INFECTION AND IMMUNITY, Feb. 1992, p. 366-373

Vol. 60, No. 2

0019-9567/92/020366-08$02.00/0 Copyright © 1992, American Society for Microbiology

Human Platelet Aggregation by Yersinia pseudotuberculosis Is Mediated by Invasin MICHEL SIMONET,'* PATRICK TRIADOU,2 CLAUDE FREHEL,1 MARIE-CHRISTINE MOREL-KOPP,3 CECILE 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 appears that this phenomenon is mediated by invasin (Inv), a 103-kDa outer membrane protein that permits bacteria to penetrate mammalian cells, since (i) an isogenic inv-deficient mutant failed to aggregate platelets compared with 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 by using a panel of anti-platelet glycoprotein monoclonal antibodies in a bacterial adhesion assay. We found that bacteria bind to platelet membrane glycoproteins Ic and Ila. Electron microscopic study of bacterium-platelet interactions also revealed that bacteria expressing invasin attach to and are phagocytized by thrombocytes, in contrast to inv-deficient bacteria, indicating that these anucleated cells are able to internalize bacteria in vitro after specific interaction with invasin.

Yersinia pseudotuberculosis is a gram-negative bacterium that causes epizootic outbreaks of disease in birds and mammals. Occasionally, humans can be infected by ingestion of food or water contaminated by this pathogen. Acute mesenteric lymphadenitis is the most common clinical manifestation of human infection, sometimes complicated by septicemia, particularly in patients with underlying diseases such as cirrhosis or hemochromatosis (37). All pathogenic Yersinia strains harbor a 70-kb plasmid called pYV (plasmid Yersinia virulence), which encodes proteins designated Yops (Yersinia outer membrane protein). When bacteria are grown at 37°C in a calcium-deficient medium, Yops are expressed at high levels, as outer membrane-associated proteins or released in culture supernatants. The synthesis of Yops is inhibited at 37°C by adding Ca2+ (2.5 mM) or by incubating bacteria at 28°C regardless of the presence of Ca2+. yop mutants of Y. pseudotuberculosis (and other pathogenic Yersinia species) are reduced in virulence in the mouse, strongly suggesting that they are involved in the pathogenic process (for reviews, see references 7, 10, and 11). The exact function of Yops in the virulence remains unknown, except for YopH, YopE, and YopM. Both YopH and YopE inhibit phagocytosis of Y. pseudotuberculosis, while YopE is cytotoxic, inducing disruption of the actin microfilament structure in eukaryotic cells (42-44). Moreover, it has been recently reported that YopH is a tyrosine phosphatase that dephosphorylates eukaryotic proteins (4, 17). Finally, Leung et al. (30) showed that YopM-containing supernatant proteins of Yersinia pestis inhibited thrombin- or ristocetin-induced platelet aggregation, whereas this was not observed with supernatant proteins from the YopM-deficient Y. pestis mutant. This finding can be explained by a significant homology between a portion of the thrombin and the von Willebrand factorbinding domains of the a chain of human platelet glycoprotein lb (GPIb) and the amino acid sequence of YopM deduced from yopM (31). *

Chromosomal genes are also involved in the virulence of Y. pseudotuberculosis. Isberg and Falkow (22) identified a 3.2-kb region on the chromosome, including the inv gene, which encodes a 103-kDa protein, invasin (Inv), promoting bacterial entry into epithelial cells in vitro (26). The expression of this gene is thermoregulated (25), and invasin is found in the outer membrane and on the surface of bacteria (26). Invasin can directly attach to mammalian cell lines (23) and recognizes multiple integrins (24). A smaller invasion locus called ail (for attachment invasion locus), first identified on the chromosome of Yersinia enterocolitica (34), is also present 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 bacterial surface (33). Although many virulence factors of Y. pseudotuberculosis are presently characterized in vitro, the events that occur in a host infected by a pathogenic strain remain poorly understood. We previously observed the presence of bacteria adhering to thrombocytes and associated to platelet aggregates in blood vessels of mice infected by a virulent strain of Y. pseudotuberculosis (46). This observation prompted us to study platelet-bacterium interactions. We report here that Y. pseudotuberculosis triggers platelet aggregate formation in vitro and that invasin mediates bacterium-platelet interaction leading to platelet activation. Plasmid-free bacteria bind to platelet membrane glycoproteins and are rapidly internalized by thrombocytes. MATERIALS AND METHODS

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

Corresponding author. 366

YERSINIA PSEUDOTUBERCULOSIS-PLATELET INTERACTIONS

VOL. 60, 1992

TABLE 1. Bacterial strains and plasmids used in this study Strain or plasmid

Yersinia pseudotuberculosis YPIII

Description or genotype

YPIIIc

Inv', pYV Inv', pYV cured

YP202 IP2790c

Inv-, pYV cured Inv', pYV

IP2790c

Inv', pYV cured

Reference or source

S 5 26

Institut Pasteur, Paris, France Institut Pasteur, Paris, France

Escherichia coli HB101

F' thr leu lac Y rpsL mtl xyl hsdR hsdM recA56

6

Plasmids pBR325 pRI203

pBR322 derivative pBR325 inv

2 26

coli were grown in LB medium containing 100 ,ug of ampicillin per ml. Overnight cultures grown at 28 or 37°C were centrifuged, and the pellets were resuspended in 0.15 M NaCl. Antibodies. Monoclonal antibody (MAb) 3A2-1 recognizing 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 and Conference on Human Leucocyte Differentiation Antigens (50). Platelet preparation. Human platelets were obtained from two healthy donors [platelet phenotype plAl Bak (a+), Br (a-)]. Blood was aseptically drawn in 130 mM trisodium citrate (ratio 10:1). Platelet-rich plasma (PRP) or plateletpoor plasma was obtained by centrifuging blood at 200 x g for 10 min at room temperature or at 3,000 x g for 30 min at 4°C, respectively. Washed platelets were prepared as follows. Blood was collected in a solution containing 0.4 mM citric acid, 0.7 mM trisodium citrate, 0.24 g of glucose per liter, 25 mg of apyrase (Sigma Chemical Co., St. Louis, Mo.) per liter, and 7 mg of prostaglandin El (Sigma) per liter (ratio 5:1) and centrifuged at 1,100 x g for 10 min at 20°C. PRP was then 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 citric acid, 0.9 g of glucose per liter, 3.5 g of bovine albumin per liter, 25 mg of apyrase per liter, and 7 mg of prostaglandin El per liter (buffer A). The washing procedure was repeated twice, and platelets were then resuspended in a solution (final pH 7.35) containing 137 mM NaCl, 2.6 mM KCl, 3.8 mM CaCl2, 1 mM MgCl2, 12 mM NaHCO3, 0.4 mM NaH2PO4, 1 g of glucose per liter, 3.5 g of bovine albumin per liter, and 25 mg of apyrase per liter (buffer B). Platelet preparations 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°C 200 of PRP (3 x 108 platelets per ml preincubated for 1 min at 37°C) with 200 ,u1 of bacterial suspensions adjusted to an optical density of 0.8 at 600 nm (109 bacteria per ml). Baseline (0%) and 100% aggregation were established by

367

light transmission through vials containing bacterial suspensions incubated with PRP or platelet-poor plasma, respectively. Aggregation curves were recorded for 15 min after starting the assay with a Chronolog graphic recorder (2 cm/min). Soluble collagen (Organon Teknika Co., Morris Plains, N.J.) and ADP (Sigma) were used as platelet agonists. Binding of bacteria to platelets. Fifty microliters of a suspension of washed platelets (4 x 108/ml) suspended in buffer B was incubated with 100 ,ul of a bacterial suspension (109 bacteria per ml) for 10 min at 37°C with stirring (1,000 rpm). Smears were then prepared and stained by the MayGrunwald-Giemsa procedure. The number of bacteria associated with platelets (association index) was determined for 100 cells. The binding assay was also performed with platelets preincubated for 20 to 30 min at room temperature with MAbs against platelet glycoproteins (7.5 ,ug of antibody per 108 platelets). Data are expressed as means + standard errors of the means of two experiments performed on different days. Differences in the mean number of bacteria bound to platelets were compared by one-way analysis of variance using computer program StatView II. Electron microscopy. (i) Fixation and embedding. After a 10-min incubation of bacteria with platelets in buffer B, cells were centrifuged at 400 x g for 7 min and then the pellet was fixed for 1 to 2 h at room temperature with 2.5% glutaraldehyde 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 cacodylate buffer, concentrated in 2% agar, and treated for 1 h with 1% uranyl acetate in Veronal buffer (pH 5.0). Samples were then dehydrated in acetone and embedded in Epon. Thin sections were stained with 2% uranyl acetate and lead acetate. (ii). Ruthenium red staining. Ruthenium red staining was performed by adding ruthenium red to fixatives (glutaraldehyde, 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, and aggregate formation was monitored by using an aggregometer. At a ratio of three bacteria per one platelet, aggregation began after 1 min and reached a plateau within 4 min (Fig. 1). Platelet aggregation was complete under these conditions

since addition of two platelet agonists, collagen (2 ,ug/ml) or

ADP (20 ,uM), could not induce further aggregation. Aggregation was dependent on the bacterium-platelet ratio, as shown by a decrease of the magnitude of platelet aggregation when the ratio was reduced (3 bacteria per 10 platelets) (data not

shown).

The platelet aggregation assay was done with strain YPIII with or without pYV grown at 28 or at 37°C in a medium containing either 2.5 mM Ca2" or no Ca2". As shown in Fig. 1, the induction of platelet aggregation was independent of the presence of the pYV plasmid in bacteria and occurred when bacteria were grown at 28°C. In contrast, microorganisms grown at 37°C did not significantly aggregate platelets. Similar results were observed with wild-type strain IP2790 and its pYV-cured derivative IP2790c grown at 28°C (data not shown). However, in contrast with strain YPIII, strain IP2790 cultivated at 37°C aggregated platelets. Involvement of Y. pseudotuberculosis invasin in platelet aggregation. We studied the role in platelet aggregate formation of invasin, a 103-kDa outer membrane protein of Y. pseudotuberculosis, known to be maximally produced at

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INFECT. IMMUN.

SIMONET ET AL.

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