MXIG, A MEMBRANE-PROTEIN REQUIRED FOR SECRETION OF SHIGELLA SPP LPA INVASINS - INVOLVEMENT IN ENTRY INTO EPITHELIAL-CELLS AND IN INTERCELLULAR DISSEMINATION

Entry of Shigella flexneri into epithelial cells involves secretory pr oteins, the Ipa proteins, and their dedicated secretion apparatus, the Mxi-Spa translocon, which is encoded by the mxi and spa operons. We h ave characterized the mxiG gene that is located at the proximal part o f the mxi operon. Inactivation of mxiG abolished Ipa secretion, which indicates that MxiG is an essential component of the Mxi-Spa transloco n. Immunoblotting analysis of membrane fractions suggests that the 42 kDa MxiG protein is associated with both the inner and outer membranes . Taking advantage of the complementation of the mxiG mutant by a plas mid carrying a wild-type copy of mxiG (which restored Ipa secretion, e ntry into HeLa cells, and cell-to-cell spread) we mutagenized the mxiG gene carried by the complementing plasmid to replace the RGD motif of MxiG by RAD, This mutation (mxiG), which had no effect on the stabil ity of the protein, did not affect Ipa secretion in vitro or entry int o HeLa cells, but impaired intercellular dissemination. Therefore, Mxi G and possibly proteins secreted by the Mxi-Spa translocon are involve d not only in entry but also in spread of Shigella between epithelial cells.