UC Irvine

Arthropod-borne pathogens that cause severe disease in humans often cause limited, asymptomatic infections in rodent reservoirs; however, the molecular and cellular barriers that determine these species-specific outcomes remain poorly understood. Furthermore, studying rickettsial pathogenesis and dissemination in real time has historically been hindered by the genetic intractability of these obligate intracellular bacteria. This dissertation addresses these dual challenges by defining the immunological mechanisms that shape host susceptibility to the tick- borne pathogen Rickettsia parkeri and by developing novel genetic tools to track its dissemination in vivo. First, I investigated the immunological basis of host-range restriction between permissive human hosts and resistant rodent reservoirs. By comparing mouse and human macrophages, I demonstrated that inducible nitric oxide synthase (iNOS/Nos2)-driven nitric oxide (NO) production is the dominant interferon-gamma (IFN-γ)-activated effector that restricts R. parkeri in mouse macrophages. In contrast, human macrophages fail to generate the high concentrations of NO required to limit bacterial replication. Through comparative transcriptional profiling and pharmacological manipulation of NO synthesis, I identified a distinct quantitative NO "threshold" required for restriction. This threshold is readily achieved by mouse cells but not by human cells, explaining species-specific differences in infection permissiveness. Translating these findings in vivo, I established that Nos2-deficient mice infected with R. parkeri develop localized eschars, the hallmark clinical features of human cutaneous rickettsiosis. This mouse model does not develop lethal systemic disease and provides an improved, trackable, physiologically relevant model for human infection. Next, to overcome the technical limitations of tracking Rickettsia dissemination in vivo, I generated and characterized the first bioluminescent strains of R. parkeri. While strains expressing Firefly and Aka luciferase exhibited reduced virulence, the engineered NanoLuc-expressing strain maintained wild-type virulence and enabled real-time, high-resolution tracking of bacterial burdens. Using in vivo bioluminescence imaging, I mapped R. parkeri dissemination from the initial skin site to distal appendages and unexpected deep-tissue organs, including the pancreas, kidney, gastrointestinal tract, and gallbladder. Utilizing R. parkeri expressing the superbright fluorophore AausFP1, I further characterized the cellular niches of infection, revealing that while the bacteria associate with diverse immune cells in sera, a significant proportion resides in an unexpected cell-free state in the systemic infection mouse model. Ultimately, this dissertation provides key evolutionary insights into how interspecies differences in IFN-γ signaling shape pathogen host range and human disease susceptibility. Concurrently, the genetic, imaging, and animal models developed herein establish a powerful, versatile toolkit to dissect the molecular determinants of rickettsial pathogenesis, paving the way for targeted therapeutic interventions against severe spotted fever group rickettsioses.