Functional Analysis of the Quorum-Sensing Streptococcal Invasion Locus (sil)

Group A streptococcus (GAS) causes a wide variety of human diseases, and at the same time, GAS can also circulate without producing symptoms, similar to its close commensal relative, group G streptococcus (GGS). We previously identified, by transposon-tagged mutagenesis, the streptococcal invasion locus (sil). sil is a quorum-sensing regulated locus which is activated by the autoinducer peptide SilCR through the two-component system SilA-SilB. Here we characterize the DNA promoter region necessary for SilA-mediated activation. This site is composed of two direct repeats of 10 bp, separated by a spacer of 11 bp. Fusion of this site to gfp allowed us to systematically introduce single-base substitutions in the repeats region and to assess the relative contribution of various positions to promoter strength. We then developed an algorithm giving different weights to these positions, and performed a chromosome-wide bioinformatics search which was validated by transcriptome analysis. We identified 13 genes, mostly bacteriocin related, that are directly under the control of SilA. Having developed the ability to quantify SilCR signaling via GFP accumulation prompted us to search for GAS and GGS strains that sense and produce SilCR. While the majority of GAS strains lost sil, all GGS strains examined still possess the locus and ∼63% are able to respond to exogenously added SilCR. By triggering the autoinduction circle using a minute concentration of synthetic SilCR, we identified GAS and GGS strains that are capable of sensing and naturally producing SilCR, and showed that SilCR can be sensed across these streptococci species. These findings suggest that sil may be involved in colonization and establishment of commensal host-bacterial relationships

Identification of Parameters of Host Cell Vulnerability during Salmonella Infection by Quantitative Image Analysis and Modeling

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Injection of T3SS effectors not resulting in invasion is the main targeting mechanism of Shigella toward human lymphocytes

International audienceThe enteroinvasive bacterium Shigella is a facultative intracellular bacterium known, in vitro, to invade a large diversity of cells through the delivery of virulence effectors into the cell cytoplasm via a type III secretion system (T3SS). Here, we provide evidence that the injection of T3SS effectors does not necessarily result in cell invasion. Indeed, we demonstrate through optimization of a T3SS injection reporter that effector injection without subsequent cell invasion, termed the injection-only mechanism, is the main strategy used by Shigella to target human immune cells. We show that in vitro-activated human peripheral blood B, CD4+ T, and CD8+ T lymphocytes as well as switched memory B cells are mostly targeted by the injection-only mechanism. B and T lymphocytes residing in the human colonic lamina propria, encountered by Shigella upon its crossing of the mucosal barrier, are also mainly targeted by injection-only. These findings reveal that cells refractory to invasion can still be injected, thus extending the panel of host cells manipulated to the benefit of the pathogen. Future analysis of the functional consequences of the injection-only mechanism toward immune cells will contribute to the understanding of the priming of adaptive immunity, which is known to be altered during the course of natural Shigella infection

B lymphocytes undergo TLR2-dependent apoptosis upon Shigella infection mediated by the virulence factor IpaD

International audienceShigella is a gram-negative enteroinvasive bacterium and the causative agent of bacillary dysentery, an acute recto-colitis. Antibody-mediated natural immunity to Shigella requires several episodes of infection to get primed and is short-lasting, suggesting that the B cell response is functionally impaired. Here we show that upon ex vivo infection of human colonic tissue, invasive S. flexneri interacts with and invades B lymphocytes. We observe the induction of a type three secretion apparatus (T3SA)-dependent B cell death in vitro, both in lamina propria B lymphocytes and the human CL-01 B cell line. This cell death and the parallel reduction of the B cell pool can also be observed in an in vivo mouse infection model. Intriguingly, Shigella-induced B cell death does not require bacterial invasion or injection of virulence effectors via the T3SA in vitro. Instead, the virulence factor IpaD triggers mitochondrial B cell apoptosis in the presence of bacterial co-signals that render B lymphocytes prone to die. We provide evidence that IpaD binds to and induces apoptosis via TLR2, a signaling pathway that has thus far only been considered as a mitogenic stimulus for B lymphocytes. Apoptotic B lymphocytes in close contact with Shigella displaying IpaD are also detected in isolated lymphoid follicles of rectal biopsies of naturally-infected individuals. These findings reveal a novel mechanism of T3SA action to induce B cell death by the binding of a virulence factor and reveal an efficient strategy by which entero-invasive pathogens could impair the priming of a protective immune response

Glycan-Glycan Interaction Determines Shigella Tropism toward Human T Lymphocytes

International audienceDirect interactions between bacterial and host glycans have been recently reported to be involved in the binding of pathogenic bacteria to host cells. In the case of Shigella, the Gram-negative enteroinvasive bacterium responsible for acute rectocolitis, such interactions contribute to bacterial adherence to epithelial cells. However, the role of glycans in the tropism of Shigella for immune cells whose glycosylation pattern varies depending on their activation state is unknown. We previously reported that Shigella targets activated, but not nonactivated, human CD4+ T lymphocytes. Here, we show that nonactivated CD4+ T lymphocytes can be turned into Shigella-targetable cells upon loading of their plasma membrane with sialylated glycosphingolipids (also termed gangliosides). The Shigella targeting profile of ganglioside-loaded nonactivated T cells is similar to that of activated T cells, with a predominance of injection of effectors from the type III secretion system (T3SS) not resulting in cell invasion. We demonstrate that gangliosides interact with the O-antigen polysaccharide moiety of lipopolysaccharide (LPS), the major bacterial surface antigen, thus promoting Shigella binding to CD4+ T cells. This binding step is critical for the subsequent injection of T3SS effectors, a step which we univocally demonstrate to be dependent on actin polymerization. Altogether, these findings highlight the critical role of glycan-glycan interactions in Shigella pathogenesis.IMPORTANCE Glycosylation of host cell surface varies with species and location in the body, thus contributing to species specificity and tropism of microorganisms. Cross talk by Shigella, the Gram-negative enteroinvasive bacterium responsible for bacillary dysentery, with its exclusively human host has been extensively studied. However, the molecular determinants of the step of binding to host cells are poorly defined. Taking advantage of the observation that human-activated CD4+ T lymphocytes, but not nonactivated cells, are targets of Shigella, we succeeded in rendering the refractory cells susceptible to targeting upon loading of their plasma membrane with sialylated glycosphingolipids (gangliosides) that are abundantly present on activated cells. We show that interactions between the sugar polar part of gangliosides and the polysaccharide moiety of Shigella lipopolysaccharide (LPS) promote bacterial binding, which results in the injection of effectors via the type III secretion system. Whereas LPS interaction with gangliosides was proposed long ago and recently extended to a large variety of glycans, our findings reveal that such glycan-glycan interactions are critical for Shigella pathogenesis by driving selective interactions with host cells, including immune cells

B lymphocytes undergo TLR2-dependent apoptosis upon Shigella infection

International audienceAntibody-mediated immunity to Shigella, the causative agent of bacillary dysentery, requires several episodes of infection to get primed and is short-lasting, suggesting that the B cell response is functionally impaired. We show that upon ex vivo infection of human colonic tissue, invasive S. flexneri interacts with and occasionally invades B lymphocytes. The induction of a type three secretion apparatus (T3SA)-dependent B cell death is observed in the human CL-01 B cell line in vitro, as well as in mouse B lymphocytes in vivo. In addition to cell death occurring in Shigella-invaded CL-01 B lymphocytes, we provide evidence that the T3SA needle tip protein IpaD can induce cell death in noninvaded cells. IpaD binds to and induces B cell apoptosis via TLR2, a signaling receptor thus far considered to result in activation of B lymphocytes. The presence of bacterial co-signals is required to sensitize B cells to apoptosis and to up-regulate tlr2, thus enhancing IpaD binding. Apoptotic B lymphocytes in contact with Shigella-IpaD are detected in rectal biopsies of infected individuals. This study therefore adds direct B lymphocyte targeting to the diversity of mechanisms used by Shigella to dampen the host immune response

A streptococcal protease that degrades CXC chemokines and impairs bacterial clearance from infected tissues

Group A Streptococcus (GAS) causes the life-threatening infection in humans known as necrotizing fasciitis (NF). Infected subcutaneous tissues from an NF patient and mice challenged with the same GAS strain possessed high bacterial loads but a striking paucity of infiltrating polymorphonuclear leukocytes (PMNs). Impaired PMN recruitment was attributed to degradation of the chemokine IL-8 by a GAS serine peptidase. Here, we use bioinformatics approach coupled with target mutagenesis to identify this peptidase as ScpC. We show that SilCR pheromone downregulates scpC transcription via the two-component system—SilA/B. In addition, we demonstrate that in vitro, ScpC degrades the CXC chemokines: IL-8 (human), KC, and MIP-2 (both murine). Furthermore, using a murine model of human NF, we demonstrate that ScpC, but not the C5a peptidase ScpA, is an essential virulence factor. An ScpC-deficient mutant is innocuous for untreated mice but lethal for PMN-depleted mice. ScpC degrades KC and MIP-2 locally in the infected skin tissues, inhibiting PMN recruitment. In conclusion, ScpC represents a novel GAS virulence factor functioning to directly inactivate a key element of the host innate immune response