In the human intestine, target cells of enteropathogens differentiate during cell migration along the crypt-villus axis. We have recently provided evidence that intestinal cell differentiation up-regulates intestinal cell infection by the noninvasive enterotoxigenic Escherichia coli [5, 23]. Several enterovirulent bacteria can penetrate intestinal epithelial cells, which are normally nonphagocytic. To document the role of intestinal epithelial cell differentiation in the pathogenesis of enteroinvasive bacteria, we examined here the intestinal cell-association and cell-entry of Yersinin pseudotuberculosis as a function of cell differentiation. For this put-pose we used the colon carcinoma Caco-2 cell line in culture, which provides the most useful tool for the study of intestinal epithelial cell differentiation, because of its unique ability to spontaneously differentiate upon reaching confluence in normal culture condition. We report here that the thermoregulated inv and nil loci of Y. pseudotuberculosis have distinct roles in infection of Caco-2 cells. The nil locus initiates the cell-association and the inv locus initiates both the cell-association and the cell-entry processes. Moreover, we observed that: (i) both the bacterial cell-association (ail) and the bacterial cell-invasion (inv) occur at subconfluence when the Caco-2 cells are undifferentiated, and (ii) these processes are arrested when the differentiation commences. Since the integrin-beta(1), heterodimers are involved in cell-entry of Y. pseudotuberculosis in several mammalian cells, we further examined which beta(1) integrin promotes bacterial cell-entry in Caco-2 cells. Using several monoclonal antibodies (MoAbs) to integrin we observed that only the antibody directed against alpha(5) beta(1) integrin blocked the cell-entry of Y. pseudotuberculosis into undifferentiated Caco-2 cells. To investigate how cell differentiation inhibits bacterial cell-entry, we examined, by indirect immunofluorescence staining, the expression of alpha(5) beta(1) integrin by Caco-2 cells as a function of the cell differentiation. We observed that: (i) the alpha(5) beta(1) integrin is intensively expressed by the proliferative undifferentiated Caco-2 cells entirely covering the cell surface; (ii) when the cell confluency occurs alpha(5) beta(1) integrin is immediately redistributed, lining the focal cell-to-cell contacts. These results indicate that the intestinal bacterial cell receptor involved in Y. pseudotuberculosis cell-entry is accessible in undifferentiated Caco-2 cells and is made inaccessible after intestinal epithelial cell differentiation. These results are the first to demonstrate that intestinal epithelial cell differentiation down-regulates the cell accessibility of a bacterial cell receptor involved in human intestinal cell infection.
