Virulent Shigella flexneri subverts the host innate immune response through manipulation of antimicrobial peptide gene expression

Antimicrobial factors are efficient defense components of the innate immunity, playing a crucial role in the intestinal homeostasis and protection against pathogens. In this study, we report that upon infection of polarized human intestinal cells in vitro, virulent Shigella flexneri suppress transcription of several genes encoding antimicrobial cationic peptides, particularly the human β-defensin hBD-3, which we show to be especially active against S. flexneri. This is an example of targeted survival strategy. We also identify the MxiE bacterial regulator, which controls a regulon encompassing a set of virulence plasmid-encoded effectors injected into host cells and regulating innate signaling, as being responsible for this dedicated regulatory process. In vivo, in a model of human intestinal xenotransplant, we confirm at the transcriptional and translational level, the presence of a dedicated MxiE-dependent system allowing S. flexneri to suppress expression of antimicrobial cationic peptides and promoting its deeper progression toward intestinal crypts. We demonstrate that this system is also able to down-regulate additional innate immunity genes, such as the chemokine CCL20 gene, leading to compromised recruitment of dendritic cells to the lamina propria of infected tissues. Thus, S. flexneri has developed a dedicated strategy to weaken the innate immunity to manage its survival and colonization ability in the intestine

Role of Paneth cells during infection of neonatal mice by Cryptosporidium parvum

Cryptosporidium parvum is a zoonotic apicomplexan parasite responsible for a diarrheal disease named cryptosporidiosis. This protozoan parasite is found worldwide and is transmitted by contaminated water. The immature intestinal immune system in very young animals and children under 5 places them at high risk of developing severe cryptosporidiosis. Paneth cells (PC) are specialized intestinal epithelial cells located at the base of intestinal crypts producing antimicrobial peptides (AMPs) that develop and mature after birth. We and others have already described in vitro that antimicrobial peptides such as CRAMP and CCL20 can alter the viability of sporozoites of C. parvum (1). We therefore wondered whether PCs and the AMPs that they produce can participate in the protective innate immune response against the parasite. By using a mouse model of neonatal cryptosporidiosis, we investigated the role of Paneth cells in the innate immune response against C. parvum. We first compared the susceptibility to C. parvum of mice genetically modified to be depleted of PCs (Sox9flox/flox-vil-Cre mice) and observed an increased level of infection when PCs are absent, associated with a reduced expression of AMPs. We also determined the effect of Cryptosporidium parvum infection on PC development and activity. By immunofluorescence, we observed on intestinal sections that C. parvum infection decreases the number of granule-positive-PCs and lysozymepositive- PCs in neonatal mice. Altogether, these first results clearly demonstrate that PCs are important contributors of the innate protective immune response in mice and that lyzozyme, already described to be efficient in vitro on C. parvum sporozoite viability, may be involved in this effect

Role of Paneth cells during infection of neonatal mice by Cryptosporidium parvum

Cryptosporidium parvum is a zoonotic apicomplexan parasite responsible for a diarrheal disease named cryptosporidiosis. This protozoan parasite is found worldwide and is transmitted by contaminated water. The immature intestinal immune system in very young animals and children under 5 places them at high risk of developing severe cryptosporidiosis. Paneth cells (PC) are specialized intestinal epithelial cells located at the base of intestinal crypts producing antimicrobial peptides (AMPs) that develop and mature after birth. We and others have already described in vitro that antimicrobial peptides such as CRAMP and CCL20 can alter the viability of sporozoites of C. parvum (1). We therefore wondered whether PCs and the AMPs that they produce can participate in the protective innate immune response against the parasite. By using a mouse model of neonatal cryptosporidiosis, we investigated the role of Paneth cells in the innate immune response against C. parvum. We first compared the susceptibility to C. parvum of mice genetically modified to be depleted of PCs (Sox9flox/flox-vil-Cre mice) and observed an increased level of infection when PCs are absent, associated with a reduced expression of AMPs. We also determined the effect of Cryptosporidium parvum infection on PC development and activity. By immunofluorescence, we observed on intestinal sections that C. parvum infection decreases the number of granule-positive-PCs and lysozymepositive- PCs in neonatal mice. Altogether, these first results clearly demonstrate that PCs are important contributors of the innate protective immune response in mice and that lyzozyme, already described to be efficient in vitro on C. parvum sporozoite viability, may be involved in this effect

Functional genomics of Lactobacillus casei establishment in the gut

International audienceAlthough the composition of the gut microbiota and its symbiotic contribution to key host physiological functions are well established, little is known as yet about the bacterial factors that account for this symbiosis. We selected Lactobacillus casei as a model microorganism to proceed to genomewide identification of the functions required for a symbiont to establish colonization in the gut. As a result of our recent development of a transposon-mutagenesis tool that overcomes the barrier that had prevented L. casei random mutagenesis, we developed a signature-tagged mutagenesis approach combining whole-genome reverse genetics using a set of tagged transposons and in vivo screening using the rabbit ligated ileal loop model. After sequencing transposon insertion sites in 9,250 random mutants, we assembled a library of 1,110 independent mutants, all disrupted in a different gene, that provides a representative view of the L. casei genome. By determining the relative quantity of each of the 1,110 mutants before and after the in vivo challenge, we identified a core of 47 L. casei genes necessary for its establishment in the gut. They are involved in housekeeping functions, metabolism (sugar, amino acids), cell wall biogenesis, and adaptation to environment. Hence we provide what is, to our knowledge, the first global functional genomics analysis of L. casei symbiosis

Epithelial Cell Proliferation Arrest Induced by Lactate and Acetate from <i>Lactobacillus casei</i> and <i>Bifidobacterium breve</i>

<div><p>In an attempt to identify and characterize how symbiotic bacteria of the gut microbiota affect the molecular and cellular mechanisms of epithelial homeostasis, intestinal epithelial cells were co-cultured with either <i>Lactobacillus</i> or <i>Bifidobacterium</i> as <i>bona fide</i> symbionts to examine potential gene modulations. In addition to genes involved in the innate immune response, genes encoding check-point molecules controlling the cell cycle were among the most modulated in the course of these interactions. In the m-ICcl2 murine cell line, genes encoding cyclin E1 and cyclin D1 were strongly down regulated by <i>L. casei</i> and <i>B. breve</i> respectively. Cell proliferation arrest was accordingly confirmed. Short chain fatty acids (SCFA) were the effectors of this modulation, alone or in conjunction with the acidic pH they generated. These results demonstrate that the production of SCFAs, a characteristic of these symbiotic microorganisms, is potentially an essential regulatory effector of epithelial proliferation in the gut.</p></div

Acetate induces cell proliferation arrest in a concentration & pH dependent manner.

<p>A: Number of m-ICcl2 per well after incubation at different pH with 20 mM of SCFA. Number represent cell counts (x10⁵) per well. B-C: qRT-PCR of cyclin D1 (B) and cyclin E1 (C) gene expression after incubation of m-ICcl2 with or without 20 mM acetate or lactate at different pHs. qRT-PCR was analyzed by the dδCt method using m-ICcl2 alone and GAPDH as reference.</p

Impact of acetate and lactate on m-ICcl2 cyclin D1 and cyclin E1 gene expression.

<p>qRT-PCR was analyzed by the dδCt method using m-ICcl2 alone and GAPDH as reference.</p

Fold change of gene expression after overnight co-culture of m-ICcl2 with <i>L.casei</i> and <i>B.breve</i>.

<p>qRT-PCR was analyzed by the ddCt method using m-ICcl2 alone and GAPDH as reference.</p