Dissecting the Developmental Life Cycle and Developing Genetic Tools for Understanding the Biology of <i>Orientia tsutsugamushi</i>

Orientia tsutsugamushi is an obligate intracellular bacterium that is spread by mites and causes a life-threatening human disease, scrub typhus. This disease affects at least one million people annually with a high risk of mortality if not treated promptly. Orientia is poorly understood compared to many other pathogens due to genetic intractability. This bacterium only propagates and replicates within host cells. they are predominantly found in endothelial, dendritic, and monocyte/macrophage cells, and stay inside infected cells for seven days or longer before exiting by a budding mechanism. The developmental differentiation of the intracellular infection cycle of Orientia has not been studied previously. My thesis research initially focused on how Orientia differentiates into distinct subpopulations during the infection cycle, how distinct subpopulations of Orientia affect the infection in host cells, and different protein profiles of bacteria in distinct stages during the infection cycle. My research demonstrates that O. tsutsugamushi differentiates into five distinct subpopulations: early entry, pre-replicative, replicative, maturation, and extracellular, representing a new model for developmental differentiation in the intracellular cycle of Orientia tsutsugamushi. Each subpopulation relates to different degrees of metabolic activity, replication, infectivity, including morphology, presence of marker gene, and subcellular localization. The transition between the subpopulations likely results from an integration of signals from the host cell environment and the bacteria which leads to morphological and physiological changes through the regulation of genes and proteins. This work allows us to understand the fundamentals of bacterial development and the regulation mechanism of differentiation of Orientia tsutsugamushi, which could be beneficial for the improvement of diagnosis and treatment. Genetic tools have not been developed previously for Orientia, but genetic manipulation in the most closely related bacteria, rickettsial species, has been successfully established in the last decade. My work on genetic manipulation of Orientia is described in the second part of this thesis. The transposon mutagenesis vector and nucleotide analog (BNA) were used to manipulate the genome of Orientia. Unfortunately, the transposon mutagenesis system failed to integrate into the Orientia genome, whereas BNA was able to down regulate the expression of the targeted protein (TSA56). Using a nucleotide analog to target a specific gene is the first step to manipulate the genome of Orientia, and this has led to the study of other targeted genes involving virulence and pathogenicity. Due to the genetic intractability of Orientia that limits the molecular dissection of bacteria pathogenesis, virulence, and host-pathogen interaction, sequencing technologies provide a promising way to understand the molecular processes of the disease. This motivated the third part of this thesis, in which dual RNA-seq was applied to two clinical isolates grown in cultured HUVEC cells. This study revealed the transcriptomic profile of Orientia and identified the different immune response networks in response to each strain. Differential activation of the immune response in cultured cells between the two strains was shown to correlate with differences in virulence as measured in a mouse model of infection. These findings will help to understand the mechanism of bacterial pathogenesis in Orientia tsutsugamushi and may be used for characterization of other genetically intractable bacterial pathogens