Deciphering innate and adaptive immune responses to Rickettsia parkeri

PROJECT SUMMARY/ABSTRACT Tick-borne Rickettsia species cause serious human disease in the United States and worldwide, including spotted fever and typhus, with no approved vaccines and an increasing incidence of infection. R. rickettsii is the causative agent of Rocky Mountain spotted fever, and the closely related R. parkeri causes spotted fever in North and South America. However, due to their obligate intracellular nature and technical challenges, the interactions between Rickettsia and innate and adaptive immunity remain largely unclear. Elucidating how Rickettsia evade innate immunity and stimulate an adaptive response is critical for developing effective vaccines and therapeutics to better understand, prevent, and treat human disease. In preliminary work, we performed a forward genetic screen to identify for the first time the rickettsial factors required for survival in primary mouse macrophages. Two of the most attenuated mutants contained insertions in the genes rmlD and wecA, which are required for biosynthesis of O-antigen, the external component of lipopolysaccharide. We found that O-antigen is required to avoid targeting by ubiquitin and inflammasomes. However, the underlying mechanisms by which O-antigen enables R. parkeri to evade these innate immune defense systems remain unclear. Moreover, O-antigen and outer membrane proteins (OMPs) are both implicated in the adaptive response, yet it is unclear how O-antigen and OMPs protect against T cells and antibodies. Thus, key outstanding questions include: which Rickettsia factors enable survival in immune cells? What are the molecular mechanisms by which O-antigen enables R. parkeri to avoid ubiquitin and inflammasomes? And, what are the relative contributions of O-antigen and OMPs to the adaptive response? We will answer these questions by comprehensively identifying the R. parkeri virulence factors required for survival in macrophages by performing an innovative forward genetic screen. We will reveal the underlying mechanisms by which Rickettsia virulence factors including O-antigen protects from ubiquitin by identifying the E3 ligase(s) that target R. parkeri. We will use electron microscopy to determine if O-antigen composes the capsule, which may shield against E3 ligases, and will measure the degree to which O-antigen protects from autophagy and inflammasomes. In vivo, we will determine how O-antigen protects against innate immunity during acute infection and the relative contributions of O-antigen and OMPs to protection against antibodies and T cells. We will innovate by using unbiased genome-wide protein microarrays to identify novel rickettsial antigens targeted by antibodies. Such antigens may serve as novel vaccine candidates. Together, these studies will leverage our expertise in bacterial and host genetics, cell biology, and in vivo infection models to discover mechanisms by which an obligate intracellular pathogen evades innate immunity, with clinical importance of revealing mechanisms of immunity and identifying novel antigens for vaccines.