Stéphane Méresse

Peyer's Patch Dendritic Cells Sample Antigens by Extending Dendrites Through M Cell-Specific Transcellular Pores

BACKGROUND & AIMSnPeyers patches (PPs) of the small intestine are antigen sampling and inductive sites that help establish mucosal immunity. Luminal antigens are transported from the mucosal surface of PPs to the subepithelial dome (SED), through the specialized epithelial M cells of the follicle-associated epithelium. Among the SED resident dendritic cells (DCs), which are situated ideally for taking up these antigens, some express high levels of lysozyme (LysoDC) and have strong phagocytic activity. We investigated the mechanisms by which LysoDCs capture luminal antigens in vivo.nnnMETHODSnWe performed 2-photon microscopy on explants of PPs from mice in which the enhanced green fluorescent protein gene was inserted into the lysozyme M locus (lys-EGFP mice), allowing fluorescence detection of LysoDC.nnnRESULTSnLysoDC extended dendrites through M-cell-specific transcellular pores to the gut lumen. The M-cell adhesion molecules junctional adhesion molecule-A and epithelial cell adhesion molecule were recruited to sites of transcellular migration. Transcellular dendrites scanned the M-cell apical surface and the gut luminal content; they were able to take pathogenic bacteria and inert particles in the lumen before retracting back to the SED.nnnCONCLUSIONSnWe describe an antigen sampling mechanism that occurs in PPs and involves cooperation between M cells of the follicle-associated epithelium and DCs of the subepithelial dome. This process might be developed to target vaccines to the mucosa.

Redundant Hydrogen Peroxide Scavengers Contribute to Salmonella Virulence and Oxidative Stress Resistance

Salmonella enterica serovar Typhimurium is an intracellular pathogen that can survive and replicate within macrophages. One of the host defense mechanisms that Salmonella encounters during infection is the production of reactive oxygen species by the phagocyte NADPH oxidase. Among them, hydrogen peroxide (H(2)O(2)) can diffuse across bacterial membranes and damage biomolecules. Genome analysis allowed us to identify five genes encoding H(2)O(2) degrading enzymes: three catalases (KatE, KatG, and KatN) and two alkyl hydroperoxide reductases (AhpC and TsaA). Inactivation of the five cognate structural genes yielded the HpxF(-) mutant, which exhibited a high sensitivity to exogenous H(2)O(2) and a severe survival defect within macrophages. When the phagocyte NADPH oxidase was inhibited, its proliferation index increased 3.7-fold. Moreover, the overexpression of katG or tsaA in the HpxF(-) background was sufficient to confer a proliferation index similar to that of the wild type in macrophages and a resistance to millimolar H(2)O(2) in rich medium. The HpxF(-) mutant also showed an attenuated virulence in a mouse model. These data indicate that Salmonella catalases and alkyl hydroperoxide reductases are required to degrade H(2)O(2) and contribute to the virulence. This enzymatic redundancy highlights the evolutionary strategies developed by bacterial pathogens to survive within hostile environments.

Salmonella T3SSs: successful mission of the secret(ion) agents

Bacteria of the genus Salmonella express nanosyringe-like organelles called type three secretion systems (T3SSs). These systems promote the secretion of bacterial compounds and their translocation into host cells. Pathogenic Salmonella use two distinct T3SSs, with specialized functions, having the purpose to modify the biology of the host organism and to ensure a successful infection. The bacterial proteins translocated through the first T3SS (T3SS-1) facilitate the entry of Salmonella into host cells, whereas T3SS-2 is an important factor for shaping the intracellular replication niche. In addition both T3SSs have a strong impact on the host inflammation. For a long time the two T3SSs were thought to act separately. However, there is increasing evidence that their regulation depends not only on separate but also shared regulatory mechanisms and that their time of action during infection overlaps. Here, we review the current understanding of the structure and of the regulation of expression and activity of both T3SSs. The output image is multifaceted, as recent studies show that subpopulations of Salmonella present diverging patterns of expression and activity of T3SSs during important steps of infection. These diversities may advance the chances of Salmonella to outpace competitors and to well establish itself in its niche in the host.

Pathogenic Bacteria and Dead Cells Are Internalized by a Unique Subset of Peyer's Patch Dendritic Cells That Express Lysozyme

BACKGROUND & AIMSnLysozyme has an important role in preventing bacterial infection. In the gastrointestinal tract, lysozyme is thought to be mainly expressed by Paneth cells of the crypt epithelium. We investigated its expression in the Peyers patch, a major intestinal site of antigen sampling and pathogen entry.nnnMETHODSnWe performed immunostaining on normal and Salmonella Typhimurium-infected intestinal samples and analyzed them by confocal microscopy and flow cytometry.nnnRESULTSnIn Peyers patch of mouse, rat, and human, lysozyme was strongly expressed in the germinal center of follicles by tingible body macrophages and in the subepithelial dome by a subset of myeloid dendritic cells (DC). Among DC subsets from mouse Peyers patches, these lysozyme-expressing DC displayed the highest surface expression of class II major histocompatibility complex and costimulatory molecules; they were the most efficient at capturing microspheres in vitro. Moreover, they were the main DC subset involved in bacterial pathogen uptake and in dead cell clearance, including M cells.nnnCONCLUSIONSnThe subepithelial dome of Peyers patches contains a unique population of intestinal DC that secretes high levels of lysozyme and internalizes bacteria and dead cells.

A hypomorphic mutation in the Gfi1 transcriptional repressor results in a novel form of neutropenia

Using N‐ethyl‐N‐nitrosourea‐induced mutagenesis, we established a mouse model with a novel form of neutropenia resulting from a point mutation in the transcriptional repressor Growth Factor Independence 1 (Gfi1). These mice, called Genista, had normal viability and no weight loss, in contrast to mice expressing null alleles of the Gfi1 gene. Furthermore, the Genista mutation had a very limited impact on lymphopoiesis or on T‐ and B‐cell function. Within the bone marrow (BM), the Genista mutation resulted in a slight increase of monopoiesis and in a block of terminal granulopoiesis. This block occurred just after the metamyelocytic stage and resulted in the generation of small numbers of atypical CD11b+Ly‐6Gint neutrophils, the nuclear morphology of which resembled that of mature WT neutrophils. Unexpectedly, once released from the BM, these atypical neutrophils contributed to induce mild forms of autoantibody‐induced arthritis and of immune complex‐mediated lung alveolitis. They additionally failed to provide resistance to acute bacterial infection. Our study demonstrates that a hypomorphic mutation in the Gfi1 transcriptional repressor results in a novel form of neutropenia characterized by a split pattern of functional responses, reflecting the distinct thresholds required for eliciting neutrophil‐mediated inflammatory and anti‐infectious responses.

Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella.

During the course of infection, Salmonella enterica serovar Typhimurium must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments. Three low pH inducible amino acid decarboxylases were annotated in the genome of S. Typhimurium, AdiA, CadA and SpeF, which are specific for arginine, lysine and ornithine, respectively. In this study, we characterized and compared the contributions of those enzymes in response to acidic challenges. Individual mutants as well as a strain deleted for the three genes were tested for their ability (i) to survive an extreme acid shock, (ii) to grow at mild acidic pH and (iii) to infect the mouse animal model. We showed that the lysine decarboxylase CadA had the broadest range of activity since it both had the capacity to promote survival at pH 2.3 and growth at pH 4.5. The arginine decarboxylase AdiA was the most performant in protecting S. Typhimurium from a shock at pH 2.3 and the ornithine decarboxylase SpeF conferred the best growth advantage under anaerobiosis conditions at pH 4.5. We developed a GFP-based gene reporter to monitor the pH of the environment as perceived by S. Typhimurium. Results showed that activities of the lysine and ornithine decarboxylases at mild acidic pH did modify the local surrounding of S. Typhimurium both in culture medium and in macrophages. Finally, we tested the contribution of decarboxylases to virulence and found that these enzymes were dispensable for S. Typhimurium virulence during systemic infection. In the light of this result, we examined the genomes of Salmonella spp. normally responsible of systemic infection and observed that the genes encoding these enzymes were not well conserved, supporting the idea that these enzymes may be not required during systemic infection.

Kinesin regulation by Salmonella.

As the result of their adaptation to the host, intracellular pathogens have evolved mechanisms to usurp and take the control of eukaryotic processes. In the case of Salmonella, this is in part achieved through the cytoplasmic translocation of bacterial effectors capable of acting on the biology of infected cells. These bacterial effectors might have enzymatic activities or target eukaryotic proteins. We have identified two Salmonella effectors that target the plus-end directed microtubule motor kinesin-1. PipB2 is a Salmonella vacuole-specific cargo adaptor for kinesin-1 while SifA binds the host protein SKIP, which interacts with the microtubule motor. SKIP is a large, multi domain protein of unknown function. Our recent investigations show that SKIP regulates the positioning of late endosomal compartments in a microtubules and kinesin-1 dependent manner. Moreover they indicate that SKIP activates the microtubule motor both in the context of infected and non-infected cells. Here we review these recent results and propose a model for the Salmonella effector-mediated regulation of kinesin-1 activity.

In Vivo Identification and Characterization of CD4 + Cytotoxic T Cells Induced by Virulent Brucella abortus Infection

CD4+ T cells display a variety of helper functions necessary for an efficient adaptive immune response against bacterial invaders. This work reports the in vivo identification and characterization of murine cytotoxic CD4+ T cells (CD4+ CTL) during Brucella abortus infection. These CD4+ CTLs express granzyme B and exhibit immunophenotypic features consistent with fully differentiated T cells. They express CD25, CD44, CD62L ,CD43 molecules at their surface and produce IFN-γ. Moreover, these cells express neither the co-stimulatory molecule CD27 nor the memory T cell marker CD127. We show here that CD4+ CTLs are capable of cytolytic action against Brucella-infected antigen presenting cells (APC) but not against Mycobacterium-infected APC. Cytotoxic CD4+ T cell population appears at early stages of the infection concomitantly with high levels of IFN-γ and granzyme B expression. CD4+ CTLs represent a so far uncharacterized immune cell sub-type triggered by early immune responses upon Brucella abortus infection.

The infectious intracellular lifestyle of Salmonella enterica relies on the adaptation to nutritional conditions within the Salmonella-containing vacuole

ABSTRACT Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative pathogen that causes various host-specific diseases. During their life cycle, Salmonellae survive frequent exposures to a variety of environmental stresses, e.g. carbon-source starvation. The virulence of this pathogen relies on its ability to establish a replicative niche, named Salmonella-containing vacuole, inside host cells. However, the microenvironment of the SCV and the bacterial metabolic pathways required during infection are largely undefined. In this work we developed different biological probes whose expression is modulated by the environment and the physiological state of the bacterium. We constructed transcriptional reporters by fusing promoter regions to the gfpmut3a gene to monitor the expression profile of genes involved in glucose utilization and lipid catabolism. The induction of these probes by a specific metabolic change was first tested in vitro, and then during different conditions of infection in macrophages. We were able to determine that Entner-Doudoroff is the main metabolic pathway utilized by Salmonella during infection in mouse macrophages. Furthermore, we found sub-populations of bacteria expressing genes involved in pathways for the utilization of different sources of carbon. These populations are modified in presence of different metabolizable substrates, suggesting the coexistence of Salmonella with diverse metabolic states during the infection.

A Method to Introduce an Internal Tag Sequence into a Salmonella Chromosomal Gene

Epitope tags are short peptide sequences that are particularly useful for the characterization of proteins against which no antibody has been developed. Influenza hemagglutinin (HA) tag is one of the most widely used epitope tags as several valuable monoclonal and polyclonal antibodies that can be used in various techniques are commercially available. Therefore, adding a HA tag to a protein of interest is quite helpful to get rapid and cost less information regarding its localization, its expression or its biological function. In this chapter, we describe a process, derived from the Datsenko and Wanner procedure, which allows the introduction of an internal 2HA tag sequence into a chromosomal gene of the bacterial pathogen Salmonella.