SUMMARY 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 O. tsutsugamushi the ability to survive in leukocytes and endothelial cells. The bacterial mechanisms responsible are largely unknown, highlighting a gap in our knowledge of host- pathogen interactions that influence scrub typhus outcome. A family of eukaryotic-like effectors called Anks are key O. tsutsugamushi virulence factors. Most consist of an N-terminal ankyrin repeat (AR) domain that mediates protein-protein interactions with host targets and a C-terminal F-box that recruits the host SCF E3 ubiquitin ligase complex to ubiquitinate the AR-bound proteins. The interacting partners and cellular processes that the Anks modulate are mostly unknown. We discovered that O. tsutsugamushi Ank13 is a nucleomodulin. Gene expression profiles in cells ectopically expressing Ank13 recapitulate many of those observed for O. tsutsugamushi infected host cells, indicating that Ank13 contributes to the pathogen’s ability to modulate cellular processes at the transcriptional level. Both infected and Ank13-expressing cells exhibit down-regulation of genes involved in immune responses and other processes regulated by the Notch signaling pathway. A yeast two- hybrid screen coupled with co-immunoprecipitation identified host MIB1 as an Ank13 binding partner. MIB1 is a positive regulator of canonical Notch signaling. MIB1 levels are reduced in O. tsutsugamushi infected cells, and this is phenocopied in cells ectopically expressing Ank13 or an Ank13 mutant with a functionally inactivated F- box. These data suggest that Ank13 sequestration of MIB1 promotes its auto-ubiquitination and proteasomal degradation during infection. Notch ligand surface presentation on infected cells is altered and Notch-related gene expression is quiescent in these cells, indicating that O. tsutsugamushi impairs Notch signaling. Notably, these same genes are significantly downregulated in cells ectopically expressing Ank13. A preliminary yeast toxicity suppressor screen implicated yeast proteins that have human homologs involved in host transcription regulatory pathways, including ribosome and cell cycle modulation, and non-canonical Notch signaling, as being modulated by Ank13. Thus, Ank13 alters Notch-dependent and -independent transcription to manipulate multiple eukaryotic processes. Aim 1 will interrogate the hypothesis that O. tsutsugamushi Ank13 promotes MIB1 auto- ubiquitination/degradation to impede Notch-stimulated processes. As a complementary approach, Aim 2 will comprehensively define the cohort of host targets and cellular processes that Ank13 modulates during infection. Specifically, we will couple unbiased yeast suppressor screening and affinity proteomics assays to identify host proteins/pathways targeted by Ank13 and will investigate their relevance to O. tsutsugamushi pathogenesis. Overall, this proposal will advance our fundamental understanding of nucleomodulin biology and define novel pathways targeted by O. tsutsugamushi, together providing a powerful impact to the bacterial pathogenesis field.

NARRATIVE Orientia tsutsugamushi is a bacterium that is the causative agent of scrub typhus, a potentially fatal disease that afflicts one million people annually in the endemic Asia-Pacific region but also South America, Europe, and Africa. This proposal will examine the mechanisms by which O. tsutsugamushi modulates host gene expression and immune signaling. This will expand our understanding of pathogen-host manipulation and provide potential targets for treatment of this global disease.