Salmonella Typhimurium is able to invade and survive intracellularly independently of its T3SS1

International audienceSalmonella Typhimurium is a bacterial pathogen with an intestinal tropism, causing foodborne diseases. To infect its hosts Salmonella employs a wide range of virulence factors that allow it to actively invade, survive and multiply in a vacuole or in the cytosol. The Salmonella Pathogenicity Island 1-encoded type III secretion system (T3SS1) was the first virulence factor described for its involvement in the invasion process and intracellular fate of Salmonella in the host cell. T3SS1 effectors coordinately mimic various host-cells protein functions, to hijack the cellular machinery allowing, Salmonella invasion and the initiation of the Salmonella containing vacuole (SCV) maturation. Since, two virulence factors Rck and PagN were shown to be involved in the invasion process. Recently, out of fifteen cell lines tested we identified five, in which the Salmonella Typhimurium invasion process is independent of the three known invasion factors. Here, we investigated the intracellular fate of Salmonella Typhimurium in the murine hepatic cell line AML12. We demonstrated that both wild-type Salmonella and T3SS1-invalidated Salmonella followed a common pathway beginning by the formation of a Salmonella containing vacuole (SCV) without classical recruitment of Rho- GTPases ending to Salmonella multiplication in SCV harboring Salmonella inducing filament. These results demonstrate that Salmonella invasion can be completely independent of the T3SS-1, and suggest that other bacterial players allow SCV early maturation. This model is of major interest for establishing the protein composition of an independent T3SS-1 SCV. A better knowledge of these vacuoles will allow the identification of new bacterial and host players necessary for the intracellular survival of Salmonella

Seaweed extracts inhibit invasion of Salmonella Enteritidis into avian epithelial cells

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Two In Vivo Models to Study Salmonella Asymptomatic Carrier State in Chicks

International audienceIn chicken, Salmonella Enteritidis and Salmonella Typhimurium, the two main serotypes isolated in human infections, can persist in the host organism for many weeks and up to many years without causing any symptoms. This persistence generally occurs after a short systemic infection that may either lead to death of very young birds or develop into cecal asymptomatic persistence, which is often accompanied by a high level of bacterial excretion, facilitating Salmonella transmission to counterparts. Here we describe two models of chick infection. The first model reproduces well the poultry infection in farm flocks. Numerous reinfections and animal-animal recontaminations occur leading to a high level of cecal colonization and fecal excretion in all chicks in the flock, over several weeks. In the second model, these animal reinfections and recontaminations are hampered leading to heterogeneity of infection characterized by the presence of low and super-shedders. This model allows for more mechanistic studies of Salmonella/chicks interactions as animal recontaminations are lowered

Modulation de la prolifération cellulaire par <em>Salmonella</em> Typhimurium

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Modulation of cell proliferation by <em>Salmonella</em> Typhimurium

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The Salmonella containing vacuole is an environment adequate for the outer membrane protein PagN expression: PagN reveals its evasin function

International audienceIntroduction and objectives: Salmonella Typhimurium is a facultative intracellular entero-pathogen that remains a risk to public health worldwide. The tools and strategies allowing it to invade and survive in host cells are numerous. Nowadays, it is the only pathogen able to invade host cells using both a trigger and a zipper mechanism, respectively depending on the Type 3 secretion system-1, encoded mainly on Salmonella pathogenicity island I, and of the two invasins Rck (Wiedemann et al. 2016) and PagN (Barilleau et al. 2021). Intracellularly, Salmonella are contained in a Salmonella containing vacuole (SCV) whose maturation leads to an acidic and low divalent-cation environment. Nevertheless, in the last decades several studies gave some evidence for a Salmonella escape from the early SCV, leading to cytosolic hyper replication of the pathogen. As PagN expression was previously shown to dependent on acidic pH and low divalent cation concentration (Lambert and Smith 2008), we explored the potential role of PagN in the SCV.Materials and methods: Gentamicin protection assays to determine adhesion, invasion and multiplication were performed in chinese hamster ovary epithelial cell line. PagN expression was evaluated at several time post-infection by western blot. SCV escaping was considered by two ways to discriminate cytosolic vs vacuolar Salmonella: 1) a gentamicine / chloroquine assay, 2) cytometry and microscopy based on a localization-reporting dual color fluorescent plasmidexpressed by Salmonella (Noster et al 2019). Salmonella wild-type, pagN mutant and the complemented strain were used. Lastly, intracellular expression of PagN was imaged through confocal microscopy.Results, discussion and conclusion: PagN is highly expressed by Salmonella Typhimurium in the SCV and a reduced escape of the pagN mutant was observed compared to the wild-type strain leading to a reduced replication of the mutant in the cytosol. The impact of these cytosolic replicative bacteria on reinfection in vivo remains to be evaluated as described recently (Chong et al 2021)

Differential Salmonella Typhimurium intracellular replication and host cell responses in caecal and ileal organoids derived from chicken

International audienceChicken infection with Salmonella Typhimurium is an important source of foodborne human diseases. Salmonella colonizes the avian intestinal tract and more particularly the caecum, without causing symptoms. This thus poses a challenge for the prevention of foodborne transmission. Until now, studies on the interaction of Salmonella with the avian gut intestine have been limited by the absence of in vitro intestinal culture models. Here, we established intestinal crypt‐derived chicken organoids to better decipher the impact of Salmonella intracellular replication on avian intestinal epithelium. Using a 3D organoid model, we observed a significantly higher replication rate of the intracellular bacteria in caecal organoids than in ileal organoids. Our model thus recreates intracellular environment, allowing Salmonella replication of avian epithelium according to the intestinal segment. Moreover, an inhibition of the cellular proliferation was observed in infected ileal and caecal organoids compared to uninfected organoids. This appears with a higher effect in ileal organoids, as well as a higher cytokine and signaling molecule response in infected ileal organoids at 3 h post-infection (hpi) than in caecal organoids that could explain the lower replication rate of Salmonella observed later at 24 hpi. To conclude, this study demonstrates that the 3D organoid is a model allowing to decipher the intracellular impact of Salmonella on the intestinal epithelium cell response and illustrates the importance of the gut segment used to purify stem cells and derive organoids to specifically study epithelial cell - Salmonella interaction

Intestinal organoids for modeling Salmonella infection in chicken

International audienceChicken infection with Salmonella enterica, subspecies enterica serovar Typhimurium (S. Typhimurium) is an important source of foodborne human diseases. Salmonella colonizes the avian intestinal tract and more particularly the cecum, without causing symptoms. This thus poses a challenge for the prevention of foodborne transmission. Until now, studies on the interaction of Salmonella with the avian gut intestine have been limited by the absence of in vitro intestinal culture models. Our objectif was to establish ileal and cecal organoids by seeding intestinal epithelial stem cells in a 3-dimensional (3D) culture environment reproducing in vitro the stem cell niche to investigate the pathophysiology of Salmonella-avian intestinal epithelium interactions. These organoids form a monolayer of polarized epithelial cells containing the diverse differentiated epithelial cells, recapitulating the original structure and function of the native epithelium. Using this 3D avian organoid model, we were able to visualize and compare the ability of S. Typhimurium to invade and intracellularly replicate, according to the chicken intestinal segment, as well as its impact on the proliferation and differentiation of cells constituting the intestinal epithelium by transcriptomic analysis. This new approach allows a more representative study of avian intestinal epithelium-Salmonella interactions and an efficient reduction of the use of live animals for experimentation

Epithelial cell invasion by salmonella typhimurium induces modulation of genes controlled by aryl hydrocarbon receptor signaling and involved in extracellular matrix biogenesis

International audienceSalmonella is the only bacterium able to enter a host cell by the two known mechanisms: trigger and zipper. The trigger mechanism relies on the injection of bacterial effectors into the host cell through the Salmonella type III secretion system 1. In the zipper mechanism, mediated by the invasins Rck and PagN, the bacterium takes advantage of a cellular receptor for invasion. This study describes the transcriptomic reprogramming of the IEC-6 intestinal epithelial cell line to Salmonella Typhimurium strains that invaded cells by a trigger, a zipper, or both mechanisms. Using S. Typhimurium strains invalidated for one or other entry mechanism, we have shown that IEC-6 cells could support both entries. Comparison of the gene expression profiles of exposed cells showed that irrespective of the mechanism used for entry, the transcriptomic reprogramming of the cell was nearly the same. On the other hand, when gene expression was compared between cells unexposed or exposed to the bacterium, the transcriptomic reprogramming of exposed cells was significantly different. It is particularly interesting to note the modulation of expression of numerous target genes of the aryl hydrocarbon receptor showing that this transcription factor was activated by S. Typhimurium infection. Numerous genes associated with the extracellular matrix were also modified. This was confirmed at the protein level by western-blotting showing a dramatic modification in some extracellular matrix proteins. Analysis of a selected set of modulated genes showed that the expression of the majority of these genes was modulated during the intracellular life of S. Typhimurium