Superantigen bacterial toxins: state of the art

Toxicon 39 (2001) 1691±1701

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Superantigen bacterial toxins: state of the art Heide MuÈller-Alouf a, Christophe Carnoy b,c,*, Michel Simonet b, Joseph E. Alouf d b

a DeÂpartement de Microbiologie des EcosysteÁmes, Institut Pasteur de Lille, Lille, France Equipe mixte Inserm (E9919)-Universite (JE2225), Institut de Biologie de Lille, Lille, France c Faculte de Pharmacie, Lille, France d Institut Pasteur, Paris, France

Abstract Sup/erantigens (SAgs) are viral and bacterial proteins exhibiting a highly potent polyclonal lymphocyte-proliferating activity for CD4 1, CD8 1 and sometimes gd 1 T cells of human and (or) various animal species. Unlike conventional antigens, SAgs bind as unprocessed proteins to invariant regions of major histocompatibility complex (MHC) class II molecules on the surface of antigen-presenting cells (APCs) and to particular motifs of the variable region of the b chain (Vb) of T-cell receptor (TcR) outside the antigen-binding groove. As a consequence, SAgs stimulate at nano-to picogram concentrations up to 10 to 30% of host T-cell repertoire while only one in 10 5 ±10 6 T cells (0.01±0.0001%) are activated upon conventional antigenic peptide binding to TcR. SAg activation of an unusually high percentage of T lymphocytes initiates massive release of pro-in¯ammatory and other cytokines which play a pivotal role in the pathogenesis of the diseases provoked by SAg-producing microorganisms. We brie¯y describe in this review the molecular and biological properties of the bacterial superantigen toxins and mitogens identi®ed in the past decade. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Superantigen; Mitogen; cytokine; T cell receptor; Major histocompatibility complex; Vb repertoire; Enterotoxin; Staphylococcus; Streptococcus; Yersinia pseudotuberculosis; Mycoplasma arthritidis; Toxic shock syndromes

1. General features of superantigens The term superantigens (SAg) coined by White et al. (1989) designates a group of viral proteins and bacterial protein toxins or mitogens exhibiting highly potent polyclonal lymphocyte-transforming properties toward human and (or) other mammalian CD4 1, CD8 1 and sometimes gd T lymphocytes. SAgs differ in several respects from conventional protein and peptide antigens (Fleischer, 1994; Uchiyama et al., 1994; Ulrich et al., 1995; Arvand et al., 1996; Florquin and Aaldering, 1997). The latter are processed (degraded) by classical antigen presenting cells (APCs) into peptides that are presented by major histocompatibility complex (MHC) molecules expressed at the surface of APCs. The peptide-MHC molecules are then speci®cally recognized by T lymphocytes through the groove formed by the a and b variable domains of the T * Corresponding author. Institut de Biologie de Lille, 1 rue du Professeur Calmette, 59021 Lille, France. Tel.: 133-3-20-87-1181; fax: 133-3-20-87-11-83. E-mail address: [email protected] (C. Carnoy).

cell receptor (TcR). Therefore, each antigen-processed peptide is destined to be recognized in the context of self MHC molecules by a speci®c cognate TcR structure. Consequently, when an antigen is introduced into host's organism, the number of T cells that would have the proper TcR that can recognize the appropriate peptide (epitope) of the antigen is in the range of 1±100 cells in a million. Unlike conventional peptide antigens, SAgs bind to invariant regions of MHC class II molecules outside the classical antigen-binding groove (Fig. 1) and are presented as unprocessed (intact) proteins to T lymphocytes expressing appropriate motifs on the variable domain of the b chain (Vb) of the TcR with relatively little or no involvement of other TcR components. The human TcR repertoire comprises about 24 major types of Vb elements (Champagne et al., 1993). As a consequence, SAgs stimulate at nano-to picogram concentrations up to 30% of the human T cell repertoire. Each SAg interacts speci®cally with a characteristic set of Vb families, and this set can serve as a ®ngerprint for a particular superantigen. The percentage of responding T cells is dictated by the number of Vb families that can interact with a given superantigen as well as by the frequency of expression of the

0041-0101/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0041-010 1(01)00156-8

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Fig. 1. Representation of T cell activation by a conventional peptide antigen (Ag) or by a superantigenic toxin (SAg). TcR: T cell receptor; APC: antigen-presenting cell; MHC II: major histocompatibility class II complex molecule.

superantigen-speci®c Vb families in a given individual's repertoire. Important biochemical signals are transmitted as a consequence of the binding of SAgs to the MHC molecules on APCs and to the TcR of T cells (ternary complexes). These signals are ampli®ed by another set of signals delivered by costimulatory molecules on APCs interacting with their respective ligands on T cells (Florquin and Aaldering, 1997). SAgs upregulate the expression of the costimulatory molecules and, by bridging T cells and APCs, they bring costimulatory molecules closer to their ligands, resulting in more ef®cient interaction and transduction of the proper signals required for T cell activation. As a consequence of this mode of interaction, both T cells and APCs are stimulated to release massive amounts of in¯ammatory cytokines, both monokines and lymphokines. T-cell derived cytokines augment the release of monokines from APCs and vice versa, thereby launching an in¯ammatory cytokine cascade that can overwhelm the host regulatory network and cause damage to tissues and organs which may result in disease and even death (Uchiyama et al., 1994; Ulrich et al., 1995; Kotb, 1997; MuÈller-Alouf et al., 1996, 1997; Florquin and Aaldering, 1997; Alouf et al., 1999). In this review we shall brie¯y summarize our current knowledge on those bacterial proteins so far reported to exhibit superantigenic properties. These SAgs correspond to a number of protein toxins and toxin-like mitogens of about 20±30 kDa produced by the Gram positive bacterial species, Staphylococcus aureus, Streptococcus pyogenes, Clostridium perfringens, the Gram negative bacterial species Yersinia pseudotuberculosis and the wall-less Mycoplasma arthritidis.

2. S. aureus SAgs The repertoire of S. aureus SAgs includes: (i) the classical enterotoxins A, B, C (C1, C2, C3 variants), D, E (Bohach et al., 1997; Marrack and Kappler, 1990; Svensson et al., 1997), (ii) the recently discovered enterotoxins G, H, I (Ren et al., 1994; Munson et al., 1998; Monday and Bohach, 1999), (iii) the toxic shock syndrome toxin-1 (TSST-1) and its ovine variant (Prasad et al., 1993; Acharya et al., 1994; Monday and Bohach, 1999), and (iv) the exfoliative toxins ETA and ETB (Zollner et al., 1996; Vath et al., 1997; Monday et al., 1999). The enterotoxins constitute a family of eight single-chain polypeptides (26±28 kDa, 228±239 amino acid residues) with a typical disul®de loop which may contribute to the emetic activity of these SAgs. These enterotoxins can be divided into two groups based on amino acid sequence homology (Fig. 2). The ®rst group consists of SEA, SEE, SED and SEH. SEA and SEE are .80% homologous, whereas SED shares approximately 54% homology with SEA and SEE. SEH displays only 30% identity with SEA. The homology within the second group including SEB, SEC, SEG and S. pyogenes erythrogenic (pyrogenic) exotoxin A (SPEA) ranges from 46 to 68%. SEG is 38±42% identical to SPEA and streptococcal SAg SSA (Munson et al., 1998). SEI is weakly homologous to SEA, SEE and SED (~ 28%) but is most similar to streptococcal SAgs (Fig. 2). TSST-1 (22 kDa, 194 amino acid residues) lacks disul®de loop and shows no signi®cant homology to any of the other streptococcal and staphylococcal SAgs, despite their similar biological activities (Acharya et al., 1994). Exfoliative toxins A and B (also called exfoliatins or

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Fig. 2. Phylogenetic analysis of bacterial superantigenic toxins based on amino acid sequences. Multiple sequence alignment was performed with Clustal X program. Unrooted phylogenetic tree was constructed according to the neighbor-joining (NJ) method (Saitou and Nei, 1987) using TreeView software. Only signi®cant bootstrap value ( ^ 95%) obtained from 1000 bootstrapped runs were indicated. The scale bar (NJ distance) represents 10% amino acid difference. SPEJ sequence is incomplete (N-terminal region missing) (Proft et al., 1999). S. pyogenes mitogens (SPM and SPM-2) are not represented in the ®gure as the nucleotide sequence is not available yet.

epidermolytic toxins) are biologically related but immunologically distinct proteins. These toxins are implicated in the pathogenesis of the staphylococcal scalded skin syndrome (Ladhani et al., 1999; Piemont, 1999). The molecular weights of toxins A and B are 26,951 and 27,318 Da (242 and 246 amino acid residues), respectively. They share 55% sequence identity and no homology with enterotoxins and TSST-1. Enterotoxins B and C are chromosomally encoded whereas the genes encoding enterotoxins A and C are located on bacteriophages and enterotoxin E gene is plasmidic. The location of the genes of enterotoxins G, H and I is still not determined to our knowledge. The genes encoding exfoliatins A and B are chromosomal and plasmidic, respectively. To date the crystal structures of enterotoxins A (SundstroÈm et al., 1996a; Schad et al., 1995), B (Swaminathan et al., 1992), C2 (Papageorgiou et al., 1995), C3 (Hoffmann et al., 1994), D (SundstroÈm et al., 1996b), TSST-1 (Prasad et al., 1993; Acharya et al., 1994) and exfoliatin A (Vath et al., 1997; Cavarelli et al., 1997) have been reported. It was shown for the enterotoxins and TSST-1 that their overall conformations were very similar although the latter exhibits little or no sequence homology with the former. These

toxins were tightly compacted, ellipsoidal proteins, folded into two unequal sized domains comprised of mixed a±b structures. Morevover, the determination of the threedimensional (3-D) structures of some enterotoxins or TSST-1 bound to MHC class II or TcR domains provided interesting information on the topology of MHC±SAg±TcR trimolecular complex as particularly shown for enterotoxin B (Jardetzky et al., 1994; Papageorgiou et al., 1998; Li et al., 1998) and TSST-1 (Kim et al., 1994). A detailed report was published by Monday and Bohach (1999) and Arcus et al. (2000). 3. S. pyogenes SAgs The repertoire of S. pyogenes SAgs includes: (i) the classical erythrogenic (scarlet fever) toxins A and C also designated streptococcal pyrogenic exotoxins A and C (SPEA, SPEC) (Norgren and Eriksson, 1997; Musser, 1997; Alouf et al., 1999), and (ii) a series of recently discovered mitogenic exoproteins, namely SPEF (or MF) (Iwasaki et al., 1993; Norrby-Teglund et al., 1994; Toyosaki et al., 1996), streptococcal superantigen (SSA) (Mollick et al., 1993; Reda et al., 1994), S. pyogenes mitogen (SPM) (Nemoto et al., 1996)

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and SPM-2 (Rikiishi et al., 1997), SMEZ (Kamezawa et al., 1997), SPEG, SPEH, SPEJ and SMEZ-2 (Proft et al., 1999, 2000). Interestingly, SMEZ-2 is the most potent SAg discovered thus far (Proft et al., 1999; Arcus et al., 2000). Erythrogenic toxins A and C are encoded by bacteriophage genes speA and speC integrated in the bacterial chromosome of S. pyogenes (Alouf et al., 1999). The mature form of exotoxins A and C comprises 228 and 208 amino acid residues resulting in molecular weights of 25,787 and 24,354 Da, respectively. The speA and speC genes may be present simultaneously in certain strains (Reichardt et al., 1992). Exotoxin A comprises three cysteine residues with a disul®de loop likely bridging Cys-87 and Cys-98 whereas exotoxin C possesses a unique cysteine residue. Four naturally occurring speA alleles have been demonstrated on strains recovered from patients with severe invasive diseases (Nelson et al., 1991). Three of these (speA1, speA2 and speA3) encode toxins differing by a single amino acid. The toxin encoded by speA4 was 9% divergent from the other three ones with 26 amino acid changes. Almost all changes occurred in regions that are not highly conserved when toxin A is aligned with exotoxin C and S. aureus enterotoxins (SEA through SEE). Strains expressing speA2 and speA3 have caused the majority of streptococcal toxic shock syndrome (STSS) episodes in the past 12 years (Kline and Collins, 1996) suggesting that the gene products SPEA2 and SPEA3 may be the more bioactive toxic forms of SPEA. Evidence for two alleles of speC (speC1 and speC2) has been reported by Kapur et al. (1992); speC2 differed from speC1 by nucleotide changes at position 438 and 452, both of which are synonymous (silent) A±G transitions in the third position of codons for lysine. Two new alleles designated speC3 and speC4 were found by nested PCR in clinical swedish isolates. The three novel streptococcal SAg genes, speG, speH and speJ were recently identi®ed from two S. pyogenes M1 serotype genomic database at the University of Oklahoma provided by J. J. Ferretti (Proft et al., 1999). The smez-2 gene was isolated from strain 2035 based on sequence homology to smez gene. Interestingly, 24 alleles of smez have been identi®ed (Proft et al., 2000). Crystal structure of SPEC (Roussel et al., 1997), SPEA (Papageorgiou et al., 1999), SSA (Sundberg and Jardetzky, 1999), SPEH and SMEZ-2 (Arcus et al., 2000) has been established. The structures conform to the general staphylococal and streptococcal SAgs folding pattern so far determined. The binding to MHC class II and TcR regions was also well characterized by SAg crystallization with these ligands.

4. Comparative structural, biological and pathophysiological features of staphylococcal and streptococcal SAgs Apart staphylococcal exfoliatins A and B which constitute a separate group of SAgs, the ca-25 superantigenic

proteins produced by S. aureus (enterotoxins A to I, TSST-1) and by S. pyogenes (erythrogenic toxins A, C and the novel superantigens discovered) share to various degrees both structural relatedness at amino acid (Fig. 2) and 3-D structure levels as well as a long list of common biological properties. With the recent elucidation of the SAg concept, it became clear that many of the shared biological and pathophysiological features of the toxins could be attributed, in part, to a single underlying cellular process (Mehindate et al., 1995). A family tree based on protein sequences shows homologies or similarities from 18 to .90% at the amino acid level between S. aureus and S. pyogenes SAgs. Sequence comparisons for the 22 members of the family indicate that these SAgs could be classi®ed into four subfamilies (Fig. 2): (i) group A comprising streptococcal SPEC, SPEG, SPEJ, SMEZ, SMEZ-2, SPEH and staphylococcal SEI, (ii) group B containing staphylococcal SEC1±3, SEG and SEB and streptococal SSA and SPEA, (iii) group C gathering staphylococcal SEA, SEE, SED and SEH and (iv) ®nally group D including toxins which do not belong to any of the previous subfamilies i.e. TSST-1, SPEF and staphylococcal ETs. Staphylococcal and streptococcal SAgs exhibit a remarkable spectrum of biological and pharmacological activities summarized in Table 1 (Ulrich et al., 1995; Florquin and Aaldering, 1997; Norgren and Eriksson, 1997; Monday and Bohach, 1999; Alouf et al., 1999). These activities concern: (i) the pyrogenic effects elicited by those toxins as a consequence of the release of interleukin (IL-) 1 and tumour necrosis factor (TNF)-a and their action on hypothalamus (Jupin et al., 1988; Fast et al., 1989); (ii) reticuloendothelial system blockade and enhancement of host susceptibility to lethal shock by endotoxin (Monday and Bohach, 1999); (iii) immunosuppression of humoral and cell-mediated responses, particularly non speci®c suppression of immunoglobulin synthesis, deletion of T-cell repertoire, anergy and apoptosis of lymphocytes (Miethke et al., 1996; Mahlknecht et al., 1996; Stohl et al., 1998). The expression of cytokines and other mediators by immune cells stimulated by staphylococcal and streptococcal superantigenic exotoxins is a typical effect of these bacterial effectors. The activation of an unusual high proportion of T lymphocytes and APCs in vivo and in vitro upon binding the SAg molecules triggers an initial production of a variety of cytokines and other mediators by the stimulated cells. This process leads to the elicitation (through a complex of upregulated and downregulated immunological network, activation signals and the cooperation of adhesion molecules on target cells) of a cascade of events including further release of a wide array of cytokines and other pharmacologically active products. The study of the elicitation of these mediators by immune system cells in response to these SAgs has been widely investigated (Jupin et al., 1988; Fast et al., 1989; Miethke et al., 1992; Hensler et al., 1993; Hackett and Stevens, 1993; KoÈnig et al., 1994; Rink et al., 1996; Kotb, 1997; MuÈller-Alouf et al., 1994, 1996, 1997).

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Table 1 Major biological and pathophysiological activities of S. aureus and S. pyogenes superantigen toxins 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Superantigenicity: polyclonal proliferation of T lymphocytes following binding to Vb motifs of T cell receptor and to non-polymorphic domains of MHC class II molecules on the surface of antigen presenting cells (APC). Induction and release of APC and Th1/Th2-derived, pro- and anti-in¯ammatory, immunoregulatory and haematopoetic cytokines. Induction of certain cytokine receptors. Production of inducible nitric oxide synthase. Elicitation under particular conditions of T cell anergy or apoptosis. Mimicry of cognate T/B cell interaction, proliferation/differentiation of resting B cells into immunoglobulin secreting cells or suppression of Ig classes. Pyrogenicity in animals (direct action on hypothalamus, IL-1 and TNF-a release). Blockade of reticuloendothelial system (impairment of clearance functions). Enhancement of host susceptibility (experimental animals) to lethal shock by endotoxins. Lethality, shock in experimental animals, direct capillary leak, skin rash. Involvement in the pathogenesis of severe diseases including toxic shock syndrome and possibly Kawasaki disease. Emetic effects (staphylococcal enterotoxins only).

Beside the release of cytokines, streptococcal pyrogenic exotoxin A and staphylococcal TSST-1 elicited the release of nitric oxide by human peripheral blood mononuclear cells (PBMC) (Sriskandan et al., 1996). This production was also reported for murine splenocytes challenged with SPEA. Streptococcal exotoxins A and C were shown to induce nitric oxide synthase in murine macrophages (Christ et al., 1997). S. aureus and S. pyogenes represent a potent threat to both healtly and immunocompromised individuals. These Grampositive cocci release a wide array of toxins including the SAgs mentioned here as well as a high number of potentially harmful exoenzymes. The production of these effectors during host infection by these bacteria contribute to a broad spectrum of diseases ranging from mild to severe cutaneous and other tissue infections, to life-threatining septicemia and toxic shock syndromes (Bohach et al., 1997; Bronze and Dale, 1996). The SAgs produced by both staphylococci and streptococci play a pivotal role in the pathogenesis of various diseases provoked by these bacteria (Norgren and Eriksson, 1997; Kotb, 1997; Stevens, 1995; Hackett and Stevens, 1993; KoÈhler, 1990; Leung et al., 1995a,b; Schafer and Sheil, 1995; Uchiyama et al., 1994). Both enterotoxins and TSST-1 have been implicated in toxic-shock syndrome and very likely in other diseases particularly Kawasaki's syndrome and atopic dermatitis (Leung et al., 1995a; Yarwood et al., 2000). Furthermore, the enterotoxins have the additional property of causing staphylococal food poisoning (Bergdoll, 1989). Staphylococcal toxic shock syndrome is an acute illness which can affect several organ systems in patients infected or colonized by S. aureus strains expressing TSST-1 and (or) enterotoxins. In addition, the patients present hypotension, fever, rash, vomiting/diarrhea and skin desquamation during convalescence and very often neurological manifestations. This disease may occur in either of two clinical features, menstrual or nonmenstrual. In contrast to TSST-1, the enterotoxins are unlikely to contribute to menstrual cases. Several lines of experimental, epidemiological and

clinical observations lead to the concept that the streptococcal superantigenic toxins A and C and very likely the other streptococcal SAgs play a pivotal role in the pathogenesis of scarlet fever streptococcal toxic-shock syndrome and other severe diseases (Bronze and Dale, 1996; KoÈhler, 1990; Kotb, 1997; Michie et al., 1994; Nadal et al., 1993; Stevens et al., 1989; Stevens, 1995). Streptococcal SAgs and, as mentioned before, staphylococcal SAgs are thought to be involved in the pathogenesis of Kawasaki disease and acute multisystem vasculitis of unknown aetiology characterized by marked activation of T lymphocytes and monocytes and elevated levels of IL-1, IL-6, IFN-g and TNF-a in the plasma of patients in the acute stage of the disease (Leung et al., 1995a). 5. C. perfringens enterotoxin C. perfringens enterotoxin (CPET) involved in food poisoning due to C. perfringens has been reported to behave as a SAg inducing the proliferation of human T cells expressing the Vb 6±9 and Vb 22 motifs on their TcRs (Bowness et al., 1992). This contention was not con®rmed in a further report (Krakauer et al., 1997). 6. Y. pseudotuberculosis SAg The only Gram negative microorganism known so far to produce a superantigenic toxin is Y. pseudotuberculosis, a pathogenic bacterium responsible for enteritis and mesenteric lymphadenitis. Y. pseudotuberculosis is thought to be involved in postinfection complications such as reactive arthritis and erythema nodosum and has been suggested as one of the causative agent of the Kawasaki syndrome (Sato et al., 1983; Baba et al., 1991). The superantigenic toxin produced by Y. pseudotuberculosis, designated YPM for Y. pseudotuberculosis-derived mitogen, is a 14.5-kDa protein (131 amino acid residues) which selectively stimulates human T cells bearing Vb3, Vb9, Vb13.1 and Vb13.2 TcR variable regions, in an MHC class II-dependent manner

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without being processed by the APCs (Ito et al., 1995; Miyoshi-Akiyama et al., 1995). In mice, YPM expands T-cells with the Vb7 and Vb8 TcR variable regions (Miyoshi-Akiyama et al., 1997). Three variants of the Yersinia superantigenic toxin have been described so far. YPMa and YPMc only differ by the substitution at position 51 of an histidine residue by a tyrosine residue (Carnoy and Simonet, 1999). Although YPMa and YPMb display 83% identity, they both recognize the same Vb region on human TcR (Ramamurthy et al., 1997). An epidemiological study based on molecular typing of 30 superantigen-expressing Y. pseudotuberculosis strains strongly suggested that strains containing the ypmB gene were ancestral compared to Y. pseudotuberculosis strains expressing the two other variants (Carnoy and Simonet, 1999). A phylogenetic analysis of bacterial superantigenic toxins based on amino acid sequences clearly demonstrated the absence of homology with the staphylococcal and streptococcal superantigenic toxins suggesting that YPM variants represent a new type of bacterial superantigen (Fig. 2). Unlike many SAg genes, ypm, which encodes the Y. pseudotuberculosis superantigenic toxin, does not seem to be located on a mobile genetic element such a phage or a plasmid (to be published). However, the G 1 C content of ypmA (35% for ypmA vs. 47% for Y. pseudotuberculosis genome) strongly suggests an exogenous origin of that gene but the microorganism containing the original gene is not characterized yet. The ypm gene was never found among the two other pathogenic Yersinia species, Y. enterocolitica, responsible for enteritis and mesenteric lymphadenitis and Y. pestis, the causative agent of plague. However, Stuart et al. (1992, 1995) found a superantigenic substance from lysate of Y. enterocolitica (serotype O:8) able to stimulate human and murine T cells in a Vb- and MHC class IIdependent manner. To date, the mitogenic substance of Y. enterocolitica has not been characterized. There is clearly a geographical heterogeneity between Far East and Europe in the prevalence of the ypm genes among the Y. pseudotuberculosis strains. Yoshino et al. (1995) detected ypmA in all strains from Eastern Russia, in 95% of the clinical isolates from Japan but in only 17% of European clinical isolates. Clinical manifestations due to Y. pseudotuberculosis infections are frequently more severe in the Far East than in Europe, but so far there is no direct evidence of correlation between these clinical manifestations (Far-East scarlet fever, Izumi fever, Kawasaki syndrome) and the presence of the ypm genes. The molecular mechanism of interaction of YPMa with its ligands, TcR and MHC class II molecules, is still unclear. Because the 3-D structure from X-ray diffraction or NMR studies is lacking, structure-function analysis attempted to characterize the structural domains involved in the biological activity of YPMs. Yoshino et al. (1996) tested the inhibitory effect of synthetic peptides covering YPMa on YPMa-induced proliferation of PBMC and found that the peptide corresponding to the 23 ®rst amino acids of the

mature protein was the best inhibitor. However, this peptide inhibited only 50% of the proliferation induced by YPMa, indicating that the biological activity was not strictly located at the N-terminal region and that other structural domains might be involved (Yoshino et al., 1996). Mutational analysis of YPMa indicated that amino acids involved in MHC II and TcR recognition were scattered along the polypeptide chain rather than gathered into domains (Ito et al., 1999). Interestingly, Seprenyi et al. (1999) characterized point mutants speci®cally impaired in their binding to the TcR Vb region and consequently in their cytotoxic activation and production of in¯ammatory cytokines. The two cysteine residues located at position 32 and 129 of the mature YPMa form a disul®de bond critical for the biological activity of the toxin (Ito et al., 1999). In vivo production of YPM has been demonstrated by Abe et al. (1997) who detected the presence of anti-YPM IgG in the blood of 61% of acutely infected patients with Y. pseudotuberculosis, and found an increase of Vb3-bearing T cells during the acute phase of the infection. The toxin was found to alter in vitro epithelial function by reducing active ion transport and increasing epithelial permeability (Donnelly et al., 1999) and to induce lethal shock when injected into mice (Miyoshi-Akiyama et al., 1997). Finally, a recent report demonstrated that YPMa was able to exacerbate the virulence of Y. pseudotuberculosis in mice (Carnoy et al., 2000). Altogether, these data suggest that the YPM toxins impact on the pathogenesis of Y. pseudotuberculosis but their role in the triggering of pathologies such as reactive arthritis and Kawasaki syndrome remains to be evaluated.

7. M. arthritidis SAg M. arthritidis, which induces chronic form of arthritis in rodent resembling human rheumatoid arthritis, produces a 25-kDa (213 amino acid residues) superantigenic toxin designated MAM (M. arthritidis mitogen). MAM, which was ®rst described in the early eighties (Cole et al., 1981), strongly activates murine T cells bearing Vb6 and Vb8 TcR variable regions. MAM also stimulates human T cell through the Vb17 region but to a less extent compared to its activation of murine T cell or to the human T cell activation by the staphylococcal enterotoxins (Cole and Atkin, 1991). A strong human B cell stimulation was also promoted by MAM (Tumang et al., 1990) and was suggested as the trigger of autoimmune disease (Crow et al., 1992). Like Yersinia superantigenic toxins, MAM does not have signi®cant homology with staphylococcal and streptococcal SAgs (Cole et al., 1996) (Fig. 2). All M. arthritidis strains tested so far contain the mam gene whereas it was never found in other pathogenic Mycoplasma (M. pneumoniae, M. hominis, M. salivarium¼) suggesting a genetic stability of the mam gene (Cole et al., 1996). The binding domains recognized by MAM on TcR and MHC class II molecules have been extensively characterized. Unlike other superantigenic toxins, MAM recognizes two

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distinct domains on TcR. It interacts with the Vb region of the TcR, but it also binds to the complementary-determining region 3 (CDR3) of the b chain which is central in the peptide recognition by the TcR (Hodstev et al., 1998). Regarding the binding of MAM on MHC class II molecules, the interaction is Zn dependent and cytokine production by monocytes requires a probable dimerization of the MHC molecules as for enterotoxin SEA (Bernatchez et al., 1997). Although MAM binds preferentially to H-2E murine class II molecule and to HLADR, the homologous MHC in humans (Cole et al., 1990), it also interacts with H2-A and HLA-DQ molecules (Cole et al., 1997). Inhibition of MAM-induced lymphocyte proliferation by MAM synthetic peptides revealed that two peptides covering the region from residue 15 to 38 and from residue 71 to 95 inhibited T cell activation. Although MAM is not phylogenetically related to other SAgs, the domain encompassing residues 15±38 shared limited homology with biologically active domains of SEB and SEC (Cole et al., 1996). Interestingly, the MAM domain 71±95 contains a consensus motif found in all legume lectins where it is responsible for the saccharide bioactive conformation. This domain is able to partially inhibit ConA-mediated lymphocyte proliferation suggesting a second pathway for T-cell activation (Cole et al., 1996). The role of MAM in the development of disease in rodents is still unclear. MAM can induce transient arthritis in rats when injected into the joint and M. arthritidis is more virulent only in mouse strains whose lymphocytes are reactive to MAM (Cole, 1999). However, non-virulent strains of M. arthritidis produce MAM and intravenous injection of MAM in mice failed to induce arthritis. Furthermore, other Mycoplasma factors have been suggested as potent arthritis inducers. Therefore, it appears that even if MAM is important for the development of arthritis in rodents, other bacterial and host factors also participate to the development of disease. MAM was thought to be produced exclusively by a non-human pathogen but recently, antibodies to MAM have been found in patients suffering from rheumatoid arthritis suggesting that MAM or its analog might be produced by a human Mycoplasma (Sawitzke et al., 2000). Experimental data demonstrate that YPMs from Y. pseudotuberculosis and MAM from M. arthritidis display all the features of superantigenic toxins as described by Marrack and Kappler (1990) although they do not share amino acid sequence homology with other superantigenic toxins (Fig. 2). This clearly indicates that primary amino acid sequences are not suf®cient to predict the superantigenic activity of a molecule. Resolution of the 3-D structure of YPM and MAM and comparison with known SAg 3-D structures, might assign a speci®c conformational structure to the biological activity of these toxins. Acknowledgements Christophe Carnoy was supported by a grant from the

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Centre Hospitalier ReÂgional et Universitaire de Lille, by the Fondation pour La Recherche MeÂdicale and by the ReÂgion Nord-Pas de Calais.

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