How intracellular bacteria subvert the major histocompatibility complex (MHC) class I pathway is poorly understood. Here, we show that the obligate intracellular bacterium Orientia tsutsugamushi uses its effector protein, Ank5, to inhibit nuclear translocation of the MHC class I gene transactivator, NLRC5, and orchestrate its proteasomal degradation. Ank5 uses a tyrosine in its fourth ankyrin repeat to bind the NLRC5 N-terminus while its F-box directs host SCF complex ubiquitination of NLRC5 in the leucine-rich repeat region that dictates susceptibility to Orientia- and Ank5-mediated degradation. The ability of O. tsutsugamushi strains to degrade NLRC5 correlates with ank5 genomic carriage. Ectopically expressed Ank5 that can bind but not degrade NLRC5 protects the transactivator during Orientia infection. Thus, Ank5 is an immunoevasin that uses its bipartite architecture to rid host cells of NLRC5 and reduce surface MHC class I molecules. This study offers insight into how intracellular pathogens can impair MHC class I expression.

Cullin-1 (Cul1), a cullin–RING ubiquitin ligase component, represses c-MYC activity in the nucleus. Orientia tsutsugamushi causes the potentially fatal rickettsiosis, scrub typhus. The obligate intracellular bacterium encodes an arsenal of ankyrin repeat-containing effectors (Anks), many of which carry a eukaryotic-like F-box motif that binds Cul1. O. tsutsugamushi reduces Cul1 levels in the nucleus. This phenomenon is not due to an alteration in Cul1 neddylation but is bacterial burden- and protein synthesis-dependent. Five of the 11 Anks capable of binding Cul1 (Ank1, Ank5, Ank6, Ank9, Ank17) sequester it in the cytoplasm when each is ectopically expressed. Ank1 and Ank6 proteins with alanine substitutions in their F-boxes that render them unable to bind Cul1 cannot exclude Cul1 from the nucleus. Coincident with the reduction of Cul1 in the nuclei of Orientia -infected cells, c-MYC nuclear levels are elevated, and Cul1 target genes are differentially expressed. Several of these genes regulate apoptosis. The resistance of O. tsutsugamushi -infected cells to staurosporine-induced apoptosis is recapitulated in uninfected cells expressing Ank1 or Ank6 but not alanine-substituted versions thereof that cannot bind Cul1. Other F-box-containing Anks that cannot bind or exclude Cul1 from the nucleus also fail to confer resistance to apoptosis. Overall, O. tsutsugamushi modulates the Cul1:c-MYC intranuclear balance as an anti-apoptotic strategy that is functionally linked to a subset of its F-box-containing Anks.

Infections by intracellular pathogens often cause insult to host cell DNA, which stimulates responses that ultimately eliminate the damaged cell and hence the microbial niche. p53 is an innate immunity mediator that responds to DNA damage and intracellular infection by transcriptionally activating pathways that arrest the cell cycle, repair DNA, and elicit apoptosis. How pathogens counter p53 are incompletely understood. Here, we demonstrate that the endotheliotropic obligate intracellular bacterium Orientia tsutsugamushi blocks transcription of TP53 to nearly deplete p53 levels. Contrary to the unrestricted proliferation expected based on the transcriptome of p53-deficient infected cells, Orientia arrests the cell cycle at S phase to promote bacterial replication. It protects host endothelial cells from DNA damage even if induced by etoposide and delays genotoxic-dependent apoptosis until late in infection once a high bacterial load has been achieved. TP53 downregulation, protection against genotoxicity, and inhibition of DNA damage-dependent apoptosis are executed by the Orientia nucleomodulatory effector, Ank13. Therefore, O. tsutsugamushi inhibits TP53 expression and genotoxicity to reconfigure the intracellular environment of its host cell into one that favors bacterial replication.

Nuclear factor-κB (NF-κB) nuclear localization and gene accessibility is mediated by Ser22-phosphorylated lamin A/C (pSer22-lamin A/C). Here, we report that the intracellular bacterium Orientia tsutsugamushi impairs NF-κB nuclear accumulation by targeting lamin A using multiple ankyrin repeat (AR)-containing effectors (Anks). The Anks' immunomodulatory capability requires a conserved hydrophilic α-helical peptide that binds lamin A/pSer22-lamin A and lies between the AR and PRANC (pox proteins repeats of ankyrin C-terminal) domains. Orientia promotes pSer22-lamin A redistribution from the lamina to the nucleoplasm. This phenotype can be recapitulated in uninfected cells by ectopically expressing Anks that carry the lamin A-binding sequence. O. tsutsugamushi also alters chromatin accessibility at sites regulated by lamin A, NF-κB, and the NF-κB coactivator, adapter protein 1. These findings identify a mechanism by which a pathogen synergistically modulates lamin A and chromatin accessibility to counteract NF-κB, reinforce the regulatory link between lamin A and NF-κB, and indicate that pSer22-lamin A enhances chromatin accessibility to NF-κB and its coactivators.

Scrub typhus is an emerging chigger-borne disease caused by the obligate intracellular bacterium Orientia tsutsugamushi. Necroptosis is a form of programmed cell death (PCD) mediated by RIPK3 (serine/threonine kinase receptor interacting protein 3) and its downstream effector MLKL (mixed-lineage kinase domain-like). While O. tsutsugamushi modulates apoptosis, another form of PCD, its interplay with necroptosis is unknown. Much of Orientia pathobiology is linked to its ankyrin repeat (AR)-containing effectors (Anks). Two of these, Ank1 and Ank6, share similarities with the cowpox AR protein, vIRD (viral inducer of RIPK3 degradation) that prevents necroptosis. Here, we show that Ank1 and Ank6 reduce RIPK3 cellular levels although not as robustly as and mechanistically distinct from vIRD. Orientia infection lowers RIPK3 amounts and does not elicit necroptosis in endothelial cells. In HeLa cells ectopically expressing RIPK3, Orientia fails to inhibit RIPK3 and MLKL phosphorylation as well as cell death. MLKL colocalization with Orientia or Listeria monocytogenes, another intracytoplasmic pathogen, was not observed. Thus, O. tsutsugamushi reduces cellular levels of RIPK3 and does not elicit necroptosis but cannot inhibit this PCD pathway once it is induced. This study is a first step toward understanding how the relationship between Orientia and necroptosis contributes to scrub typhus pathogenesis.

Orientia tsutsugamushi is a mite-transmitted obligate intracellular bacterium that causes the potentially deadly zoonosis, scrub typhus. The absence of genetic tools for Orientia have limited studies of the microbe-host interactions that underlie scrub typhus. To address this gap, we developed a protocol for transforming and achieving allelic exchange in O. tsutsugamushi str. Ikeda. From evaluating multiple cell lines and antibiotics, we found that contact-inhibited EA.hy926 human endothelial-like cells best supported Orientia replication and that chloramphenicol was an effective selection marker. We engineered a homologous recombination cassette encoding a codon-modified version of the O. tsutsugamushi ank13 gene (OTT_RS04140) (CM ank13 ) and its promoter alongside genes for mScarlet-I and chloramphenicol acetyltransferase under control of the O. tsutsugamushi tsa22 -up and tsa56 -down promoters, respectively. A PCR product encompassing the cassette and chromosomal flanking regions was transformed into O. tsutsugamushi via electroporation or CaCl ₂ , the latter of which better preserved bacterial and host cell viability. EA.hy926 cells inoculated with transformed O. tsutsugamushi were grown in glass-bottom plates in the presence of chloramphenicol and imaged by live-cell microscopy to identify cultures containing mScarlet-I positive bacteria, which could be maintained in perpetuity. Chromosomal integration of the CM ank13 cassette and loss of wild-type ank13 were verified by PCR and nanopore sequencing. This report establishes platforms for genetically manipulating O. tsutsugamushi and building additional genetic tools to investigate this globally significant pathogen.