The role of a Coxiella burnetii effector protein that inhibits RIG-I signaling

Project Summary/Abstract Coxiella burnetii is an obligate intracellular bacterial pathogen that grows within a lysosome-derived vacuole. The formation of a replication-permissive vacuole by C. burnetii requires a type IVB secretion system called Dot/Icm, and the >130 effector proteins delivered by this system into the host cell. While it is established that the collective activities of these effector proteins remodel host signaling networks to create a replication- permissive environment, the molecular targets and biochemical mechanisms of most of these effectors are not known. My studies have established that C. burnetii inhibits the activation of the host RIG-I pathway, a cytosolic double-stranded RNA sensing pathway, and that this process requires two effectors, EmcA and EmcB. Although these proteins do not have predicted sequence or structural homology that would indicate possible biochemical functions, we established that EmcB has cysteine deubiquitinase activity. EmcB directly targets RIG-I and preferentially cleaves long, K63-linked ubiquitin chains that are potent activators of RIG-I signaling. The goal of this study is to determine how EmcB functions to cleave ubiquitin chains from RIG-I during infection and identify how C. burnetii infection is sensed by RIG-I. To achieve this goal, I will use biochemical assays, pull-down experiments, and infection studies using a newly engineered emcA, emcB double mutant to determine how EmcB functions to inhibit the RIG-I pathway during infection (Aim 1). Next, I will identify the specific RNAs sensed by RIG-I during C. burnetii infection and test the relative capacity of these RNAs to activate the RIG-I pathway (Aim 2). Together, these studies will enhance our understanding of how intracellular bacterial pathogens modulate host signaling networks for productive infection. By understanding the molecular details of how an intracellular pathogen modulates host processes, fundamental details of C. burnetii infection will be uncovered which could provide insights that lead to improved treatments. These studies will be complemented by a rigorous and comprehensive program of professional training, clinical training, and scientific skill building to provide preparations necessary to pursue a career as a physician- scientist investigating molecular mechanisms of microbial pathogenesis.