Orientia tsutsugamushi Ank-host interactions in scrub typhus pathogenesis

Scrub typhus is an emerging and potentially fatal global health threat. Approximately one million new cases are
reported annually. The etiologic agent is Orientia tsutsugamushi, an obligate intracellular bacterium that infects
leukocytes and endothelial cells resulting in vascular collapse, organ failure, and death. Treatment options are
limited and no preventative vaccine exists. The success of O. tsutsugamushi as a pathogen lies in its ability to
modulate host immunity and other pathways. The responsible mechanisms are unknown, highlighting the need
for a better understanding of scrub typhus host-pathogen interactions. The ankyrin repeat (AR) is a protein-
protein interaction motif that is prevalent throughout nature. O. tsutsugamushi has one of the largest arsenals of
AR-containing effectors (Anks) among bacteria and expresses all of them during infection, underscoring their
importance for intracellular survival and virulence. Most Orientia Anks carry a C-terminal F-box motif that co-opts
host ubiquitin ligases. We discovered that O. tsutsugamushi Ank1 and Ank6 impede the NF-κB pathway in an
AR- and F-box-dependent manner. Both bind and prevent the degradation of host NF-κB inhibitor, p105. Ank1
and Ank6 ARs mimic those of EPRAP, a host protein that stabilizes p105, and ubiquitinate Crybg3, a host kinase
that influences p105 stability. Further screening revealed that a total of 13 Anks antagonize NF-κB, some of
which bind p105 and others do not. Thus, multiple Anks inhibit NF-κB by distinct, overlapping mechanisms. We
found that O. tsutsugamushi lowers MHC-I levels by orchestrating proteasomal degradation of NLRC5, a
transactivator of MHC-I gene expression, and linked this phenomenon to Ank5. How Ank1, Ank5, and Ank6
inhibit innate and adaptive immunity is poorly characterized. We established that Orientia Anks alter the host cell
ubiquitome, but the extent of this strategy, identity of modified targets, and infection outcomes are unexplored.
Finally, other Anks target unknown eukaryotic pathways that also likely influence O. tsutsugamushi pathobiology.
To fill these knowledge gaps, we will decipher the mechanisms by which Anks inhibit NF-κB and use two
innovative screens that circumvent O. tsutsugamushi genetic intractability as part of our approach (Aim 1);
dissect how Ank5 promotes NLRC5 degradation to block MHC-I expression (Aim 2); and identify new host cell
pathways and ubiquitome changes that Anks modulate (Aim 3). The contribution of each newly discovered host-
Ank interaction to O. tsutsugamushi pathogenesis will be interrogated. Overall, we will advance fundamental
understanding of O. tsutsugamushi-host interactions, define novel mechanisms by which intracellular pathogens
modulate immunity, identify new scrub typhus therapeutic targets, and benefit the bourgeoning concept of
designed AR proteins as biomedicals to have a broad and powerful impact.

Grant Number: 5R01AI167857-05
NIH Institute/Center: NIH
Principal Investigator: Jason Carlyon