DOI
https://doi.org/10.25772/KYB3-WF54
Author ORCID Identifier
0000-0002-0783-2948
Defense Date
2022
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Microbiology & Immunology
First Advisor
Dr. Jason A. Carlyon
Abstract
Orientia tsutsugamushi is the etiologic agent of scrub typhus, the deadliest of all diseases caused by obligate intracellular bacteria with emerging global implications. Despite such an imposing threat, the mechanistic understanding behind O. tsutsugamushi pathobiology and its modulation of the immune response is still in its infancy. O. tsutsugamushi possesses an arsenal of (type 1 secretion system) T1SS ankyrin repeat-containing proteins (Anks). Here, we explore the utilization of two discrete Anks and their unique approaches to immune system modulation. We define one Ank as the effector responsible for infection-observed degradation of NLRC5, the transactivator for MHC-I genes. Yeast two-hybrid assays identified NLRC5 as a binding partner for a singular Ank. This interaction was confirmed using co-immunoprecipitation and found to be dependent on the fourth ankyrin repeat. Infection induced NLRC5 degradation is phenocopied by this Ank in a manner that is proteasome-, F-box- and SCF1 complex-dependent implicating xix post-translational marking of NLRC5 by ubiquitin as the Ank-mediated mechanism. NLRC5 isoform 3, lacking the expansive C-terminal leucine rich repeat (LRR) domain, is not reduced by this Ank, suggesting that the LRR domain is necessary for targeted ubiquitination. Importantly, we demonstrate that a non-functional F-box mutant of this discrete Ank acts as a dominant negative by binding endogenous NLRC5 and having antagonistic effects towards endogenous mechanisms. These data confirm that this Ank is the secreted effector responsible for binding and reducing NLRC5 levels during an O. tsutsugamushi infection. We also demonstrate that Ank13, an O. tsutsugamushi effector conserved among clinical isolates and expressed during infection, localizes to the nucleus in an importin b1-independent manner. Rather, Ank13 nucleotropism requires an isoleucine at the thirteenth position of its fourth ankyrin repeat, consistent with utilization of eukaryotic RaDAR (RanGDP-ankyrin repeats) nuclear import. RNA-seq analyses of cells expressing green fluorescent protein (GFP)-tagged Ank13, nucleotropism-deficient Ank13I127R, or Ank13ΔF-box, which lacks the F-box domain essential for interacting with SCF1 ubiquitin ligase, revealed Ank13 to be a nucleomodulin that predominantly downregulates transcription of more than 2,000 genes. The ability to do so involves its nucleotropism and F-box in synergistic and mutually exclusive manners. Ank13 also acts in the cytoplasm to dysregulate smaller cohorts of genes. The effector’s toxicity in yeast heavily depends on its F-box and less so on its nucleotropism. Genes negatively regulated by Ank13 include those involved in the inflammatory response, transcriptional control, and epigenetics. Importantly, the majority of genes xx that GFP-Ank13 most strongly downregulates are quiescent or repressed in O. tsutsugamushi-infected cells when Ank13 expression is strongest. Ank13 is the first nucleomodulin identified to co-opt RaDAR and a multi-faceted effector that functions in the nucleus and cytoplasm via F-box-dependent and -independent mechanisms to globally reprogram host cell transcription. Studies of these two Ank effectors expose only a fraction of the propensity that O. tsutsugamushi holds to modulate host cell pathways, evade immune system responses, and cause disease.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
2-12-2022