Orientia tsutsugamushi (Ot) is an obligate intracellular bacterial pathogen and the etiologic agent of scrub typhus, a potentially fatal spotted fever-type illness characterized by vascular leakage, myocarditis, and organ failure resulting from endothelial cell infection. MHC-I is a critical component of the immune systems defense against intracellular microbes. Yet, intracellular pathogens evade or counter immune system detection to establish an effective infection. Our laboratory discovered that Ot does so via the unique strategy of reducing MHC-I levels by targeting NLRC5, a recently identified transactivator of MHC-I gene expression, and we have begun to uncover the responsible mechanism. We identified the Ot Type 1 secretion system effector, Ank5, as an NLRC5 interacting partner. Ank5 carries two eukaryotic-like functional domains an ankyrin-repeat region and a C-terminal F-box. Ankyrin repeats mediate protein-protein interactions, while F-box motifs recruit SCF1 polyubiquitin ligases to co-opt the ubiquitin-proteasome system. Ectopically expressed Ank5 reduces NLRC5 cellular levels to phenocopy this Ot infection event and does so in an F-box-dependent manner. I hypothesize that Ank5 uses specific ankyrin repeat residues to interact with NLRC5 while utilizing its F-box to promote polyubiquitination and subsequent proteasomal degradation of NLRC5. To test this hypothesis, I propose two specific aims. Because Ot is genetically intractable, I will use transfected and Ot-infected HeLa cells for all initial experiments and extend my studies to an endothelial cell model. In Aim 1, I will employ amino acid substitution and co-immunoprecipitation to identify specific Ank5 residues that bind NLRC5. I will use nuclear fractionation assays to determine if Ot and Ank5 itself sequesters NLRC5 in the cytosol to block its nuclear translocation. In Aim 2, I will employ protein-protein interaction studies and siRNA knockdown to confirm if host SCF1 polyubiquitin ligase is essential for Ot and Ank5 to reduce NLRC5 cellular levels. Because ubiquitin linkages determine the cellular fate of proteins, I will determine the specific ubiquitin linkages on NLRC5 that Ank5 and Ot direct. Understanding the novel mechanism by which Ot modulates NLRC5 will not only be informative to the bacterial effector field, but will also be enlightening for how other pathogens could be modulating the immune response and influencing cardiovascular disease progression.
|Program type||Predoctoral Fellowship|
|Effective start/end date||01/01/2020 → 12/31/2021|