Bidirectional Comparisons Revealed Functional Patterns in Interaction between Salmonella enterica and Plants.

Plants may harbor the human pathogen Salmonella enterica. Interactions between S. enterica and different plant species have been studied in individual reports. However, disparities arising from the distinct experimental conditions may render a meaningful comparison very difficult. This study explored interaction patterns between different S. enterica strains including serovars Typhimurium 14028s and LT2 and serovar Senftenberg, and different plants (Arabidopsis, lettuce, and tomato) in one approach. Better persistence of S. enterica serovar Typhimurium strains was observed in all tested plants, whereas the resulting symptoms varied depending on plant species. Genes encoding pathogenesis-related proteins were upregulated in plants inoculated with Salmonella. Furthermore, transcriptome of tomato indicated dynamic responses to Salmonella, with strong and specific responses already 24 h after inoculation. By comparing with publicly accessible Arabidopsis and lettuce transcriptome results generated in a similar manner, constants and variables were displayed. Plants responded to Salmonella with metabolic and physiological adjustments, albeit with variability in reprogrammed orthologues. At the same time, Salmonella adapted to plant leaf-mimicking media with changes in biosynthesis of cellular components and adjusted metabolism. This study provides insights into the Salmonella-plant interaction, allowing for a direct comparison of responses and adaptations in both organisms.

From intestine to beyond: Salmonella entry factors display distinct transcription pattern upon infection in murine models.

The infectious process of bacteria of the genus Salmonella requires the finely regulated use of various virulence factors. Among them, the type 3 secretion system-1 (T3SS-1) and the Rck and PagN invasins are involved in the internalization of the pathogen within eukaryotic cells, but their precise role in the host and in the pathogenic process is still poorly understood. In this study, we aimed to determine the kinetics of expression of these entry factors in a typhoid fever-like and a gastroenteritis model in mice by in vivo imaging using bioluminescent Salmonella Typhimurium reporter strains carrying chromosomal transcriptional fusions. Only pagN and T3SS-1 transcription has been clearly identified. Independently of the pathological model, the caecum was identified as the main transcription site of both pagN and the T3SS-1-encoding gene both at early and late stages of the infection. An intense transcription of pagN was also observed in deep organs in the typhoid fever-like model, while that of T3SS-1 remained quite sporadic in these organs, and mainly focused on the intestine all along the infection. This work will help to understand the respective role of these entry factors at the cellular level in the pathogenesis of Salmonella in vivo.

Construction and characterization of a saturated Tn-seq library of Salmonella Typhimurium ATCC 14028.

Salmonella enterica is an important foodborne pathogen. Here, we present the construction and characterization of a high-density transposon sequencing library of the Salmonella Typhimurium ATCC 14028 strain. Essential, advantageous, and disadvantageous genes for growth in rich culture medium were identified on the chromosome and the pSLT plasmid.

Novel method to recover Salmonella enterica cells for Tn-Seq approaches from lettuce leaves and agricultural environments using combination of sonication, filtration, and dialysis membrane.

Salmonella enterica in agricultural environments has become an important concern, due to its potential transmission to humans and the associated public health risks. To identify genes contributing to Salmonella adaptation to such environments, transposon sequencing has been used in recent years. However, isolating Salmonella from atypical hosts, such as plant leaves, can pose technical challenges due to low bacterial content and the difficulty to separate an adequate number of bacteria from host tissues. In this study, we describe a modified methodology using a combination of sonication and filtration to recover S. enterica cells from lettuce leaves. We successfully recovered over a total of 3.5 × 106Salmonella cells in each biological replicate from two six-week old lettuce leaves, 7 days after infiltration with a Salmonella suspension of 5 × 107 colony forming units (CFU)/mL. Moreover, we have developed a dialysis membrane system as an alternative method for recovering bacteria from culture medium, mimicking a natural environment. Inoculating 107 CFU/mL of Salmonella into the media based on plant (lettuce and tomato) leaf and diluvial sand soil, a final concentration of 109.5 and 108.5 CFU/mL was obtained, respectively. One millilitre of the bacterial suspension after 24 h incubation at 28 °C using 60 rpm agitation was pelleted, corresponding to 109.5 and 108.5 cells from leaf- or soil-based media. The recovered bacterial population, from both lettuce leaves and environment-mimicking media, can adequately cover a presumptive library density of 106 mutants. In conclusion, this protocol provides an effective method to recover a Salmonella transposon sequencing library from in planta and in vitro systems. We expect this novel technique to foster the study of Salmonella in atypical hosts and environments, as well as other comparable scenarios.

In Vivo Tracking of Bacterial Colonization in Different Murine Models Using Bioluminescence: The Example of Salmonella.

Applications of bioluminescence for the in vivo study of pathogenic microorganisms are numerous, ranging from the quantification of virulence gene expression to measuring the effect of antimicrobial molecules on the colonization of tissues and organs by the pathogen. Most studies are performed in mice, but recent works demonstrate that this technique is applicable to larger animals like fish, guinea pigs, ferrets, and chickens. Here, we describe the construction and the utilization of a constitutively luminescent strain of Salmonella Typhimurium to monitor in vivo and ex vivo the colonization of mice in the gastroenteritis, typhoid fever, and asymptomatic carriage models of Salmonella infection.

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

In 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.

Intracellular Salmonella Paratyphi A is motile and differs in the expression of flagella-chemotaxis, SPI-1 and carbon utilization pathways in comparison to intracellular S. Typhimurium.

Although Salmonella Typhimurium (STM) and Salmonella Paratyphi A (SPA) belong to the same phylogenetic species, share large portions of their genome and express many common virulence factors, they differ vastly in their host specificity, the immune response they elicit, and the clinical manifestations they cause. In this work, we compared their intracellular transcriptomic architecture and cellular phenotypes during human epithelial cell infection. While transcription induction of many metal transport systems, purines, biotin, PhoPQ and SPI-2 regulons was similar in both intracellular SPA and STM, we identified 234 differentially expressed genes that showed distinct expression patterns in intracellular SPA vs. STM. Surprisingly, clear expression differences were found in SPI-1, motility and chemotaxis, and carbon (mainly citrate, galactonate and ethanolamine) utilization pathways, indicating that these pathways are regulated differently during their intracellular phase. Concurring, on the cellular level, we show that while the majority of STM are non-motile and reside within Salmonella-Containing Vacuoles (SCV), a significant proportion of intracellular SPA cells are motile and compartmentalized in the cytosol. Moreover, we found that the elevated expression of SPI-1 and motility genes by intracellular SPA results in increased invasiveness of SPA, following exit from host cells. These findings demonstrate unexpected flagellum-dependent intracellular motility of a typhoidal Salmonella serovar and intriguing differences in intracellular localization between typhoidal and non-typhoidal salmonellae. We propose that these differences facilitate new cycles of host cell infection by SPA and may contribute to the ability of SPA to disseminate beyond the intestinal lamina propria of the human host during enteric fever.

The Invasin and Complement-Resistance Protein Rck of Salmonella is More Widely Distributed than Previously Expected.

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.

Investigation of the invasion mechanism mediated by the outer membrane protein PagN of Salmonella Typhimurium.

BACKGROUND: Salmonella can invade host cells via a type three secretion system called T3SS-1 and its outer membrane proteins, PagN and Rck. However, the mechanism of PagN-dependent invasion pathway used by Salmonella enterica, subspecies enterica serovar Typhimurium remains unclear. RESULTS: Here, we report that PagN is well conserved and widely distributed among the different species and subspecies of Salmonella. We showed that PagN of S. Typhimurium was sufficient and necessary to enable non-invasive E. coli over-expressing PagN and PagN-coated beads to bind to and invade different non-phagocytic cells. According to the literature, PagN is likely to interact with heparan sulfate proteoglycan (HSPG) as PagN-mediated invasion could be inhibited by heparin treatment in a dose-dependent manner. This report shows that this interaction is not sufficient to allow the internalization mechanism. Investigation of the role of ß1 integrin as co-receptor showed that mouse embryo fibroblasts genetically deficient in ß1 integrin were less permissive to PagN-mediated internalization. Moreover, PagN-mediated internalization was fully inhibited in glycosylation-deficient pgsA-745 cells treated with anti-ß1 integrin antibody, supporting the hypothesis that ß1 integrin and HSPG cooperate to induce the PagN-mediated internalization mechanism. In addition, use of specific inhibitors and expression of dominant-negative derivatives demonstrated that tyrosine phosphorylation and class I phosphatidylinositol 3-kinase were crucial to trigger PagN-dependent internalization, as for the Rck internalization mechanism. Finally, scanning electron microscopy with infected cells showed microvillus-like extensions characteristic of Zipper-like structure, engulfing PagN-coated beads and E. coli expressing PagN, as observed during Rck-mediated internalization. CONCLUSIONS: Our results supply new comprehensions into T3SS-1-independent invasion mechanisms of S. Typhimurium and highly indicate that PagN induces a phosphatidylinositol 3-kinase signaling pathway, leading to a Zipper-like entry mechanism as the Salmonella outer membrane protein Rck.

Gut microbiota composition before infection determines the Salmonella super- and low-shedder phenotypes in chicken.

Heterogeneity of infection and extreme shedding patterns are common features of animal infectious diseases. Individual hosts that are super-shedders are key targets for control strategies. Nevertheless, the mechanisms associated with the emergence of super-shedders remain largely unknown. During chicken salmonellosis, a high heterogeneity of infection is observed when animal-to-animal cross-contaminations and reinfections are reduced. We hypothesized that unlike super-shedders, low-shedders would be able to block the first Salmonella colonization thanks to a different gut microbiota. The present study demonstrates that (i) axenic and antibiotic-treated chicks are more prone to become super-shedders; (ii) super or low-shedder phenotypes can be acquired through microbiota transfer; (iii) specific gut microbiota taxonomic features determine whether the chicks develop a low- and super-shedder phenotype after Salmonella infection in isolator; (iv) partial protection can be conferred by inoculation of four commensal bacteria prior to Salmonella infection. This study demonstrates the key role plays by gut microbiota composition in the heterogeneity of infection and pave the way for developing predictive biomarkers and protective probiotics.

IPEC-1 variable immune response to different serovars of Salmonella enterica subsp. enterica.

Salmonella is a genus of Gram-negative bacteria in the Enterobacteriaceae family causing various illnesses. The ability of the different serovars of Salmonella enterica subsp. enterica to infect a host and to induce pathology relies in part on their cellular and molecular interactions with the intestinal epithelium. In the current study, an in vitro approach using non-polarized or polarized IPEC-1 porcine intestinal epithelial cells were used in order to assess the relation between adhesion, invasion, and induction of the immune response as a function of the serotype of Salmonella. Five serovars, Choleraesuis (host-adapted), Typhimurium (ubiquitous), Typhisuis (host-restricted), which are relevant for pig infection, and Dublin and Gallinarum, which are host-restricted or host-adapted, were studied. A strong variation was observed in the percentages of adhesion and invasion amongst the S. enterica serovars used to interact with the non-polarized and polarized cells. Subsequently, differences were identified between serovars in terms of immune response induced. Serovars Typhimurium and Typhisuis induced a strong innate immune response four and half hours after the beginning of cell stimulation while Choleraesuis, Gallinarum, and Dublin did not. A strong inflammatory response could limit the spread of the porcine serovars to the gut while, with a weak response, bacteria may not be constrained by the immune response enabling severe systemic diseases. Different repertoires of adhesion factors and of secreted protein effectors between Salmonella serovars interacting with IPEC-1 cells probably explains the differences in their early pathogenic behaviours.

H-NS is the major repressor of Salmonella Typhimurium Pef fimbriae expression.

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.

Systemic Administration of Avian Defensin 7: Distribution, Cellular Target, and Antibacterial Potential in Mice.

Defensins are natural antimicrobial peptides. The avian beta-defensin AvBD7 isolated from the chicken bone marrow possess broad antibacterial spectrum and strong resistance to proteolysis. However, its ability to fight systemic infections of major concern for public health, such as salmonellosis, is unknown. As a first approach, fluorescence labeling of AvBD7 allowed to track its systemic distribution after intraperitoneal injection in mice using whole body live imaging. It was associated to peritoneal cells and to deeper organs such as the liver. In the next step, the use of labeled AvBD7 allowed to observe its interaction with murine macrophages in culture. After incubation, it was able to penetrate inside the cells through an endocytosis-like mechanism. Furthermore, natural AvBD7 contributed to the control of intracellular multiplication of a multidrug resistant Salmonella strain, after incubation with infected macrophages. Finally, administration in a model of systemic lethal Salmonella infection in mice led to significant improvement of mouse survival, consistently with significant reduction of the liver bacterial load. In conclusion, the results reveal a hitherto unknown intracellular antibacterial effect of AvBD7 in Salmonella target cells and support AvBD7 as a candidate of interest for the treatment of infectious diseases caused by multidrug-resistant pathogenic Enterobacteriaceae.

Salmonella Typhimurium Invalidated for the Three Currently Known Invasion Factors Keeps Its Ability to Invade Several Cell Models.

To establish an infection, Salmonella has to interact with eukaryotic cells. Invasion of non-phagocytic cells (i.e., epithelial, fibroblast and endothelial cells) involves either a trigger or a zipper mechanism mediated by the T3SS-1 or the invasin Rck, respectively. Another outer membrane protein, PagN, was also implicated in the invasion. However, other unknown invasion factors have been previously suggested. Our goal was to evaluate the invasion capability of a Salmonella Typhimurium strain invalidated for the three known invasion factors. Non-phagocytic cell lines of several animal origins were tested in a gentamicin protection assay. In most cells, we observed a drastic decrease in the invasion rate between the wild-type and the triple mutant. However, in five cell lines, the triple mutant invaded cells at a similarly high level to the wild-type, suggesting the existence of unidentified invasion factors. For the wild-type and the triple mutant, scanning-electron microscopy, confocal imaging and use of biochemical inhibitors confirmed their cellular uptake and showed a zipper-like mechanism of internalization involving both clathrin- and non-clathrin-dependent pathways. Despite a functional T3SS-1, the wild-type bacteria seemed to use the same entry route as the mutant in our cell model. All together, these results demonstrate the existence of unknown Salmonella invasion factors, which require further characterization.

Role of systemic infection, cross contaminations and super-shedders in Salmonella carrier state in chicken.

Carriage of Salmonella is often associated with a high level of bacterial excretion and generally occurs after a short systemic infection. However, we do not know whether this systemic infection is required or whether the carrier-state corresponds to continuous reinfection or real persistence in caecal tissue. The use of a Salmonella Enteritidis bamB mutant demonstrated that a carrier-state could be obtained in chicken in the absence of systemic infection. The development of a new infection model in isolator showed that a marked decrease in animal reinfection and host-to-host transmission between chicks led to a heterogeneity of S. Enteritidis excretion and colonization contrary to what was observed in cages. This heterogeneity of infection was characterized by the presence of super-shedders, which constantly disseminated Salmonella to the low-shedder chicks, mainly through airborne movements of contaminated dust particles. The presence of super-shedders, in the absence of host-to-host transmission, demonstrated that constant reinfection was not required to induce a carrier-state. Finally, our results suggest that low-shedder chicks do not have a higher capability to destroy Salmonella but instead can block initial Salmonella colonization. This new paradigm opens new avenues to improve understanding of the carrier-state mechanisms and to define new strategies to control Salmonella infections.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

An Updated View on the Rck Invasin of Salmonella: Still Much to Discover.

Salmonella is a facultative intracellular Gram-negative bacterium, responsible for a wide range of food- and water-borne diseases ranging from gastroenteritis to typhoid fever depending on hosts and serotypes. Salmonella thus represents a major threat to public health. A key step in Salmonella pathogenesis is the invasion of phagocytic and non-phagocytic host cells. To trigger its own internalization into non-phagocytic cells, Salmonella has developed different mechanisms, involving several invasion factors. For decades, it was accepted that Salmonella could only enter cells through a type three secretion system, called T3SS-1. Recent research has shown that this bacterium expresses outer membrane proteins, such as the Rck protein, which is able to induce Salmonella entry mechanism. Rck mimics natural host cell ligands and triggers engulfment of the bacterium by interacting with the epidermal growth factor receptor. Salmonella is thus able to use multiple entry pathways during the Salmonella infection process. However, it is unclear how and when Salmonella exploits the T3SS-1 and Rck entry mechanisms. As a series of reviews have focused on the T3SS-1, this review aims to describe the current knowledge and the limitations of our understanding of the Rck outer membrane protein. The regulatory cascade which controls Rck expression and the molecular mechanisms underlying Rck-mediated invasion into cells are summarized. The potential role of Rck-mediated invasion in Salmonella pathogenesis and the intracellular behavior of the bacteria following a Salmonella Rck-dependent entry are discussed.

Oral Uptake of Chlamydia psittaci by Ducklings Results in Systemic Dissemination.

Enteric infections caused by Chlamydia (C.) psittaci are frequent in ducks, but mostly remain subclinical under field conditions. To emulate natural infection, we investigated the pathogenic potential of a C. psittaci field strain in orally inoculated 4-day-old ducklings. Three different challenge doses were tested and seven contact animals were also mock-inoculated with buffer in each group. Over the course of ten days, the birds were monitored for clinical symptoms and chlamydial dissemination before final examination of tissues using histopathology and immunohistochemistry. While the challenge strain disseminated systemically to all internal organs, mild signs of diarrhea were confined to ducklings inoculated with the highest dose (4.3 x 108 IFU/mL, Group 1). No other clinical symptoms or histopathological lesions were seen. The chlamydial load in internal organs as measured by PCR depended on the challenge dose and was unevenly distributed, i.e. high loads in spleen, liver, and distal small and large intestinal tract (ileum, cecum and rectum) vs. ten times lower values in lungs and proximal small intestinal tract (duodenum and jejunum). Notably, the C. psittaci infection of contact birds became evident on day 10 post-infection, with bacterial loads comparable to those of experimentally-infected animals, thus suggesting rapid bird-to-bird transmission of the challenge strain.

The Salmonella effector protein SpvC, a phosphothreonine lyase is functional in plant cells.

Salmonella is one of the most prominent causes of food poisoning and growing evidence indicates that contaminated fruits and vegetables are an increasing concern for human health. Successful infection demands the suppression of the host immune system, which is often achieved via injection of bacterial effector proteins into host cells. In this report we present the function of Salmonella effector protein in plant cell, supporting the new concept of trans-kingdom competence of this bacterium. We screened a range of Salmonella Typhimurium effector proteins for interference with plant immunity. Among these, the phosphothreonine lyase SpvC attenuated the induction of immunity-related genes when present in plant cells. Using in vitro and in vivo systems we show that this effector protein interacts with and dephosphorylates activated Arabidopsis Mitogen-activated Protein Kinase 6 (MPK6), thereby inhibiting defense signaling. Moreover, the requirement of Salmonella SpvC was shown by the decreased proliferation of the ΔspvC mutant in Arabidopsis plants. These results suggest that some Salmonella effector proteins could have a conserved function during proliferation in different hosts. The fact that Salmonella and other Enterobacteriaceae use plants as hosts strongly suggests that plants represent a much larger reservoir for animal pathogens than so far estimated.

Deciphering why Salmonella Gallinarum is less invasive in vitro than Salmonella Enteritidis.

Salmonella Gallinarum and Salmonella Enteritidis are genetically closely related however associated with different pathologies. Several studies have suggested that S. Gallinarum is less invasive in vitro than S. Enteritidis. In this study we confirm that the S. Gallinarum strains tested were much less invasive than the S. Enteritidis strains tested in cells of avian or human origin. In addition, the S. Gallinarum T3SS-1-dependent ability to invade host cells was delayed by two to three hours compared to S. Enteritidis, indicating that T3SS-1-dependent entry is less efficient in S. Gallinarum than S. Enteritidis. This was neither due to a decreased transcription of T3SS-1 related genes when bacteria come into contact with cells, as transcription of hilA, invF and sipA was similar to that observed for S. Enteritidis, nor to a lack of functionality of the S. Gallinarum T3SS-1 apparatus as this apparatus was able to secrete and translocate effector proteins into host cells. In contrast, genome comparison of four S. Gallinarum and two S. Enteritidis strains revealed that all S. Gallinarum genomes displayed the same point mutations in each of the main T3SS-1 effector genes sipA, sopE, sopE2, sopD and sopA.

Direct regulation of the pefI-srgC operon encoding the Rck invasin by the quorum-sensing regulator SdiA in Salmonella†Typhimurium.

One important step for the pathogenesis of Salmonella is its ability to penetrate host cells. Recently, a new entry system involving the outer membrane protein Rck has been characterized. Previous studies have shown that the pefI-srgC locus, which contains rck, was regulated by the temperature and SdiA, the transcriptional regulator of quorum sensing in Salmonella. To decipher the regulation of rck by SdiA, we first confirmed the operon organization of the pefI-srgC locus. Using plasmid-based transcriptional fusions, we showed that only the predicted distal promoter upstream of pefI, PefIP2, displays an SdiA- and acyl-homoserine lactones-dependent activity while the predicted proximal PefIP1 promoter exhibits a very low activity independent on SdiA in our culture conditions. A direct and specific interaction of SdiA with this PefIP2 region was identified using electrophoretic mobility shift assays and surface plasmon resonance studies. We also observed that Rck expression is negatively regulated by the nucleoid-associated H-NS protein at both 25°C and 37°C. This work is the first demonstration of a direct regulation of genes by SdiA in Salmonella and will help further studies designed to identify environmental conditions required for Rck expression and consequently contribute to better characterize the role of this invasin in vivo.