Functional Characterization of Non-Ankyrin Repeat Domains of Orientia tsutsugamushi Ank Effectors Reveals Their Importance for Molecular Pathogenesis

Orientia tsutsugamushi is a genetically intractable obligate intracellular bacterium, causes scrub typhus, and has one of the largest known armamentariums of ankyrin repeat-containing effectors (Anks). Most have a C-terminal F-box presumed to interact with the SCF ubiquitin ligase complex primarily based on their ability to bind overexpressed Skpl. Whether all F-box-containing Anks bind endogenous SCF components and the F-box residues essential for such interactions has gone unexplored. Many O. tsutsugamushi Ank F-boxes occur as part of a PRANC (pox protein repeats of ankyrin-C-terminal) domain. Roles of the non-F-box portion of the PRANC and intervening sequence region (ISR) that links the ankyrin repeat and F-box/PRANC domains are unknown. The functional relevance of these effectors' non-ankyrin repeat domains was investigated. The F-box was necessary for Flag-tagged versions of most F-box-containing Anks to precipitate endogenous Skp1, Cul1, and/or Rbx1, while the ISR and PRANC were dispensable. Ank toxicity in yeast was predominantly F-box dependent. Interrogations of Ank1, AnkS, and Ank6 established that L1, P2, E4, 19, and D17 of the F-box consensus are key for binding native SCF components and for Ank1 and Ank6 to inhibit NF-kappa B. The ISR is also essential for Ank1 and Ank6 to impair NF-kappa B. Ectopically expressed Ank1 and Ank6 lacking the ISR or having a mutagenized F-box incapable of binding SCF components performed as dominant-negative inhibitors to block O. tsutsugamushi NF-kappa B modulation. This study advances knowledge of O. tsutsugamushi Ank functional domains and offers an approach for validating their roles in infection.