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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.

International audience; Fimbriae play an important role in adhesion and are therefore essential for the interaction of bacteria with the environments they encounter. Most of them are expressed in vivo but not in vitro, thus making difficult the full characterization of these fimbriae. Here, we characterized the silencing of plasmid-encoded fimbriae (Pef) expression, encoded by the pef operon, in the worldwide pathogen Salmonella Typhimurium. We demonstrated that the nucleoid-associated proteins H-NS and Hha, and their respective paralogs StpA and YdgT, negatively regulate at pH 5.1 and pH 7.1 the transcription of the pef operon. Two promoters, PpefB and PpefA, direct the transcription of this operon. All the nucleoid-associated proteins silence the PpefB promoter and H-NS also targets the PpefA promoter. While Hha and YdgT are mainly considered as acting primarily through H-NS to modulate gene transcription, our results strongly suggest that Hha and YdgT silence pef transcription at acidic pH either by interacting with StpA or independently of H-NS and StpA. We also confirmed the previously described post-transcriptional repression of Pef fimbriae by CsrA titration via the fim mRNA and CsrB and CsrC sRNA. Finally, among all these regulators, H-NS clearly appeared as the major repressor of Pef expression. These results open new avenues of research to better characterize the regulation of these bacterial adhesive proteins and to clarify their role in the virulence of pathogens

Fimbriae play an important role in adhesion and are therefore essential for the interaction of bacteria with the environments they encounter. Most of them are expressed in vivo but not in vitro, thus making difficult the full characterization of these fimbriae. Here, we characterized the repression of plasmid-encoded fimbriae (Pef) expression, encoded by the pef operon, in the worldwide pathogen Salmonella Typhimurium. We demonstrated that the nucleoid-associated proteins H-NS, Hha and YdgT negatively regulate, at pH 5.1 and pH 7.1, the transcription initiated from the promoter located upstream of pefB, the first ORF of the pef operon. While Hha and YdgT are mainly considered as acting primarily through H-NS to modulate gene transcription, our results strongly suggest that Hha and YdgT repress pef transcription independently of H-NS at acidic pH. We also confirmed the previously described posttranscriptional repression of Pef fimbriae by CsrA titration via the fim mRNA and CsrB and CsrC sRNA. Finally, compared to the repression by CsrA titration and Hha-YdgT, H-NS clearly appeared as the major repressor of Pef expression. These results open new avenues of research to better characterize the regulation of these bacterial adhesive proteins and to clarify their role in the virulence of pathogens.

International audience; Objectifs : Les gastroentérites provoquées par Salmonella Typhimurium sont déclenchées par l'adhésion des bactéries à l'épithélium intestinal conduisant ensuite à l'invasion et à la destruction de la surface mucosale. L'opéron pef de S. Typhimurium dirige la biosynthèse des fimbriae Pef (plasmidencoded fimbriae) impliqués dans l'adhésion à l'épithélium intestinal murin mais dont le rôle précis reste à déterminer. Comme la plupart des 13 fimbriae de S. Typhimurium, l'expression des Pef est fortement régulée. In vitro, leur expression n'a pu être détectée qu'après des cultures statiques en milieu riche à pH acide. Des travaux antérieurs et des analyses microarrays suggéraient l'implication des nucléoprotéines H-NS et Hha-YdgT dans la régulation négative de l'expression des fimbriae Pef. L'objectif de cette étude était de démontrer cette répression et de caractériser les mécanismes mis en jeu. Matériels et Méthodes : Les mutants hns, du fait de leur instabilité, sont reconstruits par transduction à chaque expérience. Les activités des promoteurs ont été quantifiées avec des fusions transcriptionnelles plasmidiques chez la souche sauvage et des mutants hns et/ou hha-ydgT. L'expression des fimbriae Pef a été mesurée par RT-PCR et Western blot en estimant l'expression de pefA/PefA, qui code la sous-unité majoritaire. Résultats : H-NS et, à un degré moindre, Hha-YdgT régulent négativement la transcription de l'opéron pef en agissant sur le promoteur situé en amont de pefB, le premier ORF de l'opéron. De plus, Hha-YdgT sont capables de réprimer l'expression de pef indépendamment de H-NS quand les bactéries sont cultivées en milieu acide en conditions statiques, ce qui n'est pas le cas après culture à pH neutre. Conclusions : Ce travail démontre que la faible expression des fimbriae Pef in vitro est due à l'action combinée des nucléoprotéines H-NS et Hha-YdgT sur l'activité transcriptionnelle de la région promotrice localisée en amont de l'opéron pef. Le mode d'action exact de ces nucléoprotéines est actuellement en débat dans la littérature. Des évidences expérimentales et un modèle mécanistique récemment décrit indiquent que Hha-YdgT agissent essentiellement par l'intermédiaire de H-NS pour moduler l'expression de gènes. A l'inverse, quelques études montrent que Hha peut se fixer sur des séquences régulatrices spécifiques indépendamment de H-NS. Nos résultats sur la régulation transcriptionnelle de l'opéron pef sont en faveur de l'existence de ces deux modèles.

International audience

Work on the new invasion routes has been funded by the Région Centre. N. Abed holds a Post-Doctoral fellowship funded and performed within the “SAVIRE” project, funded by the Délégation Régionale à la Recherche et à la Technologie du Centre (FEDER) number 1634-32245 and by the Région Centre number 2008-00036085. F. Namdari holds a Doctor; International audience; The Salmonella enterica species includes about 2600 diverse serotypes, most of which cause a wide range of food- and water-borne diseases ranging from self-limiting gastroenteritis to typhoid fever in both humans and animals. Moreover, some serotypes are restricted to a few animal species, whereas other serotypes are able to infect plants as well as cold- and warm-blooded animals. An essential feature of the pathogenicity of Salmonella is its capacity to cross a number of barriers requiring invasion of a large variety of phagocytic and nonphagocytic cells. The aim of this review is to describe the different entry pathways used by Salmonella serotypes to enter different nonphagocytic cell types. Until recently, it was accepted that Salmonella invasion of eukaryotic cells required only the type III secretion system (T3SS) encoded by the Salmonella pathogenicity island-1. However, recent evidence shows that Salmonella can cause infection in a T3SS-1-independent manner. Currently, two outer membrane proteins Rck and PagN have been clearly identified as Salmonella invasins. As Rck mediates a Zipper-like entry mechanism, Salmonella is therefore the first bacterium shown to be able to induce both Zipper and Trigger mechanisms to invade host cells. In addition to these known entry pathways, recent data have shown that unknown entry routes could be used according to the serotype, the host and the cell type considered, inducing either Zipper-like or Trigger-like entry processes. The new paradigm presented here should change our classic view of Salmonella pathogenicity. It could also modify our understanding of the mechanisms leading to the different Salmonella-induced diseases and to Salmonella-host specificity.

International audience

International audience

National audience

The Salmonella enterica species includes about 2600 diverse serotypes, most of which cause a wide range of food- and water-borne diseases ranging from self-limiting gastroenteritis to typhoid fever in both humans and animals. Moreover, some serotypes are restricted to a few animal species, whereas other serotypes are able to infect plants as well as cold- and warm-blooded animals. An essential feature of the pathogenicity of Salmonella is its capacity to cross a number of barriers requiring invasion of a large variety of phagocytic and nonphagocytic cells. The aim of this review is to describe the different entry pathways used by Salmonella serotypes to enter different nonphagocytic cell types. Until recently, it was accepted that Salmonella invasion of eukaryotic cells required only the type III secretion system (T3SS) encoded by the Salmonella pathogenicity island-1. However, recent evidence shows that Salmonella can cause infection in a T3SS-1-independent manner. Currently, two outer membrane proteins Rck and PagN have been clearly identified as Salmonella invasins. As Rck mediates a Zipper-like entry mechanism, Salmonella is therefore the first bacterium shown to be able to induce both Zipper and Trigger mechanisms to invade host cells. In addition to these known entry pathways, recent data have shown that unknown entry routes could be used according to the serotype, the host and the cell type considered, inducing either Zipper-like or Trigger-like entry processes. The new paradigm presented here should change our classic view of Salmonella pathogenicity. It could also modify our understanding of the mechanisms leading to the different Salmonella-induced diseases and to Salmonella-host specificity.

L’entérobactérie Salmonella est un pathogène intracellulaire facultatif, responsable des salmonelloses humaines et animales. Le caractère pathogène de Salmonella est étroitement dépendant de sa capacité à entrer et à se multiplier dans les cellules de l’hôte. Jusqu’à présent, le seul système d’entrée décrit pour les salmonelles était un appareil de sécrétion de type III (T3SS), le T3SS-1. Récemment, nous avons caractérisé in vitro un nouveau système d'entrée de Salmonella impliquant une protéine de la membrane externe, Rck. Afin de mieux comprendre les conditions physiologiques d’expression de Rck, nous avons caractérisé la transcription du locus pefI-srgC, qui porte le cadre ouvert de lecture (ORF) rck, et la régulation exercée par le régulateur transcriptionnel SdiA qui avait été préalablement observée. Après avoir confirmé la régulation transcriptionnelle du locus pefI-srgC par SdiA, le régulateur transcriptionnel du quorum sensing, nous avons montré que les six ORFs de ce locus étaient organisés en opéron. L’utilisation de fusions transcriptionnelles a permis d’identifier 2 promoteurs en amont de pefI, le promoteur distal P2 ayant une activité beaucoup plus forte que celle de P1 et dépendante de SdiA. Un site d’initiation de la transcription correspondant au promoteur P2 a été identifié 352 bases en amont de pefI et nous avons montré par des expériences de gel retard et des analyses de résonance plasmonique de surface que la protéine SdiA interagit avec la région promotrice P2. Ces résultats devraient nous aider à comprendre quand ce système d’entrée médié par Rck est exprimé in vivo.

Session Posters - Poster; Actuellement, toute souche de Listeria monocytogenes est considérée comme potentiellement virulente. Pourtant, une variabilité de la virulence observée in vivo et in vitro a été décrite dans la littérature. Un test de détection in vitro de plages de lyse suivi d’un test d’inoculation souscutanée plantaire sur souris nous ont permis de caractériser 26 souches faiblement virulentes. Ces souches sont réparties, selon une classification hiérarchique ascendante, en 4 groupes distincts selon 4 variables phénotypiques : taux de pénétration intracellulaire, nombre de plages de lyse formées et dosages des activités phospholipases (PI-PLC et PC-PLC). A partir des données phénotypiques, les gènes de virulence intervenant dans les différentes étapes du cycle d’infection et pouvant être liés à cette faible virulence ont été séquencés. Sur les 11 souches du groupe I, qui ne pénètrent pas dans les cellules, ne forment pas de plage de lyse et n’expriment aucune activité phospholipasique, 8 présentent une substitution dans le régulateur transcriptionnel PrfA et 3, un PrfA tronqué. Une des 3 souches du groupe II, qui pénètrent dans les cellules, ne forment pas de plage de lyse et n’expriment que l’activité PI-PLC, présente 4 substitutions dans PC-PLC, et 4 dans ActA. De même, 5 des 6 souches du groupe III, qui pénètrent dans les cellules, ne forment pas de plage de lyse et n’expriment que l’activité PC-PLC,présentent 3 substitutions dans PI-PLC et 22 dans ActA. Les 6 souches du groupe IV qui ne colonisent que faiblement les souris, ne se différencient quasiment pas des souches virulentes par leurs caractères phénotypiques. Ces résultats suggèrent un lien entre le phénotype des ces souches et les substitutions génotypiques observées. Ces substitutions retrouvées chez plusieurs souches suggèrent une évolution commune. Nous devrons déterminer si les substitutions observées dans les différents gènes de virulence sont la cause de la faible virulence.

National audience

Fimbriae play an important role in adhesion and are therefore essential for the interaction of bacteria with the environments they encounter. Most of them are expressed in vivo but not in vitro, thus making difficult the full characterization of these fimbriae. Here, we characterized the silencing of plasmid-encoded fimbriae (Pef) expression, encoded by the pef operon, in the worldwide pathogen Salmonella Typhimurium. We demonstrated that the nucleoid-associated proteins H-NS and Hha, and their respective paralogs StpA and YdgT, negatively regulate at pH 5.1 and pH 7.1 the transcription of the pef operon. Two promoters, PpefB and PpefA, direct the transcription of this operon. All the nucleoid-associated proteins silence the PpefB promoter and H-NS also targets the PpefA promoter. While Hha and YdgT are mainly considered as acting primarily through H-NS to modulate gene transcription, our results strongly suggest that Hha and YdgT silence pef transcription at acidic pH either by interacting with StpA or independently of H-NS and StpA. We also confirmed the previously described post-transcriptional repression of Pef fimbriae by CsrA titration via the fim mRNA and CsrB and CsrC sRNA. Finally, among all these regulators, H-NS clearly appeared as the major repressor of Pef expression. These results open new avenues of research to better characterize the regulation of these bacterial adhesive proteins and to clarify their role in the virulence of pathogens