Déterminismes moléculaires de l'invasion tissulaire par Streptococcus pyogenes

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is a Gram-positive pathogen responsible for a wide range of diseases. GAS induces superficial infections such as pharyngitis, with 700 million cases/year worldwide, life threatening invasive infections such as necrotizing fasciitis, with 160 000 deaths, and post-infectious sequelae such as rheumatic fever. Altogether, GAS is responsible for 517 000 deaths in the world annually. GAS strains are genetically diverse and their genotyping involves the sequencing of the emm gene 5' end; emm encodes the M protein, a major virulence factor. More than 250 emm-types have been identified and they harbor specific virulence factor repertoires. During the pathogenesis of GAS infections, GAS adheres to the tissue, multiplies, affects the tissue integrity and invades it, resists and controls the immune response. During my PhD, we focused on deciphering the molecular mechanisms involved in GAS early critical steps of human tissue infection, with gyneco-obstetrical sphere infections, including endometritis, as a model. The first to third most prevalent emm-types eliciting invasive infections in Europe, emm28, is associated with these infections. In a first part of my project, we sought receptors for the emm28 specific R28 surface protein and which domains are involved in promoting adhesion to cells. In the second part, we set up an innovative ex vivo model of human decidual infection and we characterized the contribution of virulence factors to the colonization and invasion of this tissue. By cell adhesion experiments, we show that the R28 N-terminal domain (R28Nt) is responsible for an increase of GAS adhesion to human primary decidual cells. We have subdivided R28Nt into two subdomains; each is involved in binding to decidual, cervical and epithelial endometrial cells. We identified several putative R28Nt receptors and focused on R28Nt interaction with integrins. R28Nt and both subdomains directly interact with the integrins alpha3beta1, alpha6beta1 and alpha6beta4. Since R28Nt also increases the binding to the surface of pulmonary and skin epithelium cells, tissues encountered in GAS induced infections, we suggest that the R28Nt-integrin interactions contribute not only to emm28 endometritis, but also to the overall prevalence of the emm28 strains. In the second part of my project, to better characterize the mechanisms involved in GAS infections, we developed an ex vivo model of tissue infection, using human decidua, a tissue encountered during endometritis. We infected the maternal side of feto-maternal membrane, i. e. decidua, from healthy caesarians with an emm28 endometritis clinical isolate and its derived mutants. Using state of the art imaging set-up, image processing and analysis, we followed and quantified in real time different early infection steps. The bacteria multiply until they colonize the entire tissue surface in up to eight hours and this multiplication is triggered by the tissue. The bacteria form a multilayer biofilm of up to 14 µm thick. GAS readily and actively invades the decidua in a time-dependent manner, which depends on the presence of the cysteine protease SpeB. GAS induces dramatic cell cytotoxicity, with up to 50% of cells killed in the first four hours of infection; Streptolysin O (Slo) is involved in this cytotoxicity, confirming the critical importance of this factor in the early steps of infection. Cytokine overexpression and secretion in the tissue after infection indicate that GAS induces a limited immune response and the inflammatory response does not critically depend on the presence of Slo or SpeB. In conclusion, this study demonstrates the importance of several virulence factors, R28, SpeB and Slo, in the GAS emm28 early steps of infection, such as colonization, biofilm formation, cytotoxicity and tissue invasion, indicating their involvement not only in endometritis, but in other emm28 infections.Streptococcus pyogenes, également appelé Streptocoque du Goupe A (SGA), est un pathogène à l'origine d'une grande diversité d'infections, allant d'infections superficielles comme l'angine aux infections invasives, comme la fasciite nécrosante et les endométrites. Au 19ème siècle, une femme sur dix mourait après l'accouchement de fièvre puerpérale, notamment d'endométrite. En France, les infections gynéco-obstétricales correspondent encore de nos jours à 27 % des infections invasives à SGA chez les femmes. Les souches de SGA présentent une forte diversité génétique et de répertoire de facteurs de virulence. Le génotype emm28 est le troisième génotype le plus prévalent en France et il est associé aux endométrites. Nous avons analysé par deux axes complémentaires les facteurs et mécanismes impliqués dans les endométrites à SGA. Par des approches de biochimie et de biologie cellulaire, nous avons caractérisé l'interaction entre les cellules de l'hôte et R28, une protéine de surface spécifique du génotype emm28. Le domaine N-terminal de R28 (R28Nt) et ses deux sous-domaines favorisent la fixation des bactéries à des cellules endométriales, cervicales et déciduales. Ils fixent de manière directe les intégrines α3β1, α6β1 et α6β4. Par ailleurs, R28Nt promeut aussi l'adhésion à des cellules épithéliales de la peau et des poumons. Ces résultats suggèrent que la fixation des intégrines par R28Nt concourt, non seulement, aux endométrites dues au génotype emm28, mais aussi, et de manière plus générale, à la prévalence de ce génotype. Afin de mieux caractériser les étapes précoces essentielles au développement des endométrites à SGA, nous avons développé un modèle original d'infection : nous infectons ex vivo la décidue humaine, qui correspond à la membrane utérine durant la grossesse. Nous avons analysé les effets de l'infection de la décidue par des techniques de microscopie et d'analyse d'image de pointes. SGA adhère au tissu et se multiplie au contact de celui-ci grâce à des éléments sécrétés par le tissu. Sur ce tissu, SGA forme des biofilms composés d'ultrastructures ressemblant, pour certains, à des fils reliant deux coques d'une même chaine et, pour d'autres, à des filaments reliant plusieurs chaînettes ; certains s'organisent en réseau. GAS envahit en profondeur le tissu, ce qui dépend de l'expression de la cystéine protéase SpeB. SGA induit la mort de la moitié des cellules en moins de 4 h à travers la sécrétion de différents facteurs, dont la Streptolysine O (SLO). Enfin, GAS est capable de restreindre la réponse immunitaire du tissu à l'échelle transcriptomique et protéique, le contrôle protéique dépendant de l'expression de SLO et de SpeB

The Molecular Basis of Human IgG-Mediated Enhancement of C4b-Binding Protein Recruitment to Group A Streptococcus

Streptococcus pyogenes infects over 700 million people worldwide annually. Immune evasion strategies employed by the bacteria include binding of the complement inhibitors, C4b-binding protein (C4BP) and Factor H in a human-specific manner. We recently showed that human IgG increased C4BP binding to the bacterial surface, which promoted streptococcal immune evasion and increased mortality in mice. We sought to identify how IgG promotes C4BP binding to Protein H, a member of the M protein family. Dimerization of Protein H is pivotal for enhanced binding to human C4BP. First, we illustrated that Protein H, IgG, and C4BP formed a tripartite complex. Second, surface plasmon resonance revealed that Protein H binds IgG solely through Fc, but not Fab domains, and with high affinity (IgG-Protein H: KD = 0.4 nM; IgG-Fc-Protein H: KD \u3c /= 1.6 nM). Each IgG binds two Protein H molecules, while up to six molecules of Protein H bind one C4BP molecule. Third, interrupting Protein H dimerization either by raising temperature to 41 degrees C or with a synthetic peptide prevented IgG-Protein H interactions. IgG-Fc fragments or monoclonal human IgG permitted maximal C4BP binding when used at concentrations from 0.1 to 10 mg/ml. In contrast, pooled human IgG enhanced C4BP binding at concentrations up to 1 mg/ml; decreased C4BP binding at 10 mg/ml occurred probably because of Fab-streptococcal interactions at these high IgG concentrations. Taken together, our data show how S. pyogenes exploits human IgG to evade complement and enhance its virulence. Elucidation of this mechanism could aid design of new therapeutics against S. pyogenes

Streptococcus pyogenes infects human endometrium by limiting the innate immune response

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The N-terminal domain of the R28 protein promotes emm28 group A Streptococcus adhesion to host cells via direct binding to three integrins

International audienceGroup A Streptococcus (GAS) is a human-specific pathogen responsible for a wide range of diseases, ranging from superficial to life-threatening invasive infections, including endometritis, and autoimmune sequelae. GAS strains express a vast repertoire of virulence factors that varies depending on the strain genotype, and many adhesin proteins that enable GAS to adhere to host cells are restricted to some genotypes. GAS emm28 is the third most prevalent genotype in invasive infections in France and is associated with gyneco-obstetrical infections. emm28 strains harbor R28, a cell wall-anchored surface protein that has previously been reported to promote adhesion to cervical epithelial cells. Here, using cellular and biochemical approaches, we sought to determine whether R28 supports adhesion also to other cells and to characterize its cognate receptor. We show that through its N-terminal domain, R28Nt, R28 promotes bacterial adhesion to both endometrial-epithelial and endometrial-stromal cells. R28Nt was further subdivided into two domains, and we found that both are involved in cell binding. R28Nt and both subdomains interacted directly with the laminin-binding α3β1, α6β1, and α6β4 integrins; interestingly, these bindings events did not require divalent cations. R28 is the first GAS adhesin reported to bind directly to integrins that are expressed in most epithelial cells. Finally, R28Nt also promoted binding to keratinocytes and pulmonary epithelial cells, suggesting that it may be involved in supporting the prevalence in invasive infections of the emm28 genotype

Human igg increases virulence of streptococcus pyogenes through complement evasion

Streptococcus pyogenes is an exclusively human pathogen that can provoke mild skin and throat infections but can also cause fatal septicemia. This gram-positive bacterium has developed several strategies to evade the human immune system, enabling S. pyogenes to survive in the host. These strategies include recruiting several human plasma proteins, such as the complement inhibitor, C4b-binding protein (C4BP), and human (hu)-IgG through its Fc region to the bacterial surface to evade immune recognition. We identified a novel virulence mechanism whereby IgG-enhanced binding of C4BP to five of 12 tested S. pyogenes strains expressed diverse M proteins that are important surface-expressed virulence factors. Importantly, all strains that bound C4BP in the absence of IgG bound more C4BP when IgG was present. Further studies with an M1 strain that additionally expressed protein H, also a member of the M protein family, revealed that binding of hu-IgG Fc to protein H increased the affinity of protein H for C4BP. Increased C4BP binding accentuated complement downregulation, resulting in diminished bacterial killing. Accordingly, mortality from S. pyogenes infection in hu-C4BP transgenic mice was increased when hu-IgG or its Fc portion alone was administered concomitantly. Electron microscopy analysis of human tissue samples with necrotizing fasciitis confirmed increased C4BP binding to S. pyogenes when IgG was present. Our findings provide evidence of a paradoxical function of hu-IgG bound through Fc to diverse S. pyogenes isolates that increases their virulence and may counteract the beneficial effects of IgG opsonization

Complete Genome Sequence of Streptococcus pyogenes emm28 Clinical Isolate M28PF1, Responsible for a Puerperal Fever

International audienceWe report the sequence of the Streptococcus pyogenes emm28 strain M28PF1, isolated from a patient with postpartum endome-tritis. The M28 protein is smaller than that of MGAS6180 (NC_007296.1). Furthermore, the 1,896,976-bp-long chromosome presents, compared to that of MGAS6180, an inversion between the two comX genes