IPAC-Mediated Invasion of Epithelial Cells by Shigella

Shigella are gram negative enteric pathogens that cause bacillary dysentery, a serious world-wide health threat. An important feature of shigellosis is bacterial entry into nonphagocytic host cells. The invasion plasmid antigens (Ipa proteins) have been identified as the invasins of this process; however, their precise biochemical functions are not known. The long-range objective of this research is to determine the structural and functional features of these invasins from Shigella flexneri. To achieve these goals, the ipa genes were cloned, expressed, their products purified, and the biochemistry of the proteins investigated. IpaC was found to possess in vitro properties related to bacterial internalization. Therefore, the specific aims of the proposed work are: 1) to define the biochemical events that follow IpaC binding to host cells; 2) to determine mechanisms by which IpaC causes uptake of virulent and avirulent strains of S. flexneri; 3) to use purified IpaB, C, and D to investigate formation of protein complexes that facilitate invasion in vivo; and 4) to identify the functional domains of IpaC. These aims will be completed using diverse methodologies in cell biology and biochemistry in conjunction with specialized fluorescence techniques. IpaC functional domains will be identified using monoclonal antibodies and polyclonal immune sera which recognize defined epitopes and are characterized with respect to inhibitory or stimulatory activities on Shigella invasiveness. These studies will enhance understanding of S. flexneri invasion and serve to identify important new biochemical targets for prevention and treatment of bacillary dysentery.