Defining the pathobiological roles of Orientia tsutsugamushi Ank proteins

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Orientia tsutsugamushi is an obligate intracellular bacterium and causative agent of scrub typhus, a severe illness with a high fatality rate that results in approximately 1 million new cases each year. Scrub typhus has long been considered endemic to the Asia-Pacific region, but non-travel related cases and detection of the pathogen on multiple continents signify the disease as an emerging global health threat. The ankyrin repeat is one of the most-common protein-protein interaction motifs in nature. O. tsutsugamushi subverts innate and adaptive immunity using its cadre of ankyrin repeat-containing effectors (Anks), many of which also carry a PRANC/F- box motif that co-opts SCF ubiquitin ligases. Ank1 and Ank6 inhibit NF-κB accumulation in the nucleus to impair NF-κB-dependent gene expression in an ankyrin repeat- and PRANC/F-box-dependent manner. Both effectors bind the host cell NF-κB inhibitor, p105, and prevent its TNFα-induced degradation. Conspicuously, Ank1 and Ank6 ubiquitinate Crybg3, a host kinase that influences p105 stability. Further screening revealed that 13 Anks antagonize NF-κB, some of which bind p105 and others of which do not. Thus, multiple O. tsutsugamushi Anks inhibit NF-κB by distinct, overlapping mechanisms. Ank5 promotes ubiquitination and proteasomal degradation of NLRC5, a transactivator of MHC-I gene expression, to potently reduce MHC-I levels. Our findings indicate that Ank immunomodulation enables O. tsutsugamushi to win, at least initially, its tug-of-war with host cells to establish infection. Yet, the detailed mechanisms by which Anks modulate NF-κB, NLRC5, and other targets remain poorly understood. Indeed, while our data establish that O. tsutsugamushi uses Anks to alter the host cell ubiquitome, the extent of this strategy, identity of modified targets, and specific functional outcomes are poorly defined. Due to the bacterium’s genetic intractability, Ank-mediated virulence in vivo has not been pursued. Finally, several additional Anks target unknown host pathways that also likely influence O. tsutsugamushi pathobiology. To fill these knowledge gaps, we propose to (1) decipher the mechanisms by which O. tsutsugamushi Anks inhibit NF-κB and will employ a novel in vivo screen as part of this approach; (2) dissect how Ank5 promotes NLRC5 degradation to block MHC-I expression; and (3) identify new host cell pathways and ubiquitome changes that Anks modulate. The culmination of our studies will define novel and previously unsurmised mechanisms by which intracellular pathogens modulate host immunity. Overall, this work will have a broad and powerful impact.