Scrub typhus is a neglected tropical disease that infects at least one million people annually, including U.S. military personnel serving in the endemic region. Untreated scrub typhus carries a mortality rate of up to 70%. The etiologic agent is Orientia tsutsugamushi (Ot), an obligate intracellular bacterium that invades leukocytes and endothelial cells. How Ot modulates host cell functions to gain necessary nutrients and dampen immune surveillance mechanisms such that it can survive and cause disease is poorly understood. The endoplasmic reticulum (ER) maintains cellular homeostasis by controlling the processing and folding of proteins. Accumulation of misfolded proteins causes physiologic stress on the ER that induces the unfolded protein response (UPR), which relieves ER stress, in part, by facilitating ER-associated degradation (ERAD). In ERAD, misfolded proteins are translocated to the 26S proteasome where they are degraded to yield free amino acids and peptides. We discovered that Ot induces the UPR. This is a promicrobial strategy, as Ot growth is pronouncedly increased in cells pharmacologically induced to undergo ER stress but is blocked in cells in which ER stress is inhibited. We determined that Ot expresses the effector, Ank5, during infection when ER stress is elicited. When ectopically expressed, Ank5 traffics to the ER, induces the UPR, and blocks cellular protein secretion. Ank5 carries an F-box, a eukaryotic-like motif that promotes proteasomal destruction of target proteins. NLRC5, a transactivator of MHC I gene expression, was identified as a potential Ank5 interacting partner. Notably, Ot infection and ectopic expression of Ank5 in host cells reduce levels of NLRC5 and MHC I.
In Aim 1, we will determine how Ot benefits from ER stress. Specifically, given that Ot is auxotrophic for amino acids, we will evaluate the hypothesis that ER stress-induced ERAD and proteasomal degradation provide the pathogen with a nutritional pool of free amino acids. Given the ability of Ank5 to invoke ER stress, we will determine if Ank5 promotes Ot growth.
In Aim 2, we will dissect how Ank5 and Ot infection modulate MHC I levels. We will confirm if Ank5 interacts with NLRC5 and if NLRC5 is degraded in an F- box/proteasome-dependent manner in Ot infected and Ank5 expressing cells. We will determine if MHC I expression and/or surface presentation is reduced in Ot primary dendritic cells, macrophages, and endothelial cells. The culmination of these studies will greatly advance knowledge of how and why intracellular bacterial pathogens modulate ER stress. Given that ER stress and the UPR contribute to the development of cancer, diabetes, and inflammatory diseases, dissecting the interplay between intracellular bacteria, the ER, and the UPR is a promising area of microbial pathogenesis research that could have an impact that extends far beyond infectious disease. A bacterial effector that targets NLRC5 has not been demonstrated or hypothesized previously. Validating our hypothesis that Ank5 modulates NLRC5 cellular levels would amount to a paradigm shift in the understanding of how intracellular microbes manipulate immune surveillance mechanisms.
Orientia tsutsugamushi is an understudied bacterium that causes the potentially deadly infection, scrub typhus, which afflicts one million people in the Asia-Pacific annually. This project will determine how O. tsutsugamushi modulates a specific cellular process called endoplasmic reticulum stress to facilitate its survival and potentially disrupt the immune response. Doing so will expand understanding of how Orientia manipulates host cell functions to cause disease and may identify novel targets for treating scrub typhus.
| Evans, Sean M; Adcox, Haley E; VieBrock, Lauren et al. (2018) Outer Membrane Protein A Conservation among Orientia tsutsugamushi Isolates Suggests Its Potential as a Protective Antigen and Diagnostic Target. Trop Med Infect Dis 3: |
| Rodino, Kyle G; VieBrock, Lauren; Evans, Sean M et al. (2017) Orientia tsutsugamushi modulates endoplasmic reticulum-associated degradation to benefit its growth. Infect Immun : |
