Early life stress in mice is a suitable model for Irritable Bowel Syndrome but does not predispose to colitis nor increase susceptibility to enteric infections.

Neonatal period is characterized by an immature intestinal barrier. Scattered evidence suggests that early life stressful events induce long lasting alterations of intestinal homeostasis mimicking Irritable Bowel Syndrome (IBS). Those observations highlighting defect of intestinal barrier by early life stress questioned its potential role as a risk factor for gastrointestinal disorders such as colitis and infections. In this study, we aimed to analyze if maternal separation (MS) in mice mimicks IBS main features. We next addressed whether MS could trigger or exacerbate colitis in genetically predisposed mice and/or enhance susceptibility to gastrointestinal infections in wild type mice. MS induced main features of IBS in adult wild type male mice i.e. intestinal hyperpermeability, visceral hypersensitivity, microbiota dysbiosis, bile acid malabsorption and low grade inflammation in intestine associated with a defect of Paneth cells and the ILC3 population. This breach in mucosal barrier functions in adults was associated with a systemic IgG response against commensal E. coli and increased IFNγ secretion by splenocytes. However, in IL10-/- mice, MS did not trigger nor worsen colitis. Furthermore, wild type mice submitted to MS did not show increase susceptibility to gastrointestinal infections (S. Typhimurium, L. monocytogenes or T. gondii) compared to controls. Altogether, our results identify MS in mice as a good experimental model for IBS mimicking all the main features. In addition, early life stress, even though it has long lasting consequences on intestinal homeostasis, does not constitute a facilitating factor to colitis in predisposed individuals nor to gastrointestinal infections in wild type mice.

Host cell protein tyrosine kinases are activated during the entry of Listeria monocytogenes. Possible role of pp60c-src family protein kinases.

Listeria monocytogenes is able to invade a wide range of cell types by inducing its own internalization. Little is known, however, about the host cell proteins affecting the entry process which involves triggering the host cell signal transduction mechanism. We report here that entry of L. monocytogenes strains (serotypes 4b and 1/2a) into Caco-2 cells induced tyrosine phosphorylation of several host cell proteins including pp60c-src substrates. Using specific synthetic peptide substrates, we showed that L. monocytogenes activates, as early as 5 min after bacteria-cell contact, the pp60c-src family-related (srcFR) proteins by an inlAB-dependent pathway. The activation of srcFR proteins seems to be crucial in the entry of L. monocytogenes into Caco-2 cells. Indeed, specific inhibition of the srcFR signal by herbimycin A blocked the entry of L. monocytogenes strains. Taken together, our data show that L. monocytogenes enhances cell tyrosine phosphorylations and activates the pp60c-src family-related proteins by an inlAB-dependent pathway.

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.

The loss of contact inhibition and anchorage-dependent growth are key steps in the acquisition of Listeria monocytogenes susceptibility phenotype by non-phagocytic cells.

We have previously demonstrated that intestinal and kidney finite cell lines were resistant to L monocytogenes invasion (ie allowed low bacterial entry and no intracellular multiplication) in contrast to the continuous cell lines which were susceptible to Listeria invasion (ie allowed high bacterial entry and intracellular multiplication) (Velge et al (1994a) Med Microbial Immunol 183, 145). The aim of this study was to discover whether epigenetic or genetic cellular modifications could convert L monocytogenes resistant cells into a susceptible phenotype and to determine the cellular steps involved in Listeria susceptibility. Among the 5-azacytidine treated finite cell lines, the untransformed immortal cell lines established remained resistant to L monocytogenes invasion whereas the weakly transformed continuous cell lines established were converted into a susceptible phenotype. Transfection of resistant cells by SV40 large T antigen induced only highly transformed continuous cell lines displaying a susceptible phenotype. Taken together these data show that cell transformation enhanced Listeria invasion. This conclusion was supported by the observation that L monocytogenes was able to induce cell foci within murine finite cell monolayers. This morphological cell transformation was completely reversible and required live bacteria inside cells. In conclusion, we may speculate that the L monocytogenes intracellular multiplication observed within cell foci could be explained by the loss of contact inhibition of the finite cell monolayer. Indeed, the loss of both contact inhibition and anchorage-dependent growth are the key steps involved in the L monocytogenes susceptibility phenotype.

Protein tyrosine kinase inhibitors block the entries of Listeria monocytogenes and Listeria ivanovii into epithelial cells.

The internalization of Listeria by intestinal epithelial cells is still poorly understood, however it is becoming apparent that microorganisms have developed the ability to interact with host cell receptor molecules to induce their own internalization. In this report we show that inhibition of cell tyrosine phosphorylation by protein tyrosine kinase (PTK) inhibitors blocks L. monocytogenes entry into both finite and immortalized intestinal cell lines. Some differences were observed between the Listeria species. L. monocytogenes entry was inhibited by between 10- to 100-fold by PTK inhibitors competing with the tyrosine residue binding of the kinase as erbstatin or by PTK inhibitors competing with the binding of ATP to the enzyme as genistein and some tyrphostins. On the other hand, L. ivanovii entry was inhibited by erbstatin as observed with L. monocytogenes but poorly by genistein and tyrphostins. The use of these several PTK inhibitors shows that even though both L. monocytogenes and L. ivanovii entered intestinal and other cell lines by stimulating PTK, it seems that L. monocytogenes stimulated a different PTK than L. ivanovii. According to the fact that the number of PTK receptors increases on immortalized cells, the higher L. monocytogenes internalization observed with immortalized cell lines could be related to a higher PTK receptor number on these cells compared to finite cell lines.

Tissue culture assays using Caco-2 cell line differentiate virulent from non-virulent Listeria monocytogenes strains.

Within the group of Listeria sp., only L. monocytogenes is pathogenic for humans and numerous studies of L. monocytogenes strains have described non-virulent isolates. In this study, the potential value of two tissue culture assays (TCA) was analysed to ascertain the virulence properties of L. monocytogenes strains, initially typed for virulence using the immunocompromised mouse model (ICMM). The first assay assessed both the penetration into, and multiplication within, Caco-2 cells (PM assay): the second was a plaque-forming assay (PF assay). All the clinical isolates (nine strains) were virulent in both TCA. Conversely, all the non-pathogenic species (seven strains) were non-virulent in PM and PF assays. Compared with the virulence obtained in the ICMM with 29 Listeria strains, including 12 non-virulent L. monocytogenes strains, the sensitivity of both TCA was equal to1. Specificity was 0.89 and 0.84 for the PF and PM assays, respectively. However, a study of strains exhibiting virulence differences in three other in vivo virulence models showed that ICMM only detected highly virulent strains. The specificity of the PF test could, therefore, be higher, and close to that obtained by the enumeration of viable bacteria in the spleen of mice infected by subcutaneous injection in the footpad and by intravenous injection. Taken together, this study confirms the existence of low-virulence L. monocytogenes strains and shows that the virulence status of some non-clinical L. monocytogenes isolates depends on the virulence models used. The data suggest that the PF assay could be used as a primary test to evaluate the virulence of Listeria strains in order to reduce the cost of testing all strains in vivo.