Elisabeth Bottreau

Invasion of Salmonella enteritidis in avian intestinal epithelial cells in vitro is influenced by short-chain fatty acids.

Fermentation reactions in the caeca of chickens, the predominant place for Salmonella colonization, result in high concentrations of short-chain fatty acids (SCFA). Thus Salmonella bacteria are in close contact with SCFA during their life cycle. A study was carried out to analyse the effects of SCFA on invasion of Salmonella enteritidis in an avian intestinal epithelial cell line. Preincubation of S. enteritidis for 4 h in growth media supplemented with various concentrations of propionate or butyrate resulted in decreased invasion compared to bacteria, preincubated in nonsupplemented media, and to bacteria, preincubated in media supplemented with formate or acetate. Incubation of the S. enteritidis bacteria in media supplemented with mixtures of SCFA mimicking the in vivo caecal concentrations resulted in increased invasion compared with butyrate-exposed bacteria, but equal invasion compared with nonexposed bacteria. Increasing the butyrate concentration in these mixtures did not modify invasion compared with the original mixtures.

Assessment of the virulence of Listeria monocytogenes: agreement between a plaque-forming assay with HT-29 cells and infection of immunocompetent mice.

Some Listeria monocytogenes strains not related to clinical cases have been found to exhibit a low virulence level in mice as well as in an in vitro test using Caco-2 cells. The purpose of this study was to validate a new in vitro test of virulence based on a plaque-forming assay (PFA) using a HT-29 cell monolayer with 118 Listeria strains. The use of HT-29 cells in 96-well tissue culture plates allowed the testing of 30 strains per day and providing results in 24 h. In addition. statistical analyses demonstrated the reproducibility and repeatability of the PFA. No quantitative relationship was observed between the virulence of the strains and the hemolytic titer or the cytotoxic effects on HT-29 cells. In contrast, good agreement was observed between virulence assessed after subcutaneous (SC) infection and virulence obtained by PFA. Three groups of L. monocytogenes strains (avirulent, hypovirulent and fully virulent) were established by comparison of the clinical origin of the strains, the number of immunocompetent contaminated mice and the numbers of Listeria strains recovered in the spleen after SC infection. With one exception, i.e. a clinical case of L. seeligeri (sensitivity 0.98), the PFA successfully detected the virulent strains only (specificity 1). Decision-tree algorithms performed by SAS and S-Plus demonstrated that this tissue culture assay discriminated between the avirulent and hypovirulent strains and the virulent strains. This test could therefore be an alternative to in vivo tests, allowing grading of virulence.

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.

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

ABSTRACT 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 ΔyfgL 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 ΔyfgL 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.

Cell proliferation enhances entry of Listeria monocytogenes into intestinal epithelial cells by two proliferation-dependent entry pathways.

Bacterial entry into intestinal host cells is the result of a fairly sophisticated manipulation of host cell machinery by the pathogens. To study further the potential cell target of Listeria spp., the in-vitro entry of L. monocytogenes strains into intestinal cells was examined in relation to the metabolism, proliferation and differentiation of the cells by the alamarBlue assay, [3H] thymidine incorporation, and brush border-associated enzyme activities, respectively. The study showed that cell metabolism was not involved in the entry of L. monocytogenes in three cell models (two human and one porcine). On the other hand, entry was closely related to the proliferation process and poorly related to the differentiation state of the cells. The use of L. monocytogenes mutants lacking invasion proteins showed that InlA and InlB acted in synergy to mediate the entry of L. monocytogenes into proliferative cells, whereas InlA alone seemed to be involved in the entry into non-proliferative cells. These two entry pathways could correspond to the two cellular processes used by L. monocytogenes to enter proliferative and non-proliferative cells, as suggested by the use of cytochalasin D, nocodazole, chloroquine and monodansylcadaverine. Taken together, we propose a hypothesis in which the entry of L. monocytogenes is mediated by interaction between randomly distributed E-cadherin on the surface of proliferative cells. In contrast, the entry into non-proliferative cells may involve pp60c-src, a proto-oncogenic tyrosine kinase signal that modifies E-cadherin localisation. In conclusion, these results suggest that L. monocytogenes may preferentially enter crypt cells in vivo by a microfilament-dependent process, whereas the few bacteria that infect villus cells enter by an E-cadherin-internalin interaction that mediates microtubule-dependent endocytosis.

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.

Bacteria-host interactions of Salmonella Paratyphi B dT(+) in poultry

In recent years, a dramatic increase in incidence of the dextro-rotatory tartrate-positive variant (dT+) of Salmonella enterica subspecies enterica serovar Paratyphi B has been observed in poultry and poultry products. In the present study the interactions of this bacterium with the host were studied in vivo and in vitro in an attempt to explain the preferential association of this serotype with poultry. The ability of this organism to invade and multiply in chicken intestinal epithelial cells and the intracellular behaviour in chicken macrophages was studied in vitro using chicken cell lines. In vivo challenge experiments in specific pathogen-free chickens were carried out to determine the level of colonization of caeca and internal organs early after experimental infection. An in vivo trial with commercial broiler chickens, using a seeder model, was performed to determine whether S. Paratyphi B dT+ could persist and spread in broilers until slaughter. S. Paratyphi B dT+ invaded and multiplied in the chicken epithelial cell line and survived in a chicken macrophage cell line. The strain used colonized caeca and internal organs of chickens to a high extent 1 week after infection with a low-dose inoculum. Moreover, the strain was efficiently transmitted within a group of broilers and persisted until slaughter. It was concluded that S. Paratyphi B dT+ was well adapted to poultry and therefore it is suggested that specific control measures against this serotype should be considered.

Expression of Toll-Like Receptor 4 and Downstream Effectors in Selected Cecal Cell Subpopulations of Chicks Resistant or Susceptible to Salmonella Carrier State

ABSTRACT 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-1β (IL-1β), 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-1β, 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.

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.

Natural infection with the porcine respiratory coronavirus induces protective lactogenic immunity against transmissible gastroenteritis

Abstract Our objective was to evaluate the level of passive protection against transmissible gastroenteritis (TGE) among 57 newborn piglets nursing from seven seropositive sows previously naturally infected with porcine respiratory coronavirus (PRCV). After challenge exposure we observed mortality rates of 44% for litters of seven PRCV-infected sows, 40% for litters of four sows orally immunized with the attenuated TGEV strain Nouzilly, and 91% for litters of seven seronegative susceptible sows. A blocking ELISA with two appropriate monoclonal antibodies distinguished serological responses of PRCV-infected sows from those of TGEV-immunized sows. The results suggest that natural infection of the sow with PRCV may induce a degree of protective lactogenic immunity against TGE.