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 with ATPase activity, at the pole of the bacterium opposite to that at which ongoing septation occurs. IcsA is also secreted into the bacterial supernatant as a 95-kDa species, after cleavage at an SSRRASS sequence which, when mutagenized, blocks processing. MAbF15, an anti-IcsA monoclonal antibody, recognizes an epitope located within repeated Gly-rich boxes in the N-terminal half of the protein. We used this monoclonal antibody to visualize the location of a noncleavable 120-kDa IcsA mutant protein expressed in S. flexneri. We found that this noncleavable IcsA protein no longer localized exclusively to the pole of the bacterium but also could be detected circumferentially. Whereas the monoclonal antibody detected the wild-type cleavable form of IcsA in only 40% of the cells expressing this protein, the noncleavable was easily detectable in all the cells carrying the icsA mutant allele. Similar aberrant localization of the IcsA mutant protein on bacteria growing within the cytoplasm of HeLa cells was observed. The strains expressing the noncleavable IcsA protein expressed abnormal intracellular movement and were often observed moving in a direction perpendicular to their longitudinal axis. The putative protease which processes IcsA may therefore play a role in achieving polar expression of this protein and providing maximum asymmetry essential to directional movement. In addition, MAbF15 allowed us to identify a 70-kDa eukaryotic protein cross-reacting with IcsA. This protein accumulated in the actin tails of motile bacteria and in membrane ruffles of the cells.
Unité de pathogenie Microbienne Moléculaire, U 389 Institut National de la Santé et de la Recherche Médicale, Paris, France.
H
H
d'Hauteville
R
R
Dufourcq Lagelouse
F
F
Nato
P J
PJ
Sansonetti
D000199:Actins / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D001426:Bacterial Proteins / Q000276:immunology / Q000378:metabolism*; D003429:Cross Reactions; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009068:Movement; D011817:Rabbits; D012763:Shigella flexneri / Q000502:physiology*; D014157:Transcription Factors / Q000276:immunology / Q000378:metabolism*
D000199:Actins; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D014157:Transcription Factors; C058483:virG protein, Shigella flexneri