Functional analysis of a rickettsial OmpA homology domain of Shigella flexneri IcsA

Shigella flexneri is a gram negative bacterium that causes diarrhea and dys entery by invasion and spread through the colonic epithelium. Bacteria spre ad by assembling actin and other cytoskeletal proteins of the host into "ac tin tails" at the bacterial pole; actin tail assembly provides the force re quired to move bacteria through the cell cytoplasm and into adjacent cells. The 120-kDa S. flexneri outer membrane protein IcsA is essential for actin assembly. IcsA is anchored in the outer membrane by a carboxy-terminal dom ain (the beta domain), such that the amino-terminal 706 amino acid residues (the alpha domain) are exposed on the exterior of the bacillus. The alpha domain is therefore likely to contain the domains that are important to int eractions with host factors. We identify and characterize a domain of IcsA within the alpha domain that bears significant sequence similarity to two r epeated domains of rickettsial OmpA, which has been implicated in rickettsi al actin tail formation. Strains of S. flexneri and Escherichia coli that c arry derivatives of IcsA containing deletions within this domain display lo ss of actin recruitment and increased accessibility to IcsA-specific antibo dy on the surface of intracytoplasmic bacteria. However, site-directed muta genesis of charged residues within this domain results in actin assembly th at is indistinguishable from that of the wild type, and in vitro competitio n of a polypeptide of this domain fused to glutathione S-transferase did no t alter the motility of the wild-type construct. Taken together, our data s uggest that the rickettsial homology domain of IcsA is required for the pro per conformation of IcsA and that its disruption leads to loss of interacti ons of other IcsA domains within the amino terminus with host cytoskeletal proteins.