AVIRULENCE OF ROUGH MUTANTS OF SHIGELLA-FLEXNERI - REQUIREMENT OF O-ANTIGEN FOR CORRECT UNIPOLAR LOCALIZATION OF ICSA IN THE BACTERIAL OUTER-MEMBRANE

Mutations in the lipopolysaccharide (LPS) of Shigella spp. result in a ttenuation of the bacteria in both in vitro and in vivo models of viru lence, although the precise block in pathogenesis is not known. We iso lated defined mutations in two genes, galU and rfe, which directly aff ect synthesis of the LPS of S. flexneri 2a, in order to determine more precisely the step in virulence at which LPS mutants are blocked. The galU and rfe mutants invaded HeLa cells but failed to generate the me mbrane protrusions (fireworks) characteristic of intracellular motilit y displayed by wild-type shigellae. Furthermore, the galU mutant was u nable to form plaques on a confluent monolayer of eucaryotic cells and the rfe mutant generated only tiny plaques. These observations indica ted that the mutants,were blocked in their ability to spread from cell to cell. Western immunoblot analysis of expression of IcsA, the prote in essential for intracellular motility and intercellular spread, demo nstrated that both mutants synthesized IcsA, although they secreted le ss of the protein to the extracellular medium than did the wild-type p arent. More strikingly, the LPS mutants showed aberrant surface locali zation of IcsA. Unlike the unipolar localization of IcsA seen in the w ild-type parent, the galU mutant expressed the protein in a circumfere ntial fashion. The rfe mutant had an intermediate phenotype in that it displayed some localization of IcsA at one pole while also showing di ffuse localization around the bacterium. Given the known structures of the LPS of wild-type S. flexneri 2a, the rfe mutant, and the galU mut ant, we hypothesize that the core and O-antigen components of LPS are critical elements in the correct unipolar localization of IcsA. These observations indicate a more precise role for LPS in Shigella pathogen esis.