Mechanisms of SFG Rickettsia-Host Interactions

Massachusetts Institute Of Technology, Cambridge MA

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Abstract

PROJECT SUMMARY/ABSTRACT Spotted Fever Group (SFG) Rickettsia species are obligate intracellular bacterial pathogens that cause mild-to- life-threatening vascular diseases in humans. To promote widespread disease, SFG Rickettsia species have evolved dynamic strategies to invade host cells, escape into and thrive within the cytosol, and spread from cell to cell. We hypothesize that SFG Rickettsia coordinate their complex life cycle by delivering an arsenal of secreted bacterial proteins (i.e., effectors) into cells to reprogram cellular processes, but the identity and targets of these secreted effectors have remained largely unknown. Furthermore, how these effectors function in the vector are a mystery. To overcome these barriers, we have used a forward genetic screen and cell-selective proteomics to identify and study secreted effectors. We discovered that the effector Sca4 promotes cell-to-cell spread in mammalian cells in contrast to its critical role regulating bacterial growth in tick cells. Each of these activities have been linked to distinct adhesion and endocytic machinery, respectively, highlighting the different contributions effectors play across environments. Then, using a bioorthogonal non-canonical amino acid tagging strategy for the first time in the Rickettsia genus, we also discovered seven new effectors we called Secreted rickettsia factors (Srfs). Most of these Srfs are annotated as hypothetical proteins and localize to distinct subcellular compartments in host cells. This proposal will leverage these key discoveries alongside our multidisciplinary approaches in cell biology, biochemistry, and genetics to examine how secreted effectors promote different aspects of the R. parkeri infectious life cycle. In Aim 1, we will investigate the functions of Sca4 across cellular environments to elucidate how Sca4 specifically targets the subcellular pool of a host cell-cell adhesion factor during R. parkeri cell-to-cell spread (Aim 1.1) and how Sca4 promotes bacterial growth in tick cells (Aim 1.2). In Aim 1.3 we will ask if these functions are also required in in vivo models of infection, highlighting critical functions of Sca4 in mammalian and tick colonization and pathogenicity. Then in Aim 2, we will use high- throughput phenotypic and interactome screens to begin to dissect the functions of the novel Srfs. Collectively, the proposed research will dramatically improve our fundamental understanding of rickettsia biology and rickettsia-host/vector interactions, which is essential to combat the growing threat of tickborne diseases.