The role of synaptopodin during bacterial infection

University Of Colorado Denver, Aurora CO

Investigators

Abstract

PROJECT SUMMARY: Shigella flexneri is a gram-negative bacterial pathogen that causes diarrhea in humans. Essential for the disease process is the ability of the bacterium to invade epithelial cells lining the colon and to spread between the cells of the epithelium. In order to spread, S. flexneri requires actin-based motility to move to the cell periphery, it deforms the plasma membrane into a protrusion enabling it to push into neighboring cells, where the protrusion is actively engulfed and resolves into a double membrane vacuole that the bacterium escapes. Although the biological steps required for spread are well-established, the molecular mechanisms that enable them to occur are poorly understood. Our preliminary data establish that the short-chain fatty-acid butyrate promotes S. flexneri spread in a manner that is dependent on the actin-binding protein synaptopodin. Synaptopodin enhances the intercellular spread of S. flexneri and Listeria monocytogenes, indicating it may be generally required among bacterial pathogens that spread between cells. We show synaptopodin localizes to S. flexneri as they initiate protrusions, but it is largely excluded from the protrusion structure as it elongates. Synaptopodin recruitment to S. flexneri is dependent on the bacterial proteins IcsA and Spa15. These data highlight that synaptopodin appearance around bacteria is not solely attributable to its actin binding properties and show its spatial location around bacteria is regulated during protrusion formation. Yet, the manner of synaptopodin regulation and the requirement of synaptopodin during protrusion formation is unclear. We anticipate that synaptopodin forms a protein complex that promotes protrusion initiation, and to our knowledge, this would be the first such complex described for intracellular pathogens that spread. We hypothesize that S. flexneri infection alters synaptopodin function in a manner that enables the generation of forces to deform the plasma membrane and initiate protrusion formation. We aim to test this model with the following aims. AIM 1: To define the functional requirement of synaptopodin during S. flexneri infection. AIM 2: To determine how S. flexneri infection alters synaptopodin function. Several important human pathogens require intercellular spread to cause disease, the proposed approaches are likely to define a new mechanistic step that enables these pathogens to cause disease in humans by defining components of a synaptopodin-dependent protrusion initiation complex, its mechanisms of regulation, and its functional contribution to protrusion formation. We anticipate these studies may also resolve long-standing gaps in our understanding as to how pathogens that do not polymerize actin in the protrusion can generate force at the membrane to form protrusion structures. Moreover, synaptopodin is a poorly characterized protein that has not been described to have a role in infection; the investigations proposed here will provide fundamental mechanistic insights into the role of synaptopodin in cells and its requirement for bacterial pathogenesis.