Functional characterization of an Orientia tsutsugamushi nucleomodulin

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Scrub typhus is a neglected potentially fatal zoonosis that afflicts 1 million persons each year in the Asia Pacific. Recent case reports outside this region signify scrub typhus as a global health threat. The causative agent is Orientia tsutsugamushi (Ot), a mite-transmitted obligate intracellular bacterium that invades leukocytes and endothelial cells. Ot dysregulates host cell gene expression to its advantage, but the responsible bacterial factors are unknown. Ankyrin repeat-containing proteins (Anks) are intracellular microbial effectors that bind specific host proteins to co-opt or subvert eukaryotic functions. Ot carries one of the largest Ank repertories of any microbe. Most Ot Anks carry a second eukaryotic-like motif called the F-box, which interacts with the SCF1 E3 ubiquitin ligase complex that polyubiquitinates proteins. The roles of most Ot Anks and the functional relevance of the F-box to Ot pathobiology are unknown. We discovered that Ot Ank13 robusty traffics into the host cell nucleus. This phenomenon does not involve the canonical importin-driven nuclear import pathway. Rather, it requires isoleucines at the 13th position of its fourth and fifth ankyrin repeats (ARs). This observation suggests that Ank13 uses the RanGDP-dependent RaDAR nuclear import pathway, which, conspicuously, is used by eukaryotic AR-containing transcriptional regulators. Yeast two-hybrid analysis identified the following host proteins as putative Ank13 interacting partners: SCF1 complex members SKP1 and CUL1, and HMG20A, a transcription factor involved in chromosome remodeling that is negatively regulated by polyubiquitination. Thus far, the Ank13-SKP1 and Ank13-CUL1 interactions have been verified by co-immunoprecipitation. Our data support the premise that Ank13 is a nucleomodulin that contributes to the ability of Ot to modulate host cell gene expression. In two specific aims, we will evaluate the hypotheses that (1) Ank13 uses RaDAR to enter the nucleus and (2) Ank13 interacts with and polyubiquitinates HMG20A or other proteins to disrupt host cell gene expression in an F-box-dependent manner to benefit Ot infection. Despite the global biomedical significance of scrub typhus, the host-pathogen interactions that drive disease progression are poorly understood. Nucleomodulin biology is a bourgeoning area of bacterial pathogenesis that is ripe for further exploration. Accomplishing the proposed aims will have a broad and powerful impact on these fields.