Susceptibility to Salmonella carrier-state: a possible Th2 response in susceptible chicks.

Infection of chicken with Salmonella may lead to a carrier-state characterized by the persistence of bacteria in the ceca for a long period of time and result in their excretion in feces. This excretion is the source of contamination of their congeners and food. During infection, enterocytes are the primary target cells for Salmonella, the producers of soluble factors which launch immune response and cells which are reciprocally responsive to surrounding immune cells. This study used microarrays to compare the gene expression profile during carrier-state of enterocytes purified from infected and control chicks which are either resistant or susceptible to Salmonella Enteritidis carrier-state. In total, we identified 271 genes significantly differentially expressed with an absolute fold change greater than 1.5. A global analysis determined interaction networks between differentially regulated genes. Using an a priori approach, our analyses focused on differentially expressed genes which were transcriptionally linked to cytokines playing a major role in the fate of the immune response. The expression of genes transcriptionally linked to type I interferon and TGF-ß was down-regulated in infected chicks from both lines. Gene expression linked to the Th1 axis suggests the latter is inhibited in both lines. Finally, the expression of genes linked to IL-4, IL-5 and IL-13 indicates that susceptibility to carrier-state could be associated with a Th2 bias. Overall, these results highlight that the response to Salmonella during the acute phase and carrier-state is different and that enterocytes play a central role in this response.

Involvement of c-Src tyrosine kinase upstream of class I phosphatidylinositol (PI) 3-kinases in Salmonella Enteritidis Rck protein-mediated invasion.

The Salmonella outer membrane protein Rck mediates a Zipper entry mechanism controlled by tyrosine phosphorylation and class I phosphatidylinositol 3-kinase (PI 3-kinase). However, the underlying mechanism leading to this signaling cascade remains unclear. The present study showed that using Rck-coated beads or Rck-overexpressing Escherichia coli, Rck-mediated actin polymerization and invasion were blocked by PP2, a Src family tyrosine kinase inhibitor. In addition, phosphorylation of Src family kinases significantly increased after stimulation with Rck. The specific contribution of c-Src, one member of the Src family kinases, was demonstrated using c-Src-deficient fibroblasts or c-Src siRNA transfected epithelial cells. We also observed that Rck-mediated internalization led to the formation of a complex between c-Src and at least one tyrosine-phosphorylated protein. Furthermore, our results revealed that the c-Src signal molecule was upstream of PI 3-kinase during the Rck-mediated signaling pathway as Rck-mediated PI 3-kinase activation was blocked by PP2, and PI 3-kinase inhibitor had no effect on the Src phosphorylation. These results demonstrate the involvement of c-Src upstream of the PI 3-kinase in the Zipper entry process mediated by Rck.

Salmonella enteritidis Rck-mediated invasion requires activation of Rac1, which is dependent on the class I PI 3-kinases-Akt signaling pathway.

The Salmonella outer membrane protein Rck mediates a Zipper-like entry mechanism controlled by Rac, the Arp2/3 complex, and actin polymerization. However, little is known about the early steps leading to Rac activation and Rck-mediated internalization. The use of pharmacological inhibitors or PI 3-kinase dominant-negative mutant induced more than 80% less invasion without affecting attachment. Moreover, Rck-mediated internalization caused an increase in the association of p85 with at least one tyrosine-phosphorylated protein, indicating that class I PI 3-kinase activity was stimulated. We also report that this PI 3-kinase activity is essential for Rac1 activation. However, Rac recruitment at the Rck-mediated entry site was independent of its activation. Using a pharmacological approach or Akt-knockout cells, we also demonstrated that Akt was phosphorylated in response to Rck-mediated internalization as demonstrated by immunoblotting analysis and that all three Akt isoforms were required during this process. Overall, our results describe a signaling pathway involving tyrosine phosphorylation, class I PI 3-kinase, Akt activation, and Rac activation, leading to Rck-dependent Zipper entry. The specificity of this signaling pathway with regard to that of the type 3 secretion system, which is the other invasion process of Salmonella, is discussed.

Expression of Toll-like receptor 4 and downstream effectors in selected cecal cell subpopulations of chicks resistant or susceptible to Salmonella carrier state.

Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharide from Gram-negative bacteria, plays a major role in resistance of mice and humans to Salmonella infection. In chickens, Salmonella may establish a carrier state whereby bacteria are able to persist in the host organism without triggering clinical signs. Based on cellular morphological parameters, we developed a method, without using antibodies, to separate three cecal cell subpopulations: lymphocytes, enterocytes, and a population encompassing multiple cell types. We analyzed the mRNA expression of TLR4, interleukin-1ß (IL-1ß), IL-8, IL-12, and lipopolysaccharide-induced tumor necrosis factor alpha factor (LITAF) in cecal subpopulations of chicks from inbred lines resistant or susceptible to the carrier state infected with Salmonella enterica serovar Enteritidis. The results showed that resistance to the carrier state in chicks is associated with a larger percentage of lymphocytes and with higher levels of expression of TLR4 and IL-8 at homeostasis in the three cell subpopulations, as well as with a higher level of expression of LITAF in lymphocytes during the carrier state. In contrast to the early phase of infection, the carrier state is characterized by no major cell recruitment differences between infected and noninfected animals and no significant modification in terms of TLR4, IL-1ß, IL-8, IL-12, and LITAF expression in all cell subpopulations measured. However, TLR4 expression increased in the lymphocytes of chicks from the susceptible line, reaching the same level as that in infected chicks from the resistant line. These observations suggest that the carrier state is characterized by a lack of immune activation and highlight the interest of working at the level of the cell population rather than that of the organ.

Heterogeneity of type III secretion system (T3SS)-1-independent entry mechanisms used by Salmonella Enteritidis to invade different cell types.

Salmonella causes a wide range of diseases from acute gastroenteritis to systemic typhoid fever, depending on the host. To invade non-phagocytic cells, Salmonella has developed different mechanisms. The main invasion system requires a type III secretion system (T3SS) known as T3SS-1, which promotes a Trigger entry mechanism. However, other invasion factors have recently been described in Salmonella, including Rck and PagN, which were not expressed under our bacterial culture conditions. Based on these observations, we used adhesion and invasion assays to analyse the respective roles of Salmonella Enteritidis T3SS-1-dependent and -independent invasion processes at different times of infection. Diverse cell lines and cell types were tested, including endothelial, epithelial and fibroblast cells. We demonstrated that cell susceptibility to the T3SS-1-independent entry differs by a factor of nine between the most and the least permissive cell lines tested. In addition, using scanning electron and confocal microscopy, we showed that T3SS-1-independent entry into cells was characterized by a Trigger-like alteration, as for the T3SS-1-dependent entry, and also by Zipper-like cellular alteration. Our results demonstrate for what is believed to be the first time that Salmonella can induce Trigger-like entry independently of T3SS-1 and can induce Zipper-like entry independently of Rck. Overall, these data open new avenues for discovering new invasion mechanisms in Salmonella.

Rck of Salmonella enterica, subspecies enterica serovar enteritidis, mediates zipper-like internalization.

Salmonella can invade non-phagocytic cells through its type III secretion system (T3SS-1), which induces a Trigger entry process. This study showed that Salmonella enterica, subspecies enterica serovar Enteritidis can also invade cells via the Rck outer membrane protein. Rck was necessary and sufficient to enable non-invasive E. coli and Rck-coated beads to adhere to and invade different cells. Internalization analysis of latex beads coated with different Rck peptides showed that the peptide containing amino acids 140-150 promoted adhesion, whereas amino acids between 150 and 159 modulated invasion. Expression of dominant-negative derivatives and use of specific inhibitors demonstrated the crucial role of small GTPases Rac1 and Cdc42 in activating the Arp2/3 complex to trigger formation of actin-rich accumulation, leading to Rck-dependent internalization. Finally, scanning and transmission electron microscopy with Rck-coated beads and E. coli expressing Rck revealed microvillus-like extensions that formed a Zipper-like structure, engulfing the adherent beads and bacteria. Overall, our results provide new insights into the Salmonella T3SS-independent invasion mechanisms and strongly suggest that Rck induces a Zipper-like entry mechanism. Consequently, Salmonella seems to be the first bacterium found to be able to induce both Zipper and Trigger mechanisms to invade host cells.

Investigation of the role of the BAM complex and SurA chaperone in outer-membrane protein biogenesis and type III secretion system expression in Salmonella.

In Escherichia coli, the assembly of outer-membrane proteins (OMP) requires the BAM complex and periplasmic chaperones, such as SurA or DegP. Previous work has suggested a potential link between OMP assembly and expression of the genes encoding type-III secretion systems. In order to test this hypothesis, we studied the role of the different lipoproteins of the BAM complex (i.e. BamB, BamC, BamD and BamE), and the periplasmic chaperones SurA and DegP, in these two phenotypes in Salmonella. Analysis of the corresponding deletion mutants showed that, as previously described with the DeltabamB mutant, BamD, SurA and, to a lesser extent, BamE play a role in outer-membrane biogenesis in Salmonella Enteritidis, while the membrane was not notably disturbed in DeltabamC and DeltadegP mutants. Interestingly, we found that BamD is not essential in Salmonella, unlike its homologues in Escherichia coli and Neisseria gonorrhoeae. In contrast, BamD was the only protein required for full expression of T3SS-1 and flagella, as demonstrated by transcriptional analysis of the genes involved in the biosynthesis of these T3SSs. In line with this finding, bamD mutants showed a reduced secretion of effector proteins by these T3SSs, and a reduced ability to invade HT-29 cells. As DeltasurA and DeltabamE mutants had lower levels of OMPs in their outer membrane, but showed no alteration in T3SS-1 and flagella expression, these results demonstrate the absence of a systematic link between an OMP assembly defect and the downregulation of T3SSs in Salmonella; therefore, this link appears to be related to a more specific mechanism that involves at least BamB and BamD.

TolC, but not AcrB, is involved in the invasiveness of multidrug-resistant Salmonella enterica serovar Typhimurium by increasing type III secretion system-1 expression.

The AcrAB-TolC efflux system is involved in multidrug and bile salt resistances. In addition, this pump has recently been suggested to increase the invasion of Salmonella enterica serovar Typhimurium (S. Typhimurium) into host cells in vitro and could therefore have an important clinical relevance for multidrug-resistant strains. The aim of this study was to investigate the role of the TolC outer membrane channel and the AcrB transporter in the interaction of multidrug-resistant S. Typhimurium strains with eukaryotic cells, especially in relation to the expression of the type III secretion system-1 (TTSS-1) required for Salmonella invasion. Deletion of tolC led to a reduced transcription of the Salmonella pathogenicity island-1 genes sipA, invF and hilA, demonstrating that all genes required for TTSS-1 biosynthesis are down-regulated in this mutant. Consequently, tolC mutants secreted smaller amounts of the TTSS-1 effector proteins SipA and SipC, and invasion tests performed with one mutant showed that it was significantly less able to invade HT-29 epithelial cells than its parental strain. This control seems specific to the TTSS-1 among the three TTSS of Salmonella as no down-regulation of expression of TTSS-2 or flagella was observed in this mutant. By contrast, acrB mutants behaved as their parents except that they secrete a slightly greater amount of SipA and SipC proteins. These data indicate that TolC but not AcrB mediates the uptake of multidrug-resistant S. Typhimurium into target host cells. Therefore, this role of TolC in the invasion of the intestine in addition to its role in bile salt resistance reinforces the interest of targeting TolC for fighting multidrug-resistant Salmonella.

The YfgL lipoprotein is essential for type III secretion system expression and virulence of Salmonella enterica Serovar Enteritidis.

Salmonella enterica, like many gram-negative pathogens, uses type three secretion systems (TTSS) to infect its hosts. The three TTSS of Salmonella, namely, TTSS-1, TTSS-2, and flagella, play a major role in the virulence of this bacterium, allowing it to cross the intestinal barrier and to disseminate systemically. Previous data from our laboratory have demonstrated the involvement of the chromosomal region harboring the yfgL, engA, and yfgJ open reading frames in S. enterica serovar Enteritidis virulence. Using microarray analysis and real-time reverse transcription-PCR after growth of bacterial cultures favorable for either TTSS-1 or TTSS-2 expression, we show in this study that the deletion in S. enterica serovar Enteritidis of yfgL, encoding an outer membrane lipoprotein, led to the transcriptional down-regulation of most Salmonella pathogenicity island 1 (SPI-1), SPI-2, and flagellar genes encoding the TTSS structural proteins and effector proteins secreted by these TTSS. In line with these results, the virulence of the DeltayfgL mutant was greatly attenuated in mice. Moreover, even if YfgL is involved in the assembly of outer membrane proteins, the regulation of TTSS expression observed was not due to an inability of the Delta yfgL mutant to assemble TTSS in its membrane. Indeed, when we forced the transcription of SPI-1 genes by constitutively expressing HilA, the secretion of the TTSS-1 effector protein SipA was restored in the culture supernatant of the mutant. These results highlight the crucial role of the outer membrane lipoprotein YfgL in the expression of all Salmonella TTSS and, thus, in the virulence of Salmonella. Therefore, this outer membrane protein seems to be a privileged target for fighting Salmonella.

Precise excision and secondary transposition of TnphoA in non-motile mutants of a Salmonella enterica serovar Enteritidis clinical isolate.

Mutagenesis with TnphoA has been widely used in many bacteria. Here, we report the excision and secondary transposition of this transposon in three non-motile (fliC, fliF and motB) mutants of Salmonella enterica serovar Enteritidis (S. Enteritidis). Isolation of motile revertants showed that they were kanamycin resistant and conserved a copy of TnphoA in their genome in an insertion site different from the initial one. They also expressed an intact flagella. Characterization of the motile revertant derived from the fliC mutant showed that TnphoA excised precisely from the fliC gene, resulting in an equivalent amount of FliC secreted protein in the revertant compared to that of the wild-type strain. These results show that TnphoA mutants should be used with care and underline the value of using transposon derivatives lacking the transposase gene.

Identification of a new Salmonella enterica serovar Enteritidis locus involved in cell invasion and in the colonisation of chicks.

Poultry products contaminated with Salmonella enterica serovar Enteritidis are a major cause of foodborne disease in industrialized countries. Knowledge of how poultry is colonised is essential for reducing contamination of these products. We have characterized the bacterial yfg-eng locus involved in chicken colonisation. Its sequencing revealed four open reading frames (ORF), yfgM, yfgL, engA and yfgJ, all transcribed in the same orientation. An yfgL mutant of S. Enteritidis colonised the caeca (P < 0.05) and the spleens (P < 0.01) of one-day-old chicks subnormally 2 and 5 days after oral inoculation. This lower virulence was correlated with reduced secretion of the SPI-1 and flagellar proteins in the yfgL mutant compared to the wild-type strain. Consistent with this, the S. Enteritidis yfgL mutant was less motile than the wild type and fewer invaded enterocytes (P < 0.05) and avian HD11 macrophages (P < 0.001). All these defects could be partially overcome by inserting the yfg-eng locus into the mutant on a recombinant plasmid.

Establishment and characterization of partially differentiated chicken enterocyte cell clones.

Three enterocyte cell clones were established in vitro from the intestine of a PA12 hen embryo. These cells exhibited epithelioid morphology and grew as monolayers. The cells were continuously propagated in culture up to 250 passages. Gradual increase in growth rate with time and in anchorage-independent growth in both agar and agarose showed that the three cell clones spontaneously transformed in vitro. The clones were heteroploid with one marker chromosome. Interestingly, they had features of partly differentiated enterocytes, especially microvilli, junctions connecting adjacent cells (tight junctions, desmosomes, hemidesmosomes, gap junctions), villin and cytokeratins. In addition, cells expressed brush border enzyme activity and transepithelial resistance. The fact that the levels of dipeptidyl peptidase IV (DPP-IV) and alkaline phosphatase activities fluctuated according to culture time and that MHC class II was induced by activation of cells with interferon suggested that the state of differentiation of the 3 cell clones could be modified in vitro. These clones are the first established avian enterocyte cell clones to be described. Because each cell clone exhibited differences in the level of differentiation and sensitivity to Salmonella infection, their use will allow comparative investigations concerning markers of differentiation of avian enterocytes and infection by host-adapted bacteria and parasites.