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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. [ABSTRACT FROM AUTHOR]

Rickettsial agents are sensed by pattern recognition receptors but lack pathogen-associated molecular patterns commonly observed in facultative intracellular bacteria. Due to these molecular features, the order Rickettsiales can be used to uncover broader principles of bacterial immunity. Here, we used the bacterium Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, to reveal a novel microbial surveillance system. Mechanistically, we discovered that upon A. phagocytophilum infection, cytosolic phospholipase A2 cleaves arachidonic acid from phospholipids, which is converted to the eicosanoid prostaglandin E2 (PGE2) via cyclooxygenase 2 (COX2) and the membrane associated prostaglandin E synthase-1 (mPGES-1). PGE2-EP3 receptor signaling leads to activation of the NLRC4 inflammasome and secretion of interleukin (IL)-1β and IL-18. Importantly, the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) was identified as a major regulator of the immune response against A. phagocytophilum. Accordingly, mice lacking COX2 were more susceptible to A. phagocytophilum, had a defect in IL-18 secretion and exhibited splenomegaly and damage to the splenic architecture. Remarkably, Salmonella-induced NLRC4 inflammasome activation was not affected by either chemical inhibition or genetic ablation of genes associated with PGE2 biosynthesis and signaling. This divergence in immune circuitry was due to reduced levels of the PGE2-EP3 receptor during Salmonella infection when compared to A. phagocytophilum. Collectively, we reveal the existence of a functionally distinct NLRC4 inflammasome illustrated by the rickettsial agent A. phagocytophilum. [ABSTRACT FROM AUTHOR]

The sterile alpha motif (SAM) and HD domain-containing protein-1 (SAMHD1) inhibits the infection of resting CD4+ T cells and myeloid cells by human and related simian immunodeficiency viruses (HIV and SIV). Vpx inactivates SAMHD1 by promoting its proteasome-dependent degradation through an interaction with CRL4 (DCAF1) E3 ubiquitin ligase and the C-terminal region of SAMHD1. However, the determinants in SAMHD1 that are required for Vpx-mediated degradation have not been well characterized. SAMHD1 contains a classical nuclear localization signal (NLS), and NLS point mutants are cytoplasmic and resistant to Vpx-mediated degradation. Here, we demonstrate that NLS-mutant SAMHD1 K11A can be rescued by wild-type SAMHD1, restoring its nuclear localization; consequently, SAMHD1 K11A became sensitive to Vpx-mediated degradation in the presence of wild-type SAMHD1. Surprisingly, deletion of N-terminal regions of SAMHD1, including the classical NLS, generated mutant SAMHD1 proteins that were again sensitive to Vpx-mediated degradation. Unlike SAMHD1 K11A, these deletion mutants could be detected in the nucleus. Interestingly, NLS-defective SAMHD1 could still bind to karyopherin-β1 and other nuclear proteins. We also determined that the linker region between the SAM and HD domain and the HD domain itself is important for Vpx-mediated degradation but not Vpx interaction. Thus, SAMHD1 contains an additional nuclear targeting mechanism in addition to the classical NLS. Our data indicate that multiple regions in SAMHD1 are critical for Vpx-mediated nuclear degradation and that association with Vpx is not sufficient for Vpx-mediated degradation of SAMHD1. Since the linker region and HD domain may be involved in SAMHD1 multimerization, our results suggest that SAMHD1 multimerization may be required for Vpx-mediation degradation. [ABSTRACT FROM AUTHOR]