INFECTION AND IMMUNITY, Feb. 1992, p. 366-3730019-9567/92/020366-08$02.00/0Copyright © 1992, American Society for Microbiology

Human Platelet Aggregation by Yersinia pseudotuberculosisIs Mediated by Invasin

MICHEL SIMONET,'* PATRICK TRIADOU,2 CLAUDE FREHEL,1 MARIE-CHRISTINE MOREL-KOPP,3CECILE KAPLAN,3 AND PATRICK BERCHE'

Laboratoire de Microbiologiel and Laboratoire d'H/matologie,2 Faculte de Medecine Necker-Enfants Malades,F-75730 Paris Cedex 15, and Institut National de Transfusion Sanguine, F-75739 Paris Cedex 15, France

Received 10 June 1991/Accepted 9 November 1991

Plasmid-free strains of Yersinia pseudotuberculosis induce aggregation of human platelets in vitro. It appearsthat this phenomenon is mediated by invasin (Inv), a 103-kDa outer membrane protein that permits bacteriato penetrate mammalian cells, since (i) an isogenic inv-deficient mutant failed to aggregate platelets comparedwith the parental strain; (ii) a monoclonal antibody directed against invasin inhibited platelet aggregation; (iii)Inv' Escherichia coli HB101 promoted platelet aggregation. Platelet receptors for invasin were identified byusing a panel of anti-platelet glycoprotein monoclonal antibodies in a bacterial adhesion assay. We found thatbacteria bind to platelet membrane glycoproteins Ic and Ila. Electron microscopic study of bacterium-plateletinteractions also revealed that bacteria expressing invasin attach to and are phagocytized by thrombocytes, incontrast to inv-deficient bacteria, indicating that these anucleated cells are able to internalize bacteria in vitroafter specific interaction with invasin.

Yersinia pseudotuberculosis is a gram-negative bacteriumthat causes epizootic outbreaks of disease in birds andmammals. Occasionally, humans can be infected by inges-tion of food or water contaminated by this pathogen. Acutemesenteric lymphadenitis is the most common clinical man-ifestation of human infection, sometimes complicated bysepticemia, particularly in patients with underlying diseasessuch as cirrhosis or hemochromatosis (37).

All pathogenic Yersinia strains harbor a 70-kb plasmidcalled pYV (plasmid Yersinia virulence), which encodesproteins designated Yops (Yersinia outer membrane pro-tein). When bacteria are grown at 37°C in a calcium-deficientmedium, Yops are expressed at high levels, as outer mem-brane-associated proteins or released in culture superna-tants. The synthesis of Yops is inhibited at 37°C by addingCa2+ (2.5 mM) or by incubating bacteria at 28°C regardlessof the presence of Ca2+. yop mutants of Y. pseudotubercu-losis (and other pathogenic Yersinia species) are reduced invirulence in the mouse, strongly suggesting that they areinvolved in the pathogenic process (for reviews, see refer-ences 7, 10, and 11). The exact function of Yops in thevirulence remains unknown, except for YopH, YopE, andYopM. Both YopH and YopE inhibit phagocytosis of Y.pseudotuberculosis, while YopE is cytotoxic, inducing dis-ruption of the actin microfilament structure in eukaryoticcells (42-44). Moreover, it has been recently reported thatYopH is a tyrosine phosphatase that dephosphorylates eu-karyotic proteins (4, 17). Finally, Leung et al. (30) showedthat YopM-containing supernatant proteins of Yersinia pes-tis inhibited thrombin- or ristocetin-induced platelet aggre-gation, whereas this was not observed with supernatantproteins from the YopM-deficient Y. pestis mutant. Thisfinding can be explained by a significant homology betweena portion of the thrombin and the von Willebrand factor-binding domains of the a chain of human platelet glycopro-tein lb (GPIb) and the amino acid sequence of YopMdeduced from yopM (31).

* Corresponding author.

Chromosomal genes are also involved in the virulence ofY. pseudotuberculosis. Isberg and Falkow (22) identified a3.2-kb region on the chromosome, including the inv gene,which encodes a 103-kDa protein, invasin (Inv), promotingbacterial entry into epithelial cells in vitro (26). The expres-sion of this gene is thermoregulated (25), and invasin is foundin the outer membrane and on the surface of bacteria (26).Invasin can directly attach to mammalian cell lines (23) andrecognizes multiple integrins (24). A smaller invasion locuscalled ail (for attachment invasion locus), first identified onthe chromosome of Yersinia enterocolitica (34), is alsopresent on the chromosome of Y. pseudotuberculosis (35).There are no sequence homologies between inv and ail (33).Like inv, ail is thermoregulated and its product in Y.enterocolitica is a protein of 17 kDa exposed on the bacterialsurface (33).Although many virulence factors of Y. pseudotuberculosis

are presently characterized in vitro, the events that occur ina host infected by a pathogenic strain remain poorly under-stood. We previously observed the presence of bacteriaadhering to thrombocytes and associated to platelet aggre-gates in blood vessels of mice infected by a virulent strain ofY. pseudotuberculosis (46). This observation prompted us tostudy platelet-bacterium interactions. We report here that Y.pseudotuberculosis triggers platelet aggregate formation invitro and that invasin mediates bacterium-platelet interac-tion leading to platelet activation. Plasmid-free bacteria bindto platelet membrane glycoproteins and are rapidly internal-ized by thrombocytes.

MATERIALS AND METHODS

Strains and plasmids. Bacterial strains and plasmids usedin this study are described in Table 1. They were obtainedfrom Stanley Falkow (Stanford University, Palo Alto, Calif.)and Henri-Hubert Mollaret (Centre National de Referencedes Yersinia, Institut Pasteur, Paris, France). Yersiniastrains were grown in Luria broth (LB) medium supple-mented with either 5 mM CaCl2 or 20 mM sodium oxalate-20mM MgCl2 (Ca2'-deficient medium). Strains of Escherichia

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YERSINIA PSEUDOTUBERCULOSIS-PLATELET INTERACTIONS 367

TABLE 1. Bacterial strains and plasmids used in this study

Strain or Description or Reference orplasmid genotype source

Yersinia pseudo-tuberculosis

YPIII Inv', pYV SYPIIIc Inv', pYV cured 5YP202 Inv-, pYV cured 26IP2790c Inv', pYV Institut Pasteur,

Paris, FranceIP2790c Inv', pYV cured Institut Pasteur,

Paris, France

Escherichia coli F' thr leu lac Y rpsL 6HB101 mtl xyl hsdR hsdM

recA56PlasmidspBR325 pBR322 derivative 2pRI203 pBR325 inv 26

coli were grown in LB medium containing 100 ,ug of ampi-cillin per ml. Overnight cultures grown at 28 or 37°C were

centrifuged, and the pellets were resuspended in 0.15 MNaCl.

Antibodies. Monoclonal antibody (MAb) 3A2-1 recogniz-ing an epitope within the last 192 amino acids of invasin (29)was kindly provided by Ralph Isberg (Tufts University,Boston, Mass.). MAbs against platelet glycoproteins GPIa(P73), GPIb (P64), GPIc (P55), GPIIa (P104), GPIIIa (P107),GPIX (P5), VLA-4 (P91), and vitronectin receptor a chain(P102) were described at the 4th International Workshop andConference on Human Leucocyte Differentiation Antigens(50).

Platelet preparation. Human platelets were obtained fromtwo healthy donors [platelet phenotype plAl Bak (a+), Br(a-)]. Blood was aseptically drawn in 130 mM trisodiumcitrate (ratio 10:1). Platelet-rich plasma (PRP) or platelet-poor plasma was obtained by centrifuging blood at 200 x gfor 10 min at room temperature or at 3,000 x g for 30 min at4°C, respectively. Washed platelets were prepared as fol-lows. Blood was collected in a solution containing 0.4 mMcitric acid, 0.7 mM trisodium citrate, 0.24 g of glucose perliter, 25 mg of apyrase (Sigma Chemical Co., St. Louis, Mo.)per liter, and 7 mg of prostaglandin El (Sigma) per liter (ratio5:1) and centrifuged at 1,100 x g for 10 min at 20°C. PRP wasthen removed and centrifuged at 1,100 x g for 10 min.Platelet-poor plasma was removed, and the platelets were

suspended in a solution (final pH 6.5) containing 5 mM KCl,2.6 mM CaCl2, 1 mM MgCl2, 100 mM NaCl, 36 mM citricacid, 0.9 g of glucose per liter, 3.5 g of bovine albumin perliter, 25 mg of apyrase per liter, and 7 mg of prostaglandin Elper liter (buffer A). The washing procedure was repeatedtwice, and platelets were then resuspended in a solution(final pH 7.35) containing 137 mM NaCl, 2.6 mM KCl, 3.8mM CaCl2, 1 mM MgCl2, 12 mM NaHCO3, 0.4 mMNaH2PO4, 1 g of glucose per liter, 3.5 g of bovine albuminper liter, and 25 mg of apyrase per liter (buffer B). Plateletpreparations were used within 3 h after blood collection.

Platelet aggregation assay. Assays were performed in a

Chronolog 550 Lumiaggregometer (Coultronics, Hialeah,Fla.), stirring at 1,000 rpm, by incubating for 15 min at 37°C200 of PRP (3 x 108 platelets per ml preincubated for 1min at 37°C) with 200 ,u1 of bacterial suspensions adjusted toan optical density of 0.8 at 600 nm (109 bacteria per ml).Baseline (0%) and 100% aggregation were established by

light transmission through vials containing bacterial suspen-sions incubated with PRP or platelet-poor plasma, respec-tively. Aggregation curves were recorded for 15 min afterstarting the assay with a Chronolog graphic recorder (2cm/min). Soluble collagen (Organon Teknika Co., MorrisPlains, N.J.) and ADP (Sigma) were used as platelet ago-nists.

Binding of bacteria to platelets. Fifty microliters of asuspension of washed platelets (4 x 108/ml) suspended inbuffer B was incubated with 100 ,ul of a bacterial suspension(109 bacteria per ml) for 10 min at 37°C with stirring (1,000rpm). Smears were then prepared and stained by the May-Grunwald-Giemsa procedure. The number of bacteria asso-ciated with platelets (association index) was determined for100 cells. The binding assay was also performed with plate-lets preincubated for 20 to 30 min at room temperature withMAbs against platelet glycoproteins (7.5 ,ug of antibody per108 platelets). Data are expressed as means + standarderrors of the means of two experiments performed ondifferent days. Differences in the mean number of bacteriabound to platelets were compared by one-way analysis ofvariance using computer program StatView II.

Electron microscopy. (i) Fixation and embedding. After a10-min incubation of bacteria with platelets in buffer B, cellswere centrifuged at 400 x g for 7 min and then the pellet wasfixed for 1 to 2 h at room temperature with 2.5% glutaralde-hyde in cacodylate buffer (pH 7.2) containing 0.1 M sucrose.The samples were washed overnight in the same buffer,postfixed for 1 h with 1% osmium tetroxide in cacodylatebuffer, concentrated in 2% agar, and treated for 1 h with 1%uranyl acetate in Veronal buffer (pH 5.0). Samples were thendehydrated in acetone and embedded in Epon. Thin sectionswere stained with 2% uranyl acetate and lead acetate.

(ii). Ruthenium red staining. Ruthenium red staining wasperformed by adding ruthenium red to fixatives (glutaralde-hyde, osmium), as previously described (41).

RESULTS

Induction of platelet aggregation by Y. pseudotuberculosis.YPIII cells were incubated with PRP for 10 min at 37°C, andaggregate formation was monitored by using an aggregome-ter. At a ratio of three bacteria per one platelet, aggregationbegan after 1 min and reached a plateau within 4 min (Fig. 1).Platelet aggregation was complete under these conditionssince addition of two platelet agonists, collagen (2 ,ug/ml) orADP (20 ,uM), could not induce further aggregation. Aggre-gation was dependent on the bacterium-platelet ratio, asshown by a decrease of the magnitude of platelet aggregationwhen the ratio was reduced (3 bacteria per 10 platelets) (datanot shown).The platelet aggregation assay was done with strain YPIII

with or without pYV grown at 28 or at 37°C in a mediumcontaining either 2.5 mM Ca2" or no Ca2". As shown in Fig.1, the induction of platelet aggregation was independent ofthe presence of the pYV plasmid in bacteria and occurredwhen bacteria were grown at 28°C. In contrast, microorgan-isms grown at 37°C did not significantly aggregate platelets.Similar results were observed with wild-type strain IP2790and its pYV-cured derivative IP2790c grown at 28°C (datanot shown). However, in contrast with strain YPIII, strainIP2790 cultivated at 37°C aggregated platelets.

Involvement of Y. pseudotuberculosis invasin in plateletaggregation. We studied the role in platelet aggregate forma-tion of invasin, a 103-kDa outer membrane protein of Y.pseudotuberculosis, known to be maximally produced at

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FIG. 1. Platelet aggregation curves obtained by incubating PRPwith Y. pseudotuberculosis YPIII harboring (A) or not (B) a pYVplasmid and grown at (1) 28°C with 2.5 mM Ca2+; (2) 28°C withoutCa2"; (3) 37°C with 2.5 mM Ca2+; (4) 37°C without Ca2+ (platelet-bacterium ratio, 1/3). It is shown that platelet aggregation is a pYVplasmid-independent phenomenon and is maximal when bacteria aregrown at 28°C with or without Ca2" in culture medium.

28°C (25). We found that invasin was required to triggerplatelet aggregation by assaying platelet aggregate formationwith (i) strain YP202, an inv-defective mutant of strainYPIIIc; (ii) strain YPIIIc preincubated with an anti-invasinMAb; (iii) E. coli HB101 harboring plasmid pRI203, aderivative plasmid of pBR325 that contains a 4.6-kb insertencompassing the inv locus from strain YPIII and expressinginvasin in its outer membrane (26). As shown in Fig. 2A, theinv-defective mutant failed to aggregate platelets, thus sug-gesting that invasin mediates platelet aggregation. This as-sumption was further supported by showing that plateletaggregation was inhibited by preincubating Inv' bacteria for1 h at room temperature with an anti-invasin MAb (1.7mg/ml) diluted 1:100 and 1:1,000 (Fig. 2B). The inhibitoryeffect of the MAb was not observed for a higher dilution(1:10,000). Finally, strain HB101 harboring plasmid pRI203,grown either at 28 or at 37°C, triggered platelet aggregation,in contrast to control strain HB101 harboring plasmidpBR325 (Fig. 2C).

Identification of platelet membrane glycoproteins bindingbacteria. To identify the platelet membrane receptors inter-acting with invasin, we incubated bacteria with washedplatelets suspended in a buffered solution containing apyrase

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FIG. 2. Platelet aggregation curves obtained by incubating bac-teria with PRP (platelet-bacterium ratio, 1/3). (A) Strain YPIIIc(Inv') grown at 28°C (1) and 37°C (2) and strain YP202 (Inv-) grownat 28°C (3) and 37°C (4). (B) Strain YPIIIc grown at 28°C (1) andincubated with anti-invasin MAb diluted 1:100 (2), 1:1,000 (3), and1:10,000 (4). (C) E. coli HB101 grown at 37°C and harboring plasmidpBR325 (1) or pRI203 (pBR325 inv) (2). Evidence is shown thatinvasin mediates platelet aggregate formation.

(5'-adenosine-diphosphatase-5'-adenosine triphosphatase).Under these experimental conditions, platelet aggregationwas inhibited, thus allowing us to visualize and to quantifyinteraction between bacteria and platelets. This was done byincubating bacteria (strain YPIIIc or YP202) with apyrase-treated platelets for 10 min at 37°C and determining the meannumber of bacteria associated to platelets by optical micro-scopic examination of 100 stained platelets. We found thatinvasin-producing bacteria were associated with platelets(2.53 + 0.16 bacteria per platelet), whereas invasin-deficientbacteria were poorly bound to these cells (0.21 ± 0.04

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YERSINIA PSEUDOTUBERCULOSIS-PLATELET INTERACTIONS 369

TABLE 2. Effect of MAbs directed against platelet glycoproteins on association of Y. pseudotuberculosis to platelets

Strain MAb(s)a Platelet glycoprotein Association indexb Inhibition of bacterialspecificity binding to plateletsc'

YP202 (Inv-) P91 None 0.12 ± 0.03

YPIIIc (Inv') P91 None 1.52 ± 0.14P73 GPIa 2.13 ± 0.15 NoP64 GPIb 1.88 ± 0.14 NoP55 GPIc 0.88 ± 0.09 YesdP104 GPIIa 1.12 ± 0.11 YesP107 GPIIIa 1.86 ± 0.13 NoP5 GPIX 1.63 ± 0.15 NoP102 Vitronectin receptor (x chain 1.86 ± 0.14 NoP55 + P104 GPIc and GPIIa 0.43 ± 0.08 Yes

a Designation according to reference 50.b Determined as described in Materials and Methods. Mean number of bacteria per platelet + SEM of two separate experiments (for each experiment, scores

were determined on 100 platelets).I One-way analysis of variance (F test).d p < 0.05.

bacteria per platelet); this difference is highly significant (P< 0.001).The same adhesion assay was used to identify platelet

membrane glycoproteins capable of binding to bacteria.Platelets were incubated for 20 to 30 min at room tempera-ture with MAbs directed against various platelet glycopro-teins (7.5 ,ug of MAb per 108 platelets), and then bacteriawere added at a ratio of three bacteria per one platelet. After10 min of incubation at 37°C, the cell suspension was stainedand the number of bacteria adherent to platelets (associationindex) was determined by optical microscopy. The results ofthis experiment are shown in Table 2. Since no VLA-4structures exist on the surface of the platelet (19), MAb 91(anti-VLA-4) was used as a negative control for the inhibi-tion platelet bacterial adhesion assay, and any antibodiescausing inhibitory affects greater than this antibody wereconsidered to be antibodies defining invasin receptors onplatelets. Only anti-GPIc and -GPIIa MAbs significantlyreduced attachment of bacteria to platelets, and when plate-lets were incubated simultaneously with these two antibod-ies, binding of Inv' Y. pseudotuberculosis to thrombocyteswas decreased at a level close to that of Inv- Y. pseudo-tuberculosis (Table 2). These results indicate that Y. pseudo-tuberculosis attaches to platelets through GPIc and GPIIaand that bacterial binding to platelet membrane is mediatedby invasin.

Electron microscopy study showing phagocytosis of Y.pseudotuberculosis by platelets. Using the same experimentalprocedure described above with apyrase-treated platelets,we performed an electron microscopy study to furtheranalyze the bacterium-platelet interactions. As expected, wefound that invasin-producing Yersinia cells were bound toplatelets (Fig. 3A), in contrast to invasin-deficient bacteria,which were not seen adhering to platelets (Fig. 3B). Surpris-ingly, examination of thin sections revealed that the Yersiniacells associated with platelets were located inside phago-somelike structures (Fig. 4A). Ruthenium red stain, whichtraces the outline of the cell surface but not the phagosomemembrane (32), was used to distinguish genuine phago-somes. A quantitative electron microscopic study (examina-tion of 50 platelets) found that 35% of the phagosomelikeformations were unlabeled by ruthenium red (Fig. 4B), thusdemonstrating that they were genuine phagosomes, whereasthe remaining phagosomelike formations did not correspondto internalized bacteria but rather to membrane platelet

folds, as illustrated in Fig. 4C. Interestingly, we also foundthat E. coli expressing invasin was adherent to plateletscompared with E. coli HB101(pBR325), but after examina-tion of 100 anucleated cells, we never saw HB101(pRI203)cells inside platelets (Fig. 4D). These results indicate thatinvasin is involved in bacterial adherence to platelets butthat platelet-mediated phagocytosis required an additionalfactor(s) expressed by Y. pseudotuberculosis and not byInv+ E. coli.

DISCUSSION

Direct contact between Y. pseudotuberculosis and humanplatelets induces activation and platelet aggregation in vitro.Platelets became activated almost immediately after additionof bacteria, and aggregation was complete within few min-utes (Fig. 1). These results are in agreement with ourprevious observation showing that there are bacterium-platelet aggregates in the capillaries of mice infected intra-venously with a high inoculum of Y. pseudotuberculosis (46).Leung et al. (30) reported that YopM from Y. pestis, alsoexpressed by Y. pseudotuberculosis (42), inhibits thrombin-or ristocetin-induced platelet aggregation. No inhibition ofaggregate formation was observed when platelets were incu-bated with Y. pseudotuberculosis(pYV) (strain IP2790)grown at 37°C under Ca2+-restriction, conditions that allowYops expression (7, 11). However, our aggregation assayusing washed bacteria might not detect an inhibitory effectinduced by Yops, which are mostly secreted (11).Many microorganisms are able to induce platelet aggrega-

tion, including Streptococcus viridans (9, 20, 49), Strepto-coccus pyogenes (9, 28), Enterococcusfaecalis (8), Strepto-coccus pneumoniae (8), Staphylococcus aureus (8, 9, 18),Listeria monocytogenes (12), E. coli (8, 27), Fusobacteriumnecrophorum (15), Candida albicans (47), and Histoplasmacapsulatum (13). Platelet aggregate formation with strepto-cocci is most often detected after a lag phase (8, 49). Themechanisms by which microorganisms trigger thrombocyteaggregation are not completely understood. The receptorsfor the Fc region of immunoglobulin G and for complementC3b are present on the surface of platelets (39), and micro-organisms that bind immunoglobulin G or activate the com-plement system through the classical or alternate pathwaytrigger platelet aggregation (16, 18, 47, 48). We found thatplatelet aggregation rapidly occurred after addition of Y.

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BFIG. 3. Thin sections of platelets after contact with Y. pseudotuberculosis YPIIIc (Inv') or YP202 (Inv-). (A) Inv' bacteria (Y) are in

close contact or apparently inside (arrow) platelets (P); (B) inv-deficient bacteria (Y) are not seen adhering to platelets. Bar, 0.5 ,um.

pseudotuberculosis to platelets, indicating that direct bacte-rium-platelet binding might be a major inductive event foraggregation. Microscopic examination confirmed that bacte-ria directly attach to thrombocytes to induce platelet activa-tion and aggregation, even in the absence of plasma factors.That plasmid-free bacteria grown at 28°C induce an opti-

mal response strongly suggests an involvement of a ther-moregulated chromosomal gene(s) in the phenomenon ofplatelet aggregation. We found that invasin, a 103-kDa outermembrane protein of Y. pseudotuberculosis which is ex-pressed at higher levels at 28°C than at 37°C, is required totrigger aggregate formation since (i) an isogenic inv-deficientmutant failed to aggregate platelets; (ii) pretreatment of Y.pseudotuberculosis with an anti-invasin MAb blocked plate-let aggregation; and (iii) E. coli expressing invasin initiatedthe platelet response, in contrast to the control strain of E.coli. The discrepancy in the ability of strains YPIII andIP2790 grown at 37°C to induce platelet aggregation can beexplained by a difference in invasin expression at thistemperature by these two strains. Indeed, we recently re-ported (45a) that invasin expression in Y. pseudotuberculosisconsiderably differs according to the strains: some strains(such as strain IP2790) produced much more invasin in vitrothan others (such as strain YPIII). The fact that platelet

aggregation can be induced by bacteria grown at 37°C alsoemphasizes the relevance of our finding for the pathogenesisof Y. pseudotuberculosis infection.By using a panel of MAbs directed against platelet mem-

brane glycoproteins, we could also demonstrate that bacteriainteract with GPIc and GPIIa but not with GPIa, GPIb,GPIIIa, GPIX, and vitronectin receptor a chain (Table 2).These platelet surface molecules act as receptors for extra-cellular ligands, including thrombin and von Willebrandfactor for GPIb, collagen for GPIa, and fibrinogen, fi-bronectin, and thrombospondin for complex GPIIb-IIIa (39).These ligands complexed with the glycoprotein receptorsultimately produce a change in shape of the anucleated,disk-shaped cells to a sphere with numerous long extensions(pseudopodia), secretion of the contents of various intracel-lular granules, and aggregation to form a thrombus. Theplatelet glycoproteins interacting with Y. pseudotuberculosisare members of the integrin superfamily of cell adhesionmolecules (1). Integrins are ubiquitous heterodimeric glyco-proteins consisting of noncovalently associated polypeptidechains (a and ,B subunits) and are divided into subfamilies,each with a common P subunit capable of associating with aspecific group of a subunits (1). Platelet glycoproteins GPIcand GPIIa are related to the p1 subfamily (VLA, very late

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FIG. 4. Ultrastructural analysis of platelet-Inv' bacterium interactions. Cells were untreated (A) or treated (B, C, and D) with rutheniumred before embedding. (A) Yersinia cells are located inside phagosomelike structures. (B) Electron-dense deposit (arrow) is observed alongthe platelet membrane but not along the membrane of vacuoles containing Yersinia cells, indicating that these vacuoles are genuinephagosomes. (C) Vacuole membranes are labeled with ruthenium red (arrow), showing that bacteria are in fact extracellular. (D) E. coliproducing invasin is in close contact with platelet membrane but is not seen intracellularly. Bar, 0.5trm.

antigens), with the a chain of VLA-6 for GPIc and the I3chain of VLA integrins for GPIIa (1, 40). Our resultsshowing the interaction between Y. pseudotuberculosis andcertain platelet integrins are in good agreement with thosereported by Isberg and Leong (24). These researchers dem-onstrated by chromatography affinity of crude detergentextracts of mammalian cell lines that integrins containing thesubunit structures of a3P1 (VLA-3), a43 (VLA-4), a51(VLA-5), and a6P1 (VLA-6) bind to immobilized invasin,whereas cx213 (VLA-2) integrins, P2-chain integrins, andvitronectin receptor (a0i33) do not.

It is known that Y. pseudotuberculosis is able to enter awide variety of nonprofessional phagocytes, including fibro-blasts and epithelial cell, nonadherent T-cell, and erythro-leukemic cell lines (21, 36). Evidence exists that invasinpromotes bacterial penetration into epithelial cells (26).Here, we demonstrate, by an electron microscopy study ofplatelet-bacterium interactions using a staining procedureenabling us to distinguish genuine phagosomes, that Y.

pseudotuberculosis is also able to penetrate platelets. Bac-teria were ingested by platelets and included inside phago-somes. To our knowledge, this is the first report showingevidence that platelets are able to internalize bacteria.Whether or not microorganisms survive after phagocytosis iscurrently under investigation in our laboratory. Invasinpermits bacterial adherence to platelets, but penetration intothese cells seems to require an additional chromosomalfactor(s) since E. coli producing invasin adhered to plateletsbut did not enter these cells. Lipopolysaccharide, which isable to activate platelets (38), might be a potential candidateon the basis of its contribution for the entry process ofSalmonella choleraesuis in MDCK cells (14). In response toplatelet activation, cytoskeletal proteins redistribute inplatelets (3) and can then interact with integrins with subse-quent bacterial internalization.The significance of the induction of platelet aggregation by

Y. pseudotuberculosis in vitro in the pathogenic process isquestionable regarding the report of Rosqvist et al. (45), who

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372 SIMONET ET AL.

showed similar virulence for the mouse of Inv' and Inv- Y.pseudotuberculosis administered intraperitoneally. How-ever, bacterial virulence in this study was assessed only bydetermining the 50% lethal dose, and we found (45b) thatwhen Swiss mice were infected intravenously with 103- Y.pseudotuberculosis (without pYV), bacterial counts in themouse spleen over a 7-day period were regularly 10-foldhigher with Inv' bacteria than with Inv- bacteria. Onehypothesis might be that during the hematogenous dissem-ination of infection, which mainly occurs in immunocompro-mised patients, bacteria adhere to and activate platelets,inducing platelet aggregate formation. Then, circulatingplatelet microaggregates would be trapped in blood capillar-ies, especially those of the spleen and liver, causing micro-vascular occlusions as previously observed in vivo (46).Bacteria sequestrated within microthrombi and inside plate-lets would be protected from the host defense mechanismsand thus might multiply and spread toward the host tissues.

ACKNOWLEDGMENTSWe thank Stanley Falkow for critical reading of the manuscript.

M. C. Cottat, C. Dars, and N. Laurent are gratefully acknowledgedfor technical assistance, C. Coustere for statistical analysis, andM. L. Fourneaux for typing the manuscript.

This work was supported by "Contrat Jeune Equipe" no. 0009-1991 from the Universitd Paris V and by "Contrat Jeune Forma-tion" no. 90-04 from INSERM.

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Valle, N. Flores, and F. Bolivar. 1986. Plasmid vector pBR322and its special-purpose derivatives: a review. Gene 50:3-40.

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