Multifunctional roles of an Orientia tsutsugamushi nucleomodulin

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.

Grant Number: 5F32AI174656-02
NIH Institute/Center: NIH
Principal Investigator: Paige Allen