Orientia tsutsugamushi Ank-host interactions in scrub typhus pathogenesis

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Scrub typhus is an emerging and potentially fatal global health threat. Approximately one million new cases are reported annually. The etiologic agent is Orientia tsutsugamushi, an obligate intracellular bacterium that infects leukocytes and endothelial cells resulting in vascular collapse, organ failure, and death. Treatment options are limited and no preventative vaccine exists. The success of O. tsutsugamushi as a pathogen lies in its ability to modulate host immunity and other pathways. The responsible mechanisms are unknown, highlighting the need for a better understanding of scrub typhus host-pathogen interactions. The ankyrin repeat (AR) is a protein- protein interaction motif that is prevalent throughout nature. O. tsutsugamushi has one of the largest arsenals of AR-containing effectors (Anks) among bacteria and expresses all of them during infection, underscoring their importance for intracellular survival and virulence. Most Orientia Anks carry a C-terminal F-box motif that co-opts host ubiquitin ligases. We discovered that O. tsutsugamushi Ank1 and Ank6 impede the NF-κB pathway in an AR- and F-box-dependent manner. Both bind and prevent the degradation of host NF-κB inhibitor, p105. Ank1 and Ank6 ARs mimic those of EPRAP, a host protein that stabilizes p105, and ubiquitinate Crybg3, a host kinase that influences p105 stability. Further screening revealed that a total of 13 Anks antagonize NF-κB, some of which bind p105 and others do not. Thus, multiple Anks inhibit NF-κB by distinct, overlapping mechanisms. We found that O. tsutsugamushi lowers MHC-I levels by orchestrating proteasomal degradation of NLRC5, a transactivator of MHC-I gene expression, and linked this phenomenon to Ank5. How Ank1, Ank5, and Ank6 inhibit innate and adaptive immunity is poorly characterized. We established that Orientia Anks alter the host cell ubiquitome, but the extent of this strategy, identity of modified targets, and infection outcomes are unexplored. Finally, other Anks target unknown eukaryotic pathways that also likely influence O. tsutsugamushi pathobiology. To fill these knowledge gaps, we will decipher the mechanisms by which Anks inhibit NF-κB and use two innovative screens that circumvent O. tsutsugamushi genetic intractability as part of our approach (Aim 1); dissect how Ank5 promotes NLRC5 degradation to block MHC-I expression (Aim 2); and identify new host cell pathways and ubiquitome changes that Anks modulate (Aim 3). The contribution of each newly discovered host- Ank interaction to O. tsutsugamushi pathogenesis will be interrogated. Overall, we will advance fundamental understanding of O. tsutsugamushi-host interactions, define novel mechanisms by which intracellular pathogens modulate immunity, identify new scrub typhus therapeutic targets, and benefit the bourgeoning concept of designed AR proteins as biomedicals to have a broad and powerful impact.