BRIGHAM AND WOMEN'S HOSPITAL, INC., THE

Pathogens have developed sophisticated mechanisms to enter cells, evade destruction inside the eukaryotic cell and multiply. The cytoskeleton of the host cell is a common target that is manipulated by bacteria to facilitate infection. Invasive bacteria control the host cell's cytoskeleton to expedite uptake into cells that are normally nonphagocytic and to evade phagocytosis and destruction. The major cytoskeletal elements, particularly actin and microtubules, associate with and are regulated by several proteins. This proposal focuses on IQGAP1, which regulates the cytoskeleton both directly by binding actin and indirectly by interacting with a number of targets, including Cdc42, Rac1 and the microtubule-binding protein CLIP-170. We observed that IQGAP1 has a fundamental role in Cdc42 cytoskeletal function and cell motility. Based on these data we hypothesize that IQGAP1 is an integral component of the cytoskeletal alterations induced by some pathogenic microbes. This proposal focuses on Salmonella. The Specific Aims are: (1) To test the hypothesis that IQGAP1 is an element of the mechanism by which Salmonella enter cells, we shall determine whether IQGAP1 modulates Salmonella invasion by coupling Cdc42/Rac1 to the actin cytoskeleton. Analysis will be performed using mutant and dominant negative IQGAP1 constructs and a specific inhibitor peptide. (2) To test the hypothesis that IQGAP1 participates in phagocytosis and intracellular trafficking of bacteria, uptake and trafficking of Salmonella will be assessed in macrophages in which IQGAP1 function has been manipulated with dominant negative constructs, overexpression and specific knockdown. These studies should indicate whether IQGAP1 participates in Salmonella infection. In addition, the data are likely to enhance our comprehension of Cdc42 and Rac1 in Salmonella pathogenesis, contributing to an understanding of Salmonella biology. Elucidation of the mechanisms of pathogen-host interactions could reveal new targets for diagnosis, therapy and vaccines.

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