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 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 pathogenic microbes.
The Specific Aims are: (1) to test the hypothesis that IQGAP1 is an element of the mechanism by which pathogens enter cells, we shall examine the effects of bacterial infection on IQGAP1 subcellular localization using immunocytochemistry, confocal microscopy and live-cell imaging. In addition, we shall determine the consequences on bacterial invasion and subsequent cytoskeletal reorganization when intracellular IQGAP1 concentrations and function are altered by overexpression, knockdown by RNA interference and using dominant negative constructs. (2) To test the hypothesis that IQGAP1 participates in phagocytosis, phagocytic bacterial uptake 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 microbial infection, with potential implications for understanding pathogen biology. Elucidation of the mechanisms of pathogen-host interactions could reveal new targets for diagnosis, therapy and vaccines.
