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International audience; The rck open reading frame (ORF) on the pefI-srgC operon encodes an outer membrane protein responsible for invasion of nonphagocytic cell lines and resistance to complement-mediated killing. Until now, the rck ORF was only detected on the virulence plasmids of three serovars of Salmonella subsp. enterica (i.e., Bovismorbificans, Enteritidis, and Typhimurium). The increasing number of Salmonella genome sequences allowed us to use a combination of reference sequences and whole-genome multilocus sequence typing (wgMLST) data analysis to probe the presence of the operon and of rck in a wide array of isolates belonging to all Salmonella species and subspecies. We established the presence of partial or complete operons in 61 subsp. enterica serovars as well as in 4 other subspecies with various syntenies and frequencies. The rck ORF itself was retrieved in 36 subsp. enterica serovars and in two subspecies with either chromosomal or plasmid-borne localization. It displays high conservation of its sequence within the genus, and we demonstrated that most of the allelic variations identified did not alter the virulence properties of the protein. However, we demonstrated the importance of the residue at position 38 (at the level of the first extracellular loop of the protein) in the invasin function of Rck. Altogether, our results highlight that rck is not restricted to the three formerly identified serovars and could therefore have a more important role in virulence than previously expected. Moreover, this work raises questions about the mechanisms involved in rck acquisition and about virulence plasmid distribution and evolution. IMPORTANCE The foodborne pathogen Salmonella is responsible for a wide variety of pathologies depending on the infected host, the infecting serovars, and its set of virulence factors. However, the implication of each of these virulence factors and their role in the specific host-pathogen interplay are not fully understood. The significance of our research is in determining the distribution of one of these factors, the virulence plasmid-encoded invasin and resistance to complement killing protein Rck. In addition to providing elements of reflection concerning the mechanisms of acquisition of specific virulence genes in certain serotypes, this work will help to understand the role of Rck in the pathogenesis of Salmonella.

International audience; Introduction 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).

International audience; Salmonella are among the top-ranked foodborne pathogens, inducing a wide variety of diseases ranging from gastroenteritis to typhoid fever, depending on the infecting serovar, the host and its genetic background. As a facultative intracellular pathogen, it can induce its own internalization in nonphagocytic cells through at least 3 different bacterial factors: the SPI-1-encoded T3SS-1, and two outer membrane proteins, Rck and PagN. The pagN gene is part of the PhoP-PhoQ regulon, and was identified, though the use of in vivo expression technology, as a gene required for Salmonella Typhimurium survival in BALB/c mice. It encodes for a widely conserved, 27 kDa protein displaying both structure and function homology with the proteins Hek and Tia of E. coli. This invasin allows Salmonella to invade cells through a Zipper-like mechanism, following interaction with heparan sulfate proteoglycans. However, its precise role in vivo remains to be determined including the cells targeted by this invasin. In this study, we aimed to precisely determine the kinetics of expression of this entry factor in mice. Bioluminescent S. Typhimurium reporter strains carrying transcriptional fusions were used to track the transcription of pagN in three murine models reproducing the different pathologies induced by Salmonella : typhoid fever, gastroenteritis and asymptomatic carriage. We observed a transcription of pagN in the intestine independently of the genetic background of the host and the inflammatory state of the animals. Moreover, pagN transcription was detected at later time points in lymphoid organs following the systemic spread of the pathogen in the typhoid fever reproducing model. Further analyses are in progress, focusing on the identification of the cells targeted by PagN.

International audience; Salmonella are facultative intracellular bacteria able to infect a broad range of hosts using several virulence factors, including the Rck outer membrane protein. This protein is responsible for (i) resistance to complement, in particular through membrane attack complex formation inhibition, (ii) bacterial adhesion, through laminin binding, and (iii) host-cell invasion, through a Zipper-like mechanism following interaction with the EGF-R. The rck ORF belongs to the pefI-srgC operon, carried on the virulence plasmid of Salmonella. Currently, rck has only been characterized on the virulence plasmid of three serovars, i.e. Enteritidis, Typhimurium and Bovismorbificans. As the literature suggested a larger distribution, our aim was to study the prevalence of the rck ORF in the genus Salmonella taking advantage of the large online database Enterobase and of its experimental data associated (e.g. in silico serovars prediction, wgMLST sequence type). 188,233 genomes belonging to more than 471 Salmonella serovars were analyzed. Our results revealed the presence of the rck gene in 39 serovars and a predominance of some alleles for some serovars. They also suggest that the gene might not be exclusively carried on plasmids. Finally, despite a relatively high number of haplotypes, amino acid sequences comparison revealed a strong conservation of the protein sequences, as the majority of the identified variants showed only minor sequence variations with the three currently described Rck proteins. In conclusion, Rck is more widely distributed in Salmonella serovars than previously expected and consequently could play a role in the virulence of all these serovars.