The candidate is a gastroenterologist with an extremely strong commitment to basic science research, and a specific interest in epithelial cell biology in the context of host-pathogen interactions. The candidate's long- term career goal is to become an independently funded investigator in the basic science of intestinal biology, and to make fundamental contributions to our understanding of the mechanisms that regulate mammalian epithelial barrier integrity during homeostasis and during interaction with intestinal microbes. The candidate's short-term career goals are 1) to become proficient in molecular, cellular and biochemical techniques useful for mechanistically investigating cytoskeletal dynamics both in vitro and in vivo; 2) to acquire expertise in conceptually and technologically cutting-edge approaches to experimental design that can elucidate host- pathogen interactions in novel ways; 3) to develop professional experience in fostering creative and highly interdisciplinary collaborations between experts in immunology, epithelial biology and microbiology; 4) to produce sufficient preliminary data and publications that will serve as the basis for competitive R01 funding as the cornerstone of a career in academic medical research. The overall theme of this project is to understand how factors that regulate the actin cytoskeleton influence the stability and dynamic function of intercellular junctions within the intestinal epithelium. The apical junction complex (the tight junction and adherens junction) is the major regulator of intestinal permeability and must be capable of rapidly fine-tuning its occlusiveness or leakiness. The Neural Wiskott-Aldrich Syndrome protein (N-WASP) is the canonical member of a family of actin nucleating molecules that, upon activation by a variety of host- or pathogen-encoded factors, interact with the Arp2/3 complex and promote the spatiotemporally restricted assembly of actin filaments. This process is central to diverse cellula processes, but there is growing evidence from polarized epithelial cell lines and Drosophila that N-WASP plays a central and specific role in regulating the dynamic protein trafficking events involved in intercellular junction maintenance, and along this line, there has been increasing recognition of pathogens, such as EPEC and C. rodentium, that specifically target N-WASP to modulate the actin cytoskeleton and disrupt intestinal barrier function. The specific goals of this study are to test two fundamental hypotheses: 1) N-WASP is an effector molecule that balances both the delivery and removal of AJC proteins through an interaction with the Par3/Par6/aPKC polarity complex, and 2) AE pathogens require N-WASP to induce epithelial barrier dysfunction, and the translocated effector protein EspF targets host N-WASP to enhance removal of AJC proteins during infection. The proposed work will be pursued within the context of an intensive and formalized career development program, which will allow the candidate to acquire expertise in both classic and leading edge molecular and cell biological approaches to studying intestinal biology and bacterial interactions, and will take advantage of the highly collaborative and complementary co-mentorship of Scott Snapper (immune and epithelial biology) and John Leong (microbiology). In addition, a formal research advisory committee, consisting of a diverse group of researchers at MGH and Harvard Medical School with deep expertise in immunology, gut histopathology, and junction biology, has been established and has overseen the candidate's progress since July 2010. The research environment, which includes the MGH Gastrointestinal Unit and the Harvard Digestive Diseases Center at Children's Hospital Boston, will provide an intellectually enriching, technically resourceful and collaborative atmosphere which will catalyze the candidate's scientific productivity. At the conclusion of the award period, the candidate will be well positioned for a career as an independent, R01-funded investigator.
The intercellular junction is a major determinant of epithelial barrier function. The Neural Wiskott-Aldrich Syndrome protein (N-WASP) is a critical regulator of the actin cytoskeleton, which, in addition to controlling the integrity of intercellular junctions,is a major target of intestinal pathogens that cause diarrhea and mortality worldwide. Understanding the dual role of N-WASP in regulating epithelial barrier function and as a target of attack by enteric pathogens may lead to therapeutic strategies that mitigate conditions of altered intestinal permeability, including inflammation and infection.
