Shigella are intracellular enteric human pathogens that move to the cell periphery via actin-based motility, whereupon they generate protrusions of the plasma membrane that penetrate into and are taken up by adjacent uninfected cells. The generation of plasma membrane protrusions is critical for Shigella spread, but the mechanisms involved are poorly understood. We have shown that cellular diaphanous formins and IRTKS are each required for both efficient formation of plasma membrane protrusions and efficient spread of Shigella through cell monolayers. Both diaphanous formins and the IRTKS homolog IRSp53 are components of the cellular fiolopodia tip complex and both are required for efficient formation of filopodia. IRTKS and IRSp53 are I-BAR domain proteins that link the plasma membrane with the cytoskeleton while inducing concave deformations of the membrane. Based on these results, we hypothesize that Shigella generation of plasma membrane protrusions utilizes the cellular filopodia formation machinery. In this application, we propose a set of exploratory investigations to test our hypothesis. <p/>Our specific aims are as follows: Aim 1. Analysis of IRTKS functions involved in Shigella protrusion formation.<p/> Aim 2. Test the role of Tyr phosphorylation of IRTKS in S. flexneri protrusion formation. <p/>Aim 3. Determine whether other interactors of I-BAR domain proteins are required for efficient generation of plasma membrane protrusions by S. flexneri. <p/>The investigations proposed in this application are highly likely to generate new insights into themechanisms of Shigella spread through the intestinal epithelium, as well as into the fundamental processes involved in cellular membrane protrusion, particularly filopodia formation.
PUBLIC HEALTH RELEVANCE: The human pathogen Shigella is a bacterium that causes diarrhea by infecting cells that line the human intestinal tract. Critical to the disease process i the ability of Shigella to spread from one cell into adjacent cells by forming protrusions of the cell membrane. Cells that are not infected also have the capacity to form these types of protrusions. We propose detailed investigations into the molecular mechanisms involved in the generation of these protrusions. Our results are likely to lead to an improved understanding of how pathogens interact with human tissue and an improved understanding of fundamental cellular processes.