Structure and Function of IpaC from Shigella flexneri

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DESCRIPTION (provided by applicant): Shigella flexneri is a gram-negative enteric pathogen that causes bacillary dysentery (shigellosis), which continues to be an important worldwide public health problem. An essential step in the pathogenesis of shigellosis is bacterial invasion of the epithelial cells of the colon. Invasion plasmid antigen C (IpaC) is the effector protein that subverts normal epithelial cell signaling to promote Shigella uptake. Our lab has contributed a number of advances in understanding the structure-function relationship of IpaC; however, there has been little focus thus far on determining the biochemistry of IpaC-mediated actin polymerization during epithelial cell invasion. This is partly due to difficulties in purifying and handling this protein. The long-range goal of our laboratory is to determine the precise functional and structural organization of IpaC and to elucidate the molecular mechanism of and the structural basis for IpaC interaction with and subversion of the host cell cytoskeleton. The specific aims of this project are: 1) to determine the outcome of IpaC's association with the host GTPase Cdc42; 2) to determine the IpaC features responsible for direct nucleation of host cell actin which may contribute to efficient Shigella entry into epithelial cells; and 3) to identify the sequences and structures located at the IpaC C-terminus that are responsible for its effector activity and its ability to promote vacuolar escape. A common theme in the pathogenesis of many gram-negative bacteria is the specific delivery of effector proteins to eukaryotic cells to subvert the normal target cell signaling mechanisms. For S. flexneri, the outcome of this cross talk is host cytoskeletal rearrangement and subsequent bacterial entry. IpaC carries out this interspecies cellular communication by a mechanism that is quite unique relative to those identified in other gram-negative bacterial pathogens. Completion of this work will reveal important new principles of the mechanism by which bacteria trigger epithelial cells to become "phagocytic" for the benefit of the pathogen. This will help in identifying targets for chemotherapeutic control of infection by Shigella and related human pathogens. Moreover, novel bacterial proteins capable of modulating eukaryotic pathways are often innovative and valuable tools for future work in cell biology. The novel nature of IpaC's signaling mechanisms provide promise that it, too, represents a new and useful cell biology tool.