LACK OF CLEAVAGE OF ICSA IN SHIGELLA-FLEXNERI CAUSES ABERRANT MOVEMENT AND ALLOWS DEMONSTRATION OF A CROSS-REACTIVE EUKARYOTIC PROTEIN

Once in the cytoplasm of mammalian cells, Shigella flexneri expresses a motile phenotype caused by polar directional assembly of actin. This process depends on accumulation of IcsA (VirG), a 120-kDa protein wit h ATPase activity, at the pole of the bacterium opposite to that at wh ich ongoing septation occurs. IcsA is also secreted into the bacterial supernatant as a 95-kDa species, after cleavage at an SSRRASS sequenc e which, when mutagenized, blocks processing. MAb15, an anti-IcsA mono clonal antibody, recognizes an epitope located within repeated Gly-ric h boxes in the N-terminal half of the protein. We used this monoclonal antibody to visualize the location of a noncleavable 120-kDa IcsA mut ant protein expressed in S. flexneri. We found that this noncleavable IcsA protein no longer localized exclusively to the pole of the bacter ium but also could be detected circumferentially. Whereas the monoclon al antibody detected the wild-type cleavable form of IcsA in only 40% of the cells expressing this protein, the noncleavable form was easily detectable in all the cells carrying the icsA mutant allele, Similar aberrant localization of the IcsA mutant protein on bacteria growing w ithin the cytoplasm of HeLa cells was observed. The strains expressing the noncleavable IcsA protein expressed abnormal intracellular moveme nt and were often observed moving in a direction perpendicular to thei r longitudinal axis. The putative protease which processes IcsA may th erefore play a role in achieving polar expression of this protein and providing maximum asymmetry essential to directional movement. In addi tion, MAbF15 allowed us to identify a 70-kDa eukaryotic protein cross- reacting with IcsA. This protein accumulated in the actin tails of mot ile bacteria and in membrane ruffles of the cells.