Pharmacological stabilization of hypoxia-inducible factor (HIF) through prolyl hydroxylase (PHD) inhibition limits mucosal damage associated with models of murine colitis. However, little is known about how PHD inhibitors (PHDi) influence systemic immune function during mucosal inflammation or the relative importance of immunological changes to mucosal protection. We hypothesized that PHDi enhances systemic innate immune responses to colitis-associated bacteremia. Mice with colitis induced by TNBS were treated with AKB-4924, a new HIF-1 isoform-predominant PHDi and clinical, immunological and biochemical endpoints were assessed. Administration of AKB-4924 led to significantly reduced weight loss and disease activity compared to vehicle controls. Treated groups were pyrexic, but did not become subsequently hypothermic. PHDi treatment augmented epithelial barrier function and led to an approximately 50-fold reduction in serum endotoxin during colitis. AKB-4924 also decreased cytokines involved in pyrogenesis and hypothermia, significantly reducing serum levels of IL-1β, IL-6 and TNF-α, while increasing IL-10. Treatment offered no protection against colitis in epithelial-specific HIF-1α deficient mice, strongly implicating epithelial HIF-1α as the tissue target for AKB-4924-mediated protection. Taken together, these results indicate that inhibition of prolyl hydroxylase with AKB-4924 enhances innate immunity and identifies the epithelium is a central site of inflammatory protection afforded by PHDi in murine colitis.

Hypoxia inducible factor-1 (HIF-1) is a transcription factor that is a major regulator of energy homeostasis and cellular adaptation to low oxygen stress. HIF-1 is also activated in response to bacterial pathogens and supports the innate immune response of both phagocytes and keratinocytes. In this work, we show that a new pharmacological compound AKB-4924 (Akebia Therapeutics) increases HIF-1α levels and enhances the antibacterial activity of phagocytes and keratinocytes against both methicillin-sensitive and -resistant strains of Staphylococcus aureus in vitro. AKB-4924 is also effective in stimulating the killing capacity of keratinocytes against the important opportunistic skin pathogens Pseudomonas aeruginosa and Acinitobacter baumanii. The effect of AKB-4924 is mediated through the activity of host cells, as the compound exerts no direct antimicrobial activity. Administered locally as a single agent, AKB-4924 limits S. aureus proliferation and lesion formation in a mouse skin abscess model. This approach to pharmacologically boost the innate immune response via HIF-1 stabilization may serve as a useful adjunctive treatment for antibiotic-resistant bacterial infections.

Full-length VP22 is necessary for efficient spread of herpes simplex virus type 1 (HSV-1) from cell to cell during the course of productive infection. VP22 is a virion phosphoprotein, and its nuclear localization initiates between 5 and 7 h postinfection (hpi) during the course of synchronized infection. The goal of this study was to determine which features of HSV-1 infection function to regulate the translocation of VP22 into the nucleus. We report the following. The molecular mechanism of herpes simplex virus type 1 (HSV-1) tegument and envelope assembly is poorly understood. Most of the major tegument proteins exhibit nuclear or perinuclear distributions late in infection (1, 8, 10-12, 26, 33, 37, 43), and there is general agreement that primary envelopment occurs as the capsid exits the nucleus (7,19,21,42,46) (for a detailed review, see reference 17).Work in our laboratory has focused on the major tegument protein VP22 and its function in virion assembly and virus replication. Full-length VP22 is necessary for efficient spread of the virus from cell to cell, inasmuch as a recombinant virus producing a truncated form of the protein exhibits a decreased plaque size in Vero cells compared to that of wild-type virus (36). In an earlier study, we reported that the nuclear localization of VP22 initiates between 5 and 7 h postinfection (hpi) during the course of synchronized infection (37). This period corresponds to the peak of viral ␤ protein production and DNA synthesis (39). Recently, computer analyses predicted two nuclear localization signals (NLS) in the primary structure of VP22 (22). Although transiently expressed VP22 can be detected in the nuclei of transfected cells (15,22), these proposed NLS have not yet been characterized in nuclear import assays. VP22 is a 301-amino-acid protein with a predicted size of 32,000 Da (28). Thus, VP22 is below the size exclusion limit (40,000 to 45,000 Da) for passive diffusion through the nuclear pore (13). That VP22 accumulates in the cytoplasm of infected cells prior to 5 hpi suggests its translocation to the nucleus is regulated during infection. One possible means of regulation could be active retention through binding of VP22 to a cytoplasmic structure.In this study, we sought to determine which features of HSV-1 replication are involved in the redistribution of VP22 during productive infection. Our investigations show the following. Regulated VP22 nuclear localization initiates after 5 hpi with HSV-1 and is independent of viral DNA and true late protein synthesis. We propose that HSV-1 induced microtubule restructuring releases VP22 from the cytoskeleton, allowing its entry into the nucleus. Stabilization of microtubules during infection or in VP22-expressing cells increases VP22 retention in the cytoplasm. Based on these results, we conclude that microtubule reorganization during herpes HSV-1 infection acts to facilitate the nuclear localization of VP22. MATERIALS AND METHODS Cells and virus.African green monkey kidney (Vero) cells were obtained from the Ameri...

Ubiquinone (coenzyme Q; abbreviation, Q) plays an essential role in electron transport in Escherichia coli when oxygen or nitrate is the electron acceptor. The biosynthesis of Q involves at least nine reactions. Three of these reactions involve hydroxylations resulting in the introduction of hydroxyl groups at positions C-6, C-4, and C-5 of the benzene nucleus of Q. The genes encoding the enzymes responsible for these hydroxylations, ubiB, ubiH, and ubiF are located at 87, 66, and 15 min of the E. coli linkage map. The ubiF encoded oxygenase introduces the hydroxyl group at carbon five of 2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinol resulting in the formation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1, 4-benzoquinol. An ubiF mutant failed to carry out this conversion. Based on the homology to UbiH, an open reading frame (orf391) was identified at the 15 min region of the chromosome, amplified using PCR, and cloned into pUC18 plasmid. The ubiF mutants, when complemented with this plasmid, regained the ability to grow on succinate and synthesize Q.

The goal of this study was to determine whether the subcellular localization patterns of other HSV-1 tegument proteins are similar to that observed with VP22. To address this, we performed a series of indirect immunofluorescence analyses using synchronously infected cells. We observed that tegument proteins VP13/14, vhs, and VP16 localized to the nucleus as early as 5 hpi and were concentrated in nuclei by 9 hpi, which differed from that seen with VP22. Microtubule reorganization was delayed during infection with HSV-1(RF177), a recombinant virus that does not produce full-length VP22. These infected cells did not begin to lose microtubuleorganizing centers until 13 hpi. Repair of the unique long 49 (U L 49) locus in HSV-1(RF177) yielded HSV-1(RF177R). Microtubule reorganization in HSV-1(RF177R)-infected cells occurred with the same kinetics as HSV-1(F). Acetylated tubulin remained unchanged during infection with either HSV-1(F) or HSV-1(RF177).Thus, while ␣-tubulin reorganized during infection, acetylated tubulin was stable, and the absence of fulllength VP22 did not affect this stability. Our findings indicate that the nuclear localizations of tegument proteins VP13/14, VP16, and vhs do not appear to require HSV-1-induced microtubule reorganization. We conclude that full-length VP22 is needed for optimal microtubule reorganization during infection. This implies that VP22 mainly functions to reorganize microtubules later, rather than earlier, in infection. That acetylated tubulin does not undergo restructuring during VP22-dependent, virus-induced microtubule reorganization suggests that it plays a role in stabilizing the infected cells. Our results emphasize that VP22 likely plays a key role in cellular cytopathology during HSV-1 infection.