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

Abstract

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 Skp1.

Orientia tsutsugamushiAnk5 directs ubiquitination and proteasomal degradation of NLRC5 to inhibit major histocompatibility complex class I expression

“…Ank5-mediated NLRC5 degradation translates to decreased HLA-A and b2M transcription, which reduces overall and cell surface MHC class I levels. The ability of Ank5 to direct NLRC5 ubiquitination and proteasomal degradation requires its F-box and host Skp1, findings that are consistent with the observation that Ank5 interactions with Skp1 and two other SCF complex proteins, Cul1 and RING box 1, are F-box-dependent 55. Overexpression of dominant negative Ank5-F-boxAAAAA that can bind but not degrade NLRC5 protects it from Orientia-induced degradation by outcompeting bacterial-derived Ank5.…”

“…O. tsutsugamushi Ikeda Ank5 is a 345-amino acid effector protein of unknown function that has an N-terminal domain consisting of four tandemly arranged ARs and a C-terminal F-box that occurs within a PRANC ( p ox proteins r epeats of an kyrin- C terminal) domain (Extended Data Figure 3A), the latter of which is a feature shared only with other O. tsutsugamushi Anks and Anks of vertebrate poxviruses, Wolbachia species, and wasps that Wolbachia species infect. 42,50,53,55,69,76,77 The Ikeda chromosome encodes three identical Ank5 copies (OTT_RS01000, OTT_RS01950, and OTT_RS02325). 42,50 We assessed ank 5 transcription over the course of O. tsutsugamushi growth in HeLa cells and EA.hy926 human vascular endothelial cells.…”

“…In-Fusion Mutagenesis protocol and previously described Ank5∆F-box 55 F and tsa56-Full-R primers. The subsequent amplicon was cloned into TOPO vector pCR4.0 to generate pCR4.0::Ot_tsa56.…”

“…The Ank5 F-box domain binds Skp1 to assemble the SCF E3 ubiquitin ligase complex. 55 To confirm if the F-box and Skp1 contribute to the effector's ability to reduce NLRC5 cellular levels, HeLa cells that had been treated with Skp1 targeting small interfering RNA (siRNA) or non-targeting siRNA were transfected to co-express myc-NLRC5 and either Flag-Ank5 or Flag-Ank5∆F-box. Flag-Ank5 failed to degrade myc-NLRC5 in Skp1 knockdown cells (Figure 4C and 4D).…”

“…53,54 Most O. tsutsugamushi Anks carry a C-terminal eukaryotic-like F-box domain, which interacts with host Skp1 (S-phase kinase-associated protein 1) and Cul1 (cullin 1) to nucleate the SCF (Skp1-Cul1-F-box) E3 ubiquitin ligase complex that mediates polyubiquitination of substrates to destine them for 26S proteasomal degradation. 55,56 This AR-F-box bipartite architecture is exhibited by numerous other intracellular bacterial effectors and viral proteins. 53,[57][58][59][60][61][62] Strikingly, however, nearly all of the host cell target proteins that get ubiquitinated and degraded due to interacting with microbial F-box-containing Anks remain unidentified.…”