Orientia tsutsugamushi uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation

Abstract

Orientia tsutsugamushi causes scrub typhus, a potentially fatal infection that threatens over one billion people. Nuclear translocation of the transcription factor, NF-κB, is the central initiating cellular event in the antimicrobial response. Here, we report that NF-κB p65 nuclear accumulation and NF-κB-dependent transcription are inhibited in O. tsutsugamushi infected HeLa cells and/or primary macrophages, even in the presence of TNFα. The bacterium modulates p65 subcellular localization by neither degrading it nor inhibiting IκBα degradation. Rather, it exploits host exportin 1 to mediate p65 nuclear export, as this phenomenon is leptomycin B-sensitive. O. tsutsugamushi antagonizes NF-κB-activated transcription even when exportin 1 is inhibited and NF-κB consequently remains in the nucleus. Two ankyrin repeat-containing effectors (Anks), Ank1 and Ank6, each of which possess a C-terminal F-box and exhibit 58.5% amino acid identity, are linked to the pathogen’s ability to modulate NF-κB. When ectopically expressed, both translocate to the nucleus, abrogate NF-κB-activated transcription in an exportin 1-independent manner, and pronouncedly reduce TNFα-induced p65 nuclear levels by exportin 1-dependent means. Flag-tagged Ank 1 and Ank6 co-immunoprecipitate p65 and exportin 1. Both also bind importin β1, a host protein that is essential for the classical nuclear import pathway. Importazole, which blocks importin β1 activity, abrogates Ank1 and Ank6 nuclear translocation. The Ank1 and Ank6 regions that bind importin β1 also mediate their transport into the nucleus. Yet, these regions are distinct from those that bind p65/exportin 1. The Ank1 and Ank6 F-box and the region that lies between it and the ankyrin repeat domain are essential for blocking p65 nuclear accumulation. These data reveal a novel mechanism by which O. tsutsugamushi modulates the activity and nuclear transport of NF-κB p65 and identify the first microbial proteins that co-opt both importin β1 and exportin 1 to antagonize a critical arm of the antimicrobial response.

Dual RNA-seq provides insight into the biology of the neglected intracellular human pathogenOrientia tsutsugamushi

“…The protein products of all of these ank genes localise to the endoplasmic reticulum or host cell cytoplasm when ectopically expressed 63 . Ank6 interferes with NFkB translocation to the nucleus and inhibits its transcriptional activation 64 . The activity of the other differentially expressed Anks is not known.…”

Silkworm pupa oil attenuates acetaminophen‐induced acute liver injury by inhibiting oxidative stress‐mediated NF‐κB signaling

“…The levels of IL-6 and IL-12 F I G U R E 2 Effects of silkworm pupa oil on hepatic histology (original magnification 100×). NF-κB subunits p50/p65 are bound to the inhibitory protein IκBα and exist in cytosol as an inactive form (Evans, Rodino, Adcox, & Carlyon, 2018;Whitman & Barber, 2015). n = 6 are increased in primary human hepatocytes and mice with APAP treatment (Cho et al, 2015;Kim et al, 2017).…”

“…Furthermore, the activation of NF-κB signaling can induce the production of proinflammatory cytokines and hepatic inflammation. NF-κB subunits p50/p65 are bound to the inhibitory protein IκBα and exist in cytosol as an inactive form (Evans, Rodino, Adcox, & Carlyon, 2018;Whitman & Barber, 2015).…”

Bacterial Factors Targeting the Nucleus: The Growing Family of Nucleomodulins

“…Ank1 and Ank6 are transported into the nucleus via the classical importin-dependent pathway β1, and then induce export of the p65 subunit of NF-κB via exportin 1. Thus, Ank1 and Ank6 inhibit the accumulation of p65 in the nucleus and the transcription of pro-inflammatory genes of the NF-κB, pathway [101].…”