Orientia tsutsugamsuhi promotes interplay between circadian rhythm and hypoxic responses - SUMMARY Converting the host cell into a supportive niche is essential for intracellular pathogen survival and replication. Orientia tsutsugamushi is a genetically intractable obligate intracellular bacterium that causes scrub typhus, a globally emerging infection with a high fatality rate. Disease progression depends on bacterial-driven modulation of host antimicrobial responses that affords Orientia the ability to replicate to high loads in endothelial cells. The bacterial mechanisms responsible are largely unknown, highlighting a gap in our knowledge of host-pathogen interactions that influence scrub typhus outcome. Circadian rhythm and the hypoxic response are universal and essential pathways that influence innate immunity (e.g., ROS, DNA damage responses, and apoptosis) through gene expression regulation. We discovered that O. tsutsugamushi promotes the hypoxic response. In infected cells, HIF2⍺, a transcription factor that stimulates hypoxic response-regulated gene expression, is stabilized and nuclear. Moreover, the circadian rhythm-related transcription factor pair, BMAL1-CLOCK, is also nuclear and stimulates robust expression of circadian rhythm genes. Remarkably, Orientia uniquely promotes HIF2⍺ interaction with BMAL1-CLOCK indicating that it encourages interplay between circadian rhythm and the hypoxic response likely to combat host innate immune defenses. We surmised that O. tsutsugamushi executes this strategy, at least in part, using its effector, Ank13, which is a nucleomodulin that pronouncedly alters host cell gene expression. The abilities of Ank13 to modulate host gene expression, prevent host cell genotoxicity, and DNA damage-dependent apoptosis are nucleotropism-dependent. The Ank13 mechanisms of action are undetermined. Conspicuously, the circadian rhythm genes, PER1 and CRY1, are upregulated and BMAL1 expression is decreased in uninfected cells ectopically expressing Ank13 indicating that circadian rhythm-related transcription is active and that Ank13 alone recapitulates these Orientia infection associated phenomena. Yeast two-hybrid screening and co-immunoprecipitation both identified host HIF2⍺ and FIH (negative regulator of HIF2⍺) as Ank13 binding partners. Together, these data establish the model where O. tsutsugamushi uses Ank13 to orchestrate the interplay between circadian rhythm and hypoxic responses as a promicrobial strategy. Aim 1 will test the hypothesis that O. tsutsugamushi promotes interaction between the hypoxic response and circadian rhythm to counter innate immune defenses. Aim 2 will validate the ability of Ank13 to bind FIH and, in turn, stabilize HIF2⍺ allowing it to regulate BMAL1-CLOCK activity to manipulate host cell transcription and combat ROS production, DNA damage, and apoptosis. Achieving these aims will have a powerful impact by advancing fundamental understanding of O. tsutsugamushi pathobiology, dissecting a new microbial strategy for modulating host immunity, and revealing host-directed therapeutic targets for treating scrub typhus.
