virulence, e.g., Yops (Yersinia outer membrane proteins) and YadA (Yersinia adhesin). Upon contact of Yersinia with host target cell, Yops are secreted via a type III secretion system 7 . The Yops fall into two major groups of proteins: YopB and YopD, which form a delivery apparatus, and translocate several other effector proteins (YopE, YopH, YpkA/YopO, and YopM) directly into the eukaryotic cytosol through the plasma membrane 8 . Although the function of all effector Yops has not been elucidated, some of them display antiphagocytic (YopH and YopE) and cytotoxic (YopE) activities or are involved in inducing macrophage apoptosis (YopJ), all activities that are probably implicated in the extracellular survival of yersiniae and contribute to delay the development of a cell-mediated immune response 9, 10, 11 .

It is established that T cells play an essential role in defense against Yersinia in mice 12 . The protective role of IFN-{gamma}-producing CD4+ T cells has been demonstrated 13, 14 . Likewise, CD8+ lymphocytes were shown to mediate protection in adoptive transfer experiments 13 . However, the specificity and function of those protective CD8+ cells have not been clearly defined. In particular, there was no demonstration that Yersinia infection elicits a class I-restricted specific CTL response in vivo, although such a possibility was suggested using an indirect experimental approach 15 . Cytotoxic CD8+ T cells that recognize Yersinia-derived Ag presented in a class I-restricted fashion have been obtained from the joints of patients suffering from reactive arthritis. However, direct evidence that those T cells were actually generated in response to Yersinia infection is lacking 16, 17 . One major issue with the presentation of Ag derived from an extracellularly located bacterium by class I molecules is that such Ag presumably need to penetrate the cell to gain access to class I presentation pathway 18 . To date, class I presentation has been shown to work efficiently for a limited number of bacteria that survive intracellularly, such as Listeria, Mycobacteria, Salmonella, and Chlamydia 19, 20 .

In the present work we provide evidence that infection of rat with Y. pseudotuberculosis, an extracellularly living bacteria, elicits a bacteria-specific, class I-restricted, cytotoxic CD8+ T cell response. The roles played by invasin and virulence plasmid-encoded proteins in this response are further characterized, and its functional implications are discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Rats

Inbred Lewis (LEW), and Dark Agouti females, 2–3 mo old, were purchased from Iffa Credo (L’Arbresle, France) and housed under conventional conditions. Study procedures were approved by the institutional animal care committee.

Bacterial strains and growth conditions

Y. pseudotuberculosis strains (Table IGo) used in this study were grown in Luria broth (LB) medium, sometimes supplemented with 20 mM sodium oxalate/20 mM MgCl2 (Ca2+-deficient LB medium) at 28 or 37°C. Strains of Escherichia coli 25922 obtained from American Type Culture Collection (Manassas, VA) and Salmonella typhimurium (patient isolate) were grown in LB medium at 37°C. The actual number of bacteria inoculum was determined by plating serial dilutions of the inoculum on LB agar and counting CFU after an incubation period of 48 h at 28 or 37°C.


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  		Table I. Y. pseudotuberculosis strains used in this study

 
Tissue culture medium, mAbs, and other reagents

Tissue cultures were performed in RPMI 1640 medium with Glutamax I (Life Technologies, Eragny-sur-Oise, France) supplemented with 5% FCS, penicillin G (100 U/ml), streptomycin (100 µg/ml), 0.02 mM 2-ME, and 5 mM HEPES unless otherwise stated. Murine anti-rat mAbs used are listed in Table IIGo. The anti-Yersina invasin mAb 3A2 (murine IgG2a) 39 was a gift from R. R. Isberg (Tufts University, Boston, MA). Irrelevant murine mAbs were {gamma}C11 (anti-human CD82, IgG1; a gift from H. Conjeaud, Paris, France) and OKT3 (anti-human CD3, IgG2a; Cilag, Boulogne, France). Goat anti-mouse IgG-FITC was purchased from Eurobio (Les Ulis, France). Cytochalasin D (CCD) was obtained from Sigma (St. Quentin Fallavier, France).


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  		Table II. Murine anti-rat Ag mAbs used

 
Infection of animals

Y. pseudotuberculosis grown overnight at 28°C in Ca2+-deficient LB medium were centrifuged and resuspended in sterile PBS. For intragastric infection, rats were given 109 bacteria in 0.5 ml of PBS through a gastric tube for 3 consecutive days. For i.p. infection, rats received a single inoculum of 105 bacteria in 0.5 ml of PBS.

Generation of anti-Yersinia CTLs

Peripheral and mesenteric LN cells harvested from infected rats were resuspended in culture medium containing 10% Con A-stimulated rat spleen and LN cell supernatant and 50 mM {alpha}-methyl-D-mannoside and restimulated for 4–5 days in 96-well U-bottom culture dishes (105 cells/well) with LN cells from naive rats (3 x 105 cells/well) that had been in vitro infected with Y. pseudotuberculosis as follows. Bacteria grown overnight at 28°C in LB medium followed by 4-h incubation at 37°C in Ca2+-deficient LB medium to induce expression of virulence plasmid proteins were washed and resuspended in tissue culture medium without antibiotics and incubated with stimulator cells at a ratio of 10-90 bacteria/cell for 2 h at 37°C. Thereafter, infected stimulator cells were treated for 1 h at 37°C with gentamicin (100 µg/ml), washed, and irradiated (3000 rad) before use.

Anti-H-Y CTLs

CTLs specific for the rat male H-Y Ag were generated as previously described 40 . Briefly, LEW females were primed in vivo by s.c. and i.p. injection of 2 x 108 LEW male pooled LN and spleen cells. LN cells harvested from LEW female primed >3 wk previously were resuspended in culture medium containing 10% Con A supernatant and 50 mM {alpha}-methyl-D-mannosid and restimulated for 5 days in 96-well U-bottom culture dishes (105 cells/well) with irradiated (3000 rad) LN cells from LEW male (3 x 105 cells/well).

Cell-mediated cytotoxicity assay

Rat LN cell blast targets were generated by culture for 40 h with Con A as previously described 40 . In some experiments Con A-stimulated blasts that had been kept frozen at -80°C or in liquid nitrogen at 107 cells/ml in 90% FCS and 10% DMSO were thawed, resuspended at 2.5 x 106 cells/ml in the same medium used for restimulation, and incubated for 18 h before being used. Bone marrow-derived macrophage (BMDM) targets were obtained as previously described 41 . Briefly, bone marrow cells harvested from rat long bones were resuspended at 107 cells/ml in tissue culture medium without FCS and plated in bacteriological grade culture dishes. After incubation for 2 h at 37°C, nonadherent cells were removed, replated at 2 x 106 cells/ml in the complete medium supplemented with 10% FCS and 20% supernatant from L929 cells as a source of macrophage CSF, and reincubated. Cells were mechanically harvested on day 7 of culture. Infection of target cells was performed immediately before labeling, using a procedure similar to that described above for infection of stimulator cells. For labeling of target cells, 106 cells were centrifuged at 200 x g, the supernatant was removed by aspiration, 50 µCi of sodium [51Cr]chromate (DuPont-New England Nuclear, Boston, MA) was added to the cell pellet, and the cells were incubated for 3 h at room temperature (Con A blasts) or for 2 h at 37°C (BMDM). Labeled cells were washed three times by centrifugation at 200 x g for 7 min, counted, and resuspended in culture medium containing 10% FCS.

For cytotoxicity assays, restimulated LN cells harvested after 5 days of culture were washed and resuspended at 5 x 106 cells/ml in culture medium containing 10% FCS. Labeled target cells were diluted in the same medium to 5 x 104 cells/ml. Threefold dilutions of effectors (100 µl) and targets (100 µl) were dispensed in triplicate into 96-well U-bottom culture dishes. Medium without effectors (100 µl) or with 1 N HCl (100 µl) was also added to triplicate wells of targets to determine spontaneous and maximal lysis, respectively. Effectors and targets in the plates were centrifuged at 65 x g for 2 min, and the plates were incubated for 4 h at 37°C and then recentrifuged in the same manner. Supernatant was harvested from each well (50 µl) and counted in a 1450 Microbeta counter (Wallac, Turku, Finland). The percent specific lysis was computed by the formula 100 x [(isotope release by effectors - spontaneous release)/(maximum release - spontaneous release)]. The ratio of spontaneous to maximal 51Cr release from lysed targets was routinely <30% and averaged 16%. The SD among triplicate assays was always <5% specific lysis.

Flow cytometric analysis of cell surface Ags

All procedures were conducted at 4°C in PBS/2% FCS/0.01% NaN3. Cells (5 x 105) were incubated with saturating concentrations of the appropriate mAb for 30 min, washed, then incubated with FITC-conjugated monoclonal goat anti-mouse IgG for 30 min. After washing, the cells were analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).

In vitro magnetic cell depletions

Selected subsets of T cells were obtained after in vitro restimulation by negative selection using the following combinations of mouse mAbs, OX6, OX17, OX33, OX42, and 3.2.3, to purify all T cells plus either OX8 or OX35 to purify CD4+ or CD8+ T cells, respectively. All procedures were conducted at 4°C in PBS/4% FCS as follows. Cells were incubated with saturating concentrations of mAb for 15 min, washed, and further incubated with goat anti-mouse IgG-conjugated microbeads (Dynabeads M-450, Dynal, Oslo, Norway) at a ratio of 20 microbeads/cell. Cells were washed again, resuspended in PBS-FCS, and sorted with a magnet (Dynal MPC).

Ab inhibition assay

Target cells were preincubated with dilutions of mAb at 4°C for 15 min in assay medium and then combined in 96-well plates as described above at an E:T cell ratio of 33. Triplicate wells were also plated containing target cells incubated with Ab at each concentration but lacking effectors. Results were expressed as the percent inhibition = [1 - (% lysis with mAb/% lysis without mAb)] x 100.

Invasion assays

For invasion assays, 106 blast target cells were incubated in 1 ml of culture medium without antibiotics in 24-well culture plates with live Yersinia at a ratio of 50 bacteria/cell in the same conditions as those used for the cell-mediated cytotoxicity assay (i.e., 2 h at 37°C followed by 1 h at 37°C with gentamicin) and washed to eliminate gentamicin. Internalized bacteria were released from infected cells by adding 1% Triton X-100 (Sigma) and measuring viable counts on agar plates. The entry of Y. pseudotuberculosis was expressed as the percentage of intracellular bacteria = (number of bacteria surviving gentamicin treatment/number of input bacteria) x 100.

Light microscopy of cytospin smears

To prepare cytospins, blast cells were incubated with live bacteria at a ratio of 50 bacteria/cell for 1 h at 37°C followed by 1 h at 37°C with gentamicin and were spun down at 72.3 x g for 8 min in a Cytospin III (Shandon, Eragny sur Oise, France). Bacteria and lymphoblasts were examined under light microscope after May-Grünwald-Giemsa staining.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rats infected with Y. pseudotuberculosis mount a bacteria-specific CTL response

To determine whether a CTL response is primed during infection with Y. pseudotuberculosis, LEW rats were infected intragastrically or i.p. with strains of Y. pseudotuberculosis harboring its virulence plasmid pYV. Rats were killed from 1–8 wk after infection, and LN cells were restimulated on irradiated Y. pseudotuberculosis-infected LN cells as a source of APCs. As shown, CTLs were generated after intragastric infection that efficiently killed LN blasts or BMDM targets infected in vitro with wild-type (pYV+), but not plasmid-cured (pYV-) Y. pseudotuberculosis (Fig. 1Go, A and B). Infection with either strain YPIII (Fig. 1GoA) or strain IP2777 (Fig. 1GoB) could efficiently prime rats for this specific CTL response. Furthermore, infection with Y. pseudotuberculosis strain IP2777 cross-primed the CTL response against the YPIII strain (Fig. 1GoB). In kinetics studies the CTL response peaked between 2 and 4 wk after infection (not shown). Likewise, a CTL response was generated in rats infected i.p. (not shown).



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  		FIGURE 1. Evidence for CTL response against Y. pseudotuberculosis in rats. A and B, LN cells from LEW rats infected intragastrically with the wild-type (pYV+) strain of Y. pseudotuberculosis YPIII (A) or IP2777 (B) were restimulated in vitro with syngeneic LN cells infected with the same strain of Y. pseudotuberculosis that was used for priming. These CTL effectors were tested 5 days later for lysis of LEW Con A blast LN (A and B) and BMDM (A) targets sensitized in vitro by infection with Y. pseudotuberculosis strains YPIII (pYV+; A and B), YPIIIc (pYV-; A and B), IP2777 (pYV+; B), or IP2777c (pYV-; B) or uninfected. The level of lysis at an E:T cell ratio of 100 targets sensitized with wild-type Y. pseudotuberculosis varied from 20–91% in 18 separate experiments and averaged 45 ± 5% (mean ± SEM). C, Mesenteric LN (MLN) and peripheral LN (PLN) cells from LEW rats infected intragastrically with Y. pseudotuberculosis strain YPIII (pYV+) were separately restimulated in vitro as described above and tested for lysis of LEW lymphoblast targets infected with strain YPIII (pYV+) or uninfected. D, LN cells from LEW female rats primed against male rat H-Y Ag were restimulated in vitro with LN cells from LEW male rats and tested 5 days later for lysis of LEW male and female lymphoblast targets infected with strain YPIII (pYV+) or uninfected.

 
In vivo infection with Y. pseudotuberculosis was required to prime rat LN cells for this anti-Yersinia CTL response. Indeed, LN cells from uninfected rats that were restimulated in vitro on Y. pseudotuberculosis-infected LN cells failed to lyse Y. pseudotuberculosis-infected blast targets (not shown). Furthermore, following intragastric infection a significant CTL response was detectable in mesenteric LN cells but not in peripheral LN cells, indicating specificity of priming for LN cells afferent to the bacterial entry site (Fig. 1GoC). Finally, CTLs primed against the male H-Y minor histocompatibility Ag failed to lyse female targets infected with virulent Y. pseudotuberculosis (Fig. 1GoD).

This anti-Yersinia CTL response was specific for syngeneic cells infected with Y. pseudotuberculosis, since neither targets from another inbred strain of rats (Fig. 2GoA) nor targets infected with unrelated bacteria, such as E. coli or S. typhimurium, were efficiently killed (Fig. 2GoB).



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  		FIGURE 2. CTL response against Y. pseudotuberculosis in rat is genetically restricted and Yersinia-specific. A, CTL effectors were generated from LEW rats infected intragastrically with wild-type Y. pseudotuberculosis strain YPIII (pYV+) as described in Fig. 1Go and tested for lysis of LEW or Dark Agouti lymphoblast targets infected with strain YPIII (pYV+) or uninfected. B, CTL effectors generated as above were tested for lysis of LEW lymphoblast targets infected with strain YPIII (pYV+), E. coli 25922, or S. typhimurium or were uninfected. Similar results were observed in two to four separate experiments.

 
The Yersinia-specific CTL response is mediated by CD8+ T cells and is restricted by rat RT1-A

In vitro restimulated LN cells (>90% CD2+, TCR {alpha}/ß+ T cells by flow cytometry) were sorted by immunomagnetic depletion to analyze which subset of T cells was responsible for the CTL response. Sorting experiments revealed that CD8+ T cells were responsible for >90% of the CTL response (Fig. 3GoA). Furthermore, blocking experiments using preincubation of target cells with mAbs showed that the CTL response was dependent on rat class I, but not class II, MHC molecules (Fig. 3GoB). Complete blocking of the CTL response with anti-MHC class I mAbs OX 18 (not shown) or F16-4-4-11 (Fig. 3GoB), the latter being specific for rat RT1-A, argues for a conventional mechanism of the CTL response.



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  		FIGURE 3. Anti-Y. pseudotuberculosis CTL effectors are CD8+ T cells and lyse infected lymphoblast targets in a class I-restricted fashion. A, CTL effectors were generated from LEW rats infected intragastrically with wild-type Y. pseudotuberculosis strain YPIII (pYV+) and tested for lysis of LEW lymphoblast targets infected with strain YPIII (pYV+; solid bars) or uninfected (hatched bars). Immunomagnetic sorting was performed to negatively select for CD4+ T cells and CD8+ T cells that were compared with unsorted T cells. This experiment was repeated twice with similar results. B, CTL effectors generated as described above were tested for lysis of infected LEW lymphoblast targets that were preincubated with 2, 5, 15, or 30 µg/ml of anti-class I (F16-4-4-11), anti-class II (OX6), or control ({gamma}C11) mAb at an E:T cell ratio of 33. Maximal percent specific lysis in this experiment was 23%. Percent inhibition = [1 - (% lysis with mAb/% lysis without mAb)] x 100. This experiment is representative of two.

 
Target cells sensitization for killing by CTLs requires contact with viable Yersinia

When Y. pseudotuberculosis used for sensitization was killed with gentamicin before contact with blast target cells, the CTL response was completely abolished (Fig. 4Go), indicating that interaction of blast targets with metabolically active Yersinia was required for further lysis by CTLs.



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  		FIGURE 4. Sensitization of blast targets for cytolysis by anti-Y. pseudotuberculosis CTLs requires contact of target cells with live Yersinia and is independent of endocytosis. CTL effectors were generated from LEW rats infected intragastrically with wild-type Y. pseudotuberculosis strain IP2777 (pYV+; same experiment as in Fig. 1GoB) and tested for lysis of LEW lymphoblast targets. Targets were infected in vitro with Y. pseudotuberculosis strain IP2777 (pYV+; solid symbols) or uninfected (open circles). Infected targets were left untreated (solid circles) or were treated with 5 µg/ml CCD during infection (solid squares) or during the chromium release test (CRT; solid diamonds) or with 100 µg/ml gentamicin, which was added throughout target infection (solid triangles). Similar results were observed in two separate experiments.

 
Possible interpretation for this result is that Yersinia need to invade blast targets to sensitize them for lysis. This possibility was examined by culturing bacteria released from blast cells incubated with Yersinia. These experiments showed the lack of invasion of rat lymphoblasts by live Yersinia (Table IIIGo). By contrast, flow cytometry using an anti-invasin mAb demonstrated the presence of Yersinia at the surface of >40% of blast targets (Fig. 5Go), readily detectable upon contact of intact Yersinia with target cells, suggesting that interaction of live Yersinia with the target cell surface was necessary to sensitize targets for killing and occurred via attachment of the bacteria to the cell membrane. In addition, experiments using CCD indicated that sensitization of blast targets for lysis was independent of endocytosis (Fig. 4Go).


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  		Table III. Entry of Y. pseudotuberculosis into LEW rat lymphoblast targets1

 


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  		FIGURE 5. Detection of Yersinia at the surface of lymphoblast target cells. LEW Con A blast targets (106/ml) were infected in vitro with Y. pseudotuberculosis at an infection ratio of 50 bacteria/cell for 2 h at 37°C followed by 1 h in the presence of gentamicin. The presence of Yersinia at the surface of infected blast cells was detected by flow cytometry using the anti-invasin mAb 3A2 (thick solid trace). The percentage refers to positive cells defined by the binding of the control mAb OKT3 (dashed trace) and the binding of mAb 3A2 at the surface of uninfected blasts (broken trace).

 
Y. pseudotuberculosis invasin is required for sensitization of target cells

We tested the level of cytotoxicity by CTLs generated in the foregoing experimental conditions on blast target cells infected with Y. pseudotuberculosis mutant lacking invasin expression. The CTL response against target cells sensitized with plasmid harboring invasin-deficient strain YP212 (Fig. 6Go) was completely abolished. By contrast, YadA, which is a virulence plasmid-encoded protein also involved in bacterial adhesion, was not required to sensitize blast targets for lysis by CTLs, as shown using the YPIII(pIB102) yadA mutant (Fig. 6Go). A likely explanation for this result is that invasin is necessary for interaction of Y. pseudotuberculosis with blast targets. Indeed, the surface of all blast cells infected with wild-type YPIII (pYV+; Fig. 7GoA), plasmid-cured YPIIIc (pYV-; Fig. 7GoB), or E. coli 25922 (Fig. 7GoD) strains was covered by numerous bacteria, whereas only an occasional bacterium was attached to rat lymphoblasts infected with the YP212 inv mutant (Fig. 7GoC), as shown by microscopic examination.



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  		FIGURE 6. Invasin and virulence plasmid, but not YadA or YopH, are required for sensitization of blast targets for cytolysis by anti-Y. pseudotuberculosis CTLs. CTL effectors were generated from LEW rats infected with wild-type Y. pseudotuberculosis strain YPIII (pYV+) and tested for lysis of LEW lymphoblast targets infected with YPIII (pYV+), YPIIIc (pYV-), YP212 inv mutant, YPIII(pIB102) yadA mutant, or YP15 yopH mutant. Similar results were observed in two to four separate experiments for each mutant tested.

 


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  		FIGURE 7. Invasin is required for attachment of Yersinia at the surface of rat lymphoblast. LEW lymphoblasts were incubated in vitro with wild-type Y. pseudotuberculosis strain YPIII (pYV+; A), plasmid-cured derivative YPIIIc (pYV-; B), YP212 inv mutant (C), or E. coli 25922 (D). Cytospins were prepared, and the contact of bacteria (arrow) with blast cells was examined under light microscope after treatment with May-Grünwald-Giemsa stain. Magnification, x640.

 
An intact Yop delivery apparatus and YopE are both required to sensitize blast targets for killing

The presence of a functional virulence plasmid was also an absolute requirement to sensitize rat lymphoblasts for cytolysis by CTLs, since Y. pseudotuberculosis strains cured from their virulence plasmid failed to sensitize those blast targets for killing upon in vitro infection (Figs. 1Go, A and B, 6, and 8A). The lack of cytolysis of targets infected with plasmid-cured strains could indicate that proteins encoded by the corresponding plasmidic genes are actual antigenic targets for Yersinia-specific CTLs, that those proteins are required for efficient class I presentation of Yersinia Ags, or both.

The level of cytotoxicity against infected targets was similar whether targets were sensitized with Y. pseudotuberculosis cultured overnight at 28°C in LB medium or in conditions inducing Yops secretion in the culture medium (e.g., subculture at 37°C in Ca2+-deficient medium; not shown). Hence, we speculated that the secretion apparatus of Yersinia would allow effector Yops to be presented in a class I-restricted fashion by delivering such proteins directly into the target cytoplasm. To examine the contributions of several Yops encoded by Yersinia virulence plasmid to the CTL response, we tested this response against blast targets sensitized with a series of isogenic Y. pseudotuberculosis mutants selectively defective for expression of those proteins (Figs. 6Go and 8Go). Targets sensitized with the YP15 deletion mutant of YopH (Fig. 6Go) were killed at similar levels as targets infected with wild-type strain YPIII (pYV+). Similar results were observed with any of two ypkA mutants (e.g., YPIII(pIB43) insertion mutant, in which the YpkA C-terminal 548–731 amino acid residues are deleted and the YopJ production is abolished, and YPIII(pIB44) in-frame deletion mutant, in which amino acids 207–388 of YpkA are deleted; Fig. 8GoA). Large differences were observed with the two yopE mutants tested. Targets sensitized with the YPIII(pIB518) mutant of Y. pseudotuberculosis producing a nonfunctional truncated form of YopE (deletion of the C-terminal 91 amino acid residues) were killed at levels comparable to those of targets infected with wild-type strain (Fig. 8Go, A and B), whereas targets sensitized with the YPIII(pIB522) complete deletion mutant of YopE were not killed (Fig. 8GoB).



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  		FIGURE 8. Sensitization of blast targets for cytolysis by anti-Y. pseudotuberculosis CTLs requires the presence of an intact Yersinia translocation apparatus and YopE. A, CTL effectors were generated from LEW rats infected with wild-type Y. pseudotuberculosis strain YPIII (pYV+) and were tested for lysis of LEW lymphoblast targets infected with YPIII (pYV+), YPIIIc (pYV-), or YPIII isogenic mutants of Yops. B, CTL effectors generated as described above were tested for lysis of LEW lymphoblast targets infected with YPIII (pYV+), YPIII(pIB518) yopE{Delta}128–219 mutant, or YPIII(pIB522) complete deletion mutant of YopE (same experiment as in Fig. 2GoB). Similar results were observed in two to four separate experiments for each mutant tested.

 
Finally, sensitizing targets with isogenic mutants of YPIII deficient in YopB, YopD, or YopB and YopD (both proteins are critically involved in Yops translocation through eukaryotic plasma membrane) completely abolished the CTL response (Fig. 8GoA).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates for the first time that infection of rats with one of the three Yersinia species pathogenic for humans, Y. pseudotuberculosis, elicits a CD8+ T cell-mediated cytotoxic response. Furthermore, this response appears to depend on the RT1-A locus that is a major locus for conventional class I presentation in rats 42 . Previous work suggested the possibility that such a class I-restricted T cell response would occur in the setting of infection with Yersinia 15 . However, a demonstration that such a prediction actually occurred in vivo had not yet been provided.

One of the major issues regarding the possibility to mount a class I-restricted response against extracellular bacteria such as Y. pseudotuberculosis is the capacity of eukaryotic cells to process those bacteria components for class I presentation. The extracellular survival of Yersinia in the host environment is thought to result from a virulence plasmid-encoded apparatus of Yop secretion, by which Y. pseudotuberculosis, can neutralize phagocytic cells 8 . Once Yersinia attach to the target cell, the secretion system is triggered, and Yops are secreted and directly injected into the eukaryotic target cytoplasm. Several of these Yops exert an action inside their target, contributing to the paralysis of phagocytosis by these cells. This system seems to compromise possible processing of Yersinia via phagocytosis by professional APCs, a mechanism important for class I presentation in the case of intracellularly living bacteria 43 . However, it can lead to an alternate mechanism of class I presentation that would potentially trigger a CTL response. It is indeed likely that Yops that have access to the cytoplasm can be presented through a class I-mediated pathway 15 .

In our experiments sensitization of blast target cells required contact with Yersinia that failed to invade those cells, suggesting that the whole bacteria were not penetrating the cell. The possibility that soluble proteins released from Yersinia were internalized by target cells before being processed and presented by class I molecules is unlikely, since Yersinia presentation required the living state of the bacteria, at least upon contact with the target cell, and was not blocked by addition of CCD. Furthermore, sensitizing targets with Yersinia cultured in conditions inducing Yops secretion in the culture medium did not enhance cytolysis. Our observations are consistent with involvement of the Yop-secretion apparatus. This secretion system is triggered by tight contact of Yersinia with the eukaryotic cell membrane. Indeed, inv mutants that failed to bind to blast targets were unable to sensitize those targets either. Although other Yersinia proteins, such as YadA, can substitute for invasin in adhesion to epithelial cells and allow the Yop secretion apparatus to operate 44 , this alternate pathway may not be relevant to lymphoblast cells. Indeed, the presence of YadA was not required for blast target sensitization.

The absolute requirement for virulence plasmid to sensitize target cells is also consistent with the involvement of a Yop secretion apparatus. Our observation that mutants deficient in YopB or YopD failed to sensitize blast cells supports this hypothesis, since both proteins are necessary to translocate effector Yops inside eukaryotic cells. Hence, the most likely antigenic targets in our system are effector Yops. Among the effector Yops tested in our experiments, neither YopH, which was previously shown to be presented in a class I-restricted manner by eukaryotic cells 15 , nor YpkA and YopJ were implicated. Interestingly, the CTL response against blast cells sensitized with the YPIII(pIB518) partial deletion mutant of YopE was retained, as opposed to its abolition observed with the YPIII(pIB522) yopE complete deletion mutant. YPIII(pIB518) mutant produces a truncated form of YopE associated with a loss of YopE function 44 . Therefore, it is unlikely that the effect of YopE that was observed on the CTL response is explained by its known interference with eukaryotic cell metabolism 44 . Rather, our data indicate the 23-kDa YopE as a potential antigenic target for anti-Y. pseudotuberculosis CTLs in LEW rats.

This study is interesting in several aspects. To our knowledge, this is the first direct demonstration that CTLs can be efficiently primed against extracellularly living Yersinia. The mechanism by which the secretion apparatus of Yersinia is involved in this system may indicate that other bacteria displaying type III secretion apparatus, such as Salmonella, enteropathogenic E. coli, Shigella, or Pseudomonas aeruginosa, could also trigger a CTL response 45, 46, 47, 48 . This newly described system may also suggest original vaccination strategies to prime CD8+ T cells against infectious agents or even tumoral cells, using engineered bacterial vectors. However, we cannot rule out from the experiments presented in this study that Yersinia proteins other than Yops are candidate Ag for class I presentation. This could particularly be the case if professional APCs were used as targets (which could handle Ag differently than nonphagocytic lymphoblast cells). However, we believe that the system described herein is predominant, since Yersinia infection usually blocks phagocytosis. Finally, one can speculate upon the efficiency of this CTL mechanism in the host’s fight against Yersinia infection. The production of cytokines by activated CD8+ T cells could contribute to increase bactericidal capacities of macrophages. However, it is not obvious that CTL activity displayed against cells that are not actually invaded by Yersinia is an efficacious defense system. On the contrary, this system could be detrimental to the host, by destroying those cells to which only bacteria bind.


   Acknowledgments
 
We thank Dr. Hans Wolf-Watz for providing us with several Y. pseudotuberculosis mutant strains used in the study, and Dr. Jim B. Bliska for providing us with Y. pseudotuberculosis strain YP15. We thank Dr. C. Fournier for critical review of the manuscript.


   Footnotes
 
1 This work was supported by a Socíeté Française de Rhumatologie grant and in part by a grant from Association de Recherche Clinique en Rhumatologie/1996 (to G.F.). Back

2 Address correspondence and reprints requests to Dr. Maxime Breban, Institut de Rhumatologie, Hôpital Cochin, 27 rue du Faubourg Saint-Jacques, 75014 Paris, France. E-mail address: Back

3 Abbreviations used in this paper: LN, lymph node; LEW, Lewis; LB, Luria broth; CCD, cytochalasin D; BMDM, bone marrow-derived macrophage. Back

Received for publication May 12, 1998. Accepted for publication November 17, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Brubaker, R. R.. 1991. Factors promoting acute and chronic diseases caused by yersiniae. Clin. Microbiol. Rev. 4:309.[Abstract/Free Full Text]
  2. Toivanen, A., K. Granfors, R. Lahesmaa-Rantala, R. Leino, T. Stahlberg, R. Vuento. 1985. Pathogenesis of Yersinia-triggered reactive arthritis: immunological, microbiological and clinical aspects. Immunol. Rev. 86:47.[Medline]
  3. Burmester, G. R., A. Daser, T. Kamradt, A. Krause, N. A. Mitchison, J. Sieper, N. Wolf. 1995. Immunology of reactive arthritides. Annu. Rev. Immunol. 13:229.[Medline]
  4. Isberg, R. R., J. M. Leong. 1990. Multiple ß1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells. Cell 60:861.[Medline]
  5. Simonet, M., S. Falkow. 1992. Invasin expression in Yersinia pseudotuberculosis. Infect. Immun. 60:4414.[Abstract/Free Full Text]
  6. Pepe, J. C., V. L. Miller. 1993. Yersinia enterocolitica invasin: a primary role in the initiation of infection. Proc. Natl. Acad. Sci. USA 90:6473.[Abstract/Free Full Text]
  7. Silhavy, T. J.. 1997. Death by lethal injection. Science 278:1085.[Free Full Text]
  8. Cornelis, G. R., H. Wolf-Watz. 1997. The Yersinia Yop virulon: a bacterial system for subverting eukaryotic cells. Mol. Microbiol. 23:861.[Medline]
  9. Simonet, M., S. Richard, P. Berche. 1990. Electron microscopic evidence for in vivo extracellular localization of Yersinia pseudotuberculosis harboring the pYV plasmid. Infect. Immun. 58:841.[Abstract/Free Full Text]
  10. Straley, S. C., E. Skrzypek, G. V. Plano, J. B. Bliska. 1993. Yops of Yersinia spp. pathogenic for humans. Infect. Immun. 61:3105.[Free Full Text]
  11. Monack, D. M., J. Mecsas, N. Ghori, S. Falkow. 1997. Yersinia signals macrophages to undergo apoptosis and YopJ is necessary for this cell death. Proc. Natl. Acad. Sci. USA 94:10385.[Abstract/Free Full Text]
  12. Autenrieth, I. B., U. Vogel, S. Preger, B. Heymer, J. Heesemann. 1993. Experimental Yersinia enterocolitica infection in euthymic and T-cell-deficient athymic nude C57BL/6 mice: comparison of time course, histomorphology, and immune response. Infect. Immun. 61:2585.[Abstract/Free Full Text]
  13. Autenrieth, I. B., A. Tingle, A. Reske-Kunz, J. Heesemann. 1992. T lymphocytes mediate protection against Yersinia enterocolitica in mice: characterization of murine T-cell clones specific for Y. enterocolitica. Infect. Immun. 60:1140.[Abstract/Free Full Text]
  14. Bohn, E., I. B. Autenrieth. 1996. IL-12 is essential for resistance against Yersinia enterocolitica by triggering IFN-{gamma} production in NK cells and CD4+ T cells. J. Immunol. 156:1458.[Abstract]
  15. Starnbach, M. N., M. J. Bevan. 1994. Cells infected with Yersinia present an epitope to class I MHC-restricted CTL. J. Immunol. 153:1603.[Abstract]
  16. Hermann, E., D. T. Y. Yu, K.-H. Meyer zum Büschenfelde, B. Fleisher. 1993. HLA-B27-restricted CD8 T cells derived from synovial fluids of patients with reactive arthritis and ankylosing spondylitis. Lancet 342:646.[Medline]
  17. Ugrinovic, S., A. Mertz, P. Wu, J. Braun, J. Sieper. 1997. A single nonamer from the Yersinia 60-kDa heat shock protein is the target of HLA-B27-restricted CTL response in Yersinia-induced reactive arthritis. J. Immunol. 159:5715.[Abstract]
  18. Rock, K. L.. 1996. A new foreign policy: MHC class I molecules monitor the outside world. Immunol. Today 17:131.[Medline]
  19. Starnbach, M. N., M. J. Bevan, M. F. Lampe. 1994. Protective cytotoxic T lymphocytes are induced during murine infection with Chlamydia trachomatis. J. Immunol. 153:5183.[Abstract]
  20. Kaufmann, S. H. E.. 1995. Immunity to intracellular microbial pathogens. Immunol. Today 16:338.[Medline]
  21. Bölin, I., L. Norlander, H. Wolf-Watz. 1982. Temperature-inducible outer membrane of Yersinia pseudotuberculosis and Yersinia enterocolitica is associated with the virulence plasmid. Infect. Immun. 37:506.[Abstract/Free Full Text]
  22. Isberg, R. R.. 1989. Determinants for thermoinducible cell binding and plasmid-encoded penetration detected in the absence of the Yersinia pseudotuberculosis invasin protein. Infect. Immun. 57:1998.[Abstract/Free Full Text]
  23. Bölin, I., H. Wolf-Watz. 1984. Molecular cloning of the temperature-inducible outer membrane protein 1 of Yersinia pseudotuberculosis. Infect. Immun. 43:72.[Abstract/Free Full Text]
  24. Forsberg, A., H. Wolf-Watz. 1988. The virulence protein Yop5 of Yersinia pseudotuberculosis is regulated at transcriptional level by plasmid-pIB1-encoded trans-acting elements controlled by temperature and calcium. Mol. Microbiol. 2:121.[Medline]
  25. Forsberg, A., H. Wolf-Watz. 1990. Genetic analysis of the yopE region of Yersinia spp.: identification of a novel conserved locus, yerA, regulating yopE expression. J. Bacteriol. 172:1547.[Abstract/Free Full Text]
  26. Galyov, E. E., S. Hakansson, A. Forsberg, H. Wolf-Watz. 1993. A secreted protein kinase of Yersinia pseudotuberculosis is an indispensable virulence determinant. Nature 361:730.[Medline]
  27. Persson, C., R. Nordfelth, A. Homström, S. Hakansson, R. Rosqvist, H. Wolf-Watz. 1995. Cell-surface-bound Yersinia translocate the protein tyrosine phosphatase YopH by a polarized mechanism into the target cell. Mol. Microbiol. 18:135.[Medline]
  28. Fällman, M., K. Andersson, S. Hakansson, K.-M. Magnusson, O. Stendahl, H. Wolf-Watz. 1995. Yersinia pseudotuberculosis inhibits Fc receptor-mediated phagocytosis in J774 cells. Infect. Immun. 63:3117.[Abstract]
  29. Hunig, T., H.-J. Wallny, J. K. Hartley, A. Lawetzky, G. Tiefenthaler. 1989. A monoclonal antibody to a constant determinant of the rat T cell antigen receptor that induces T cell activation. J. Exp. Med. 169:73.[Abstract/Free Full Text]
  30. Beyers, A. D., A. N. Barclay, D. A. Law, Q. He, A. F. Williams. 1989. Activation of T lymphocytes via monoclonal antibodies against rat cell surface antigens with particular reference to CD2 antigen. Immunol. Rev. 111:59.[Medline]
  31. Brideau, R. J., P. B. Carter, W. R. McMaster, D. W. Mason, A. F. Williams. 1980. Two subsets of rat T lymphocytes defined with monoclonal antibodies. Eur. J. Immunol. 10:609.[Medline]
  32. Jefferies, W. A., J. R. Green, A. F. Williams. 1985. Authentic T helper CD4 (W3/25) antigen on rat peritoneal macrophages. J. Exp. Med. 162:117.[Abstract/Free Full Text]
  33. Woollett, G. R., A. N. Barclay, M. Puklavec, A. F. Williams. 1985. Molecular and antigenic heterogeneity of the rat leukocyte-common antigen from thymocytes and T and B lymphocytes. Eur. J. Immunol. 15:168.[Medline]
  34. Fukumoto, T., W. R. McMaster, A. F. Williams. 1982. Mouse monoclonal antibodies against rat major histocompatibility antigens: two Ia antigens and expression of Ia and class I antigens in rat thymus. Eur. J. Immunol. 12:237.[Medline]
  35. Hart, D. N. J., J. W. Fabre. 1981. MHC antigens in rat kidney, ureter, and bladder: localisation with monoclonal antibodies and demonstration of Ia positive dendritic cells. Transplantation 31:318.[Medline]
  36. McMaster, W. R., A. F. Williams. 1979. Identification of Ia glycoproteins in rat thymus and purification from rat spleen. Eur. J. Immunol. 9:426.[Medline]
  37. Robinson, A. P., T. M. White, D. W. Mason. 1986. Macrophage heterogeneity in the rat as delineated by two monoclonal antibodies MRC OX-41 and MRC OX-42, the latter recognizing complement receptor type 3. Immunology 57:239.[Medline]
  38. van den Brink, M. R. M., L. E. Hunt, J. C. Hiserodt. 1990. In vivo treatment with monoclonal antibody 3.2.3 selectively eliminates natural killer cells in rats. J. Exp. Med. 171:197.[Abstract/Free Full Text]
  39. Leong, J. M., R. S. Fournier, R. R. Isberg. 1990. Identification of the integrin binding domain of the Yersinia pseudotuberculosis invasin protein. EMBO J. 9:1979.[Medline]
  40. Livingstone, A. M., S. J. Powis, A. G. Diamond, G. W. Butcher, J. C. Howard. 1989. A trans-acting major histocompatibility complex-linked gene whose alleles determine gain and loss changes in the antigenic structure of a classical class I molecule. J. Exp. Med. 170:777.[Abstract/Free Full Text]
  41. Keller, R., R. Keist. 1987. Induction of interleukin 1 secretion and of tumoricidal activity in macrophages are not closely related phenomena. Eur. J. Immunol. 17:1665.[Medline]
  42. Günther, E., W. Wurst. 1984. Cytotoxic T lymphocytes of the rat are predominantly restricted by RT1. A and not RT1.C-determined major histocompatibility class I antigens. Immunogenetics 20:1.[Medline]
  43. Harding, C. V., R. Song, J. Griffin, J. France, M. J. Wick, J. D. Pfeifer, H. J. Geuze. 1995. Processing of bacterial antigens for presentation to class I and II MHC-restricted T lymphocytes. Infect. Agent Dis. 4:1.[Medline]
  44. Rosqvist, R., A. Forsberg, M. Rimpiläinen, T. Bergman, H. Wolf-Watz. 1990. The cytotoxic protein YopE of Yersinia obstructs the primary host defence. Mol. Microbiol. 4:657.[Medline]
  45. Fu, Y., J. E. Galan. 1998. The Salmonella typhimurium tyrosine phosphatase SptP is translocated into host cells and disrupts the actin cytoskeleton. Mol. Microbiol. 27:359.[Medline]
  46. Donnenberg, M. S., J. B. Kaper, B. B. Finlay. 1997. Interactions between enteropathogenic Escherichia coli and host epithelial cells. Trends Microbiol. 5:109.[Medline]
  47. Ménard, R., C. Dehio, P. J. Sansonetti. 1996. Bacterial entry into epithelial cells: the paradigm of Shigella. Trends Microbiol. 4:220.[Medline]
  48. Yahr, T. L., J. Goranson, D. W. Franck. 1996. Exoenzyme S of Pseudomonas aeruginosa is secreted by a type III pathway. Mol. Microbiol. 22:991.[Medline]



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