EVIDENCE FOR A VAPID, DIRECT EFFECT ON EPITHELIAL MONOLAYER INTEGRITYAND TRANSEPITHELIAL TRANSPORT IS RESPONSE TO SALMONELLA INVASION

In cultured monolayers of high-resistance Ma din-Darby Canine Kidney ( MDCK) cells, infection with Salmonella typhimurium SL1344 resulted in a dose- and time-dependent increase in transepithelial conductance (G( t)) and short-circuit current (I-sc). There was a direct linear relati onship between the S. typhimurium-induced increments in I-sc and G(t) suggesting that this early change in epithelial parameters is, in part , the result of a cellular conductance change most probably at the api cal membrane. An additional wild-type S. typhimurium strain, SR11, and an invasion-deficient isogenic mutant SB111 carrying a non-polar muta tion in invA were used to confirm that the S. typhimurium-induced chan ge in epithelial electrical parameters is directly linked to the invas ion process. The S. typhimurium-induced change in epithelial electrica l parameters was markedly attenuated in Na+-free choline medium. Addit ion of piretanide (10(-4) M, basal side) failed to affect the increase d epithelial conductance and I-sc after a 40-min incubation with S. ty phimurium. NPPB (5x10(-4) M) added to the apical medium reduced the S. typhimurium-stimulated I-sc by 28%, but G(t) was not significantly re duced. It is unlikely that the S. typhimurium-induced I-sc is due to C l- secretion. Staining of S. typhimurium-infected MDCK I monolayers wi th TRITC-phalloidin revealed marked alterations of F-actin; diffuse in tracellular accumulations of F-actin corresponding to the presence of invading bacteria were observed by 15 min. After 60 min, prominent ext rusions of the apical membrane corresponding to previously described ' 'membrane ruffles'' were noted. Marked accumulations of perijunctional F-actin in infected cells corresponded to contraction of the perijunc tional actin ring at the apical pole. In adjacent cells marked distort ion and stretch of the apical surface was evident. The invasion defici ent invA mutant SB111 failed to induce these morphological changes. Th ese data demonstrate that S. typhimurium invasion induces increased tr anscellular conductance which does not result from stimulation of Cl- secretion but instead appears to be predominantly due to increased Na permeability. The increased membrane conductance is coincident with i ncreased transepithelial inulin permeability indicating that the incre ment in G(t) has an additional ''paracellular'' component. The S. typh imurium-induced alterations in epithelial parameters may be related to ''membrane ruffling'' and/or to the accompanying changes in cell shap e.