Exploring the role of bacteria in viral reactivation and pathogenesis

Herpesviral sequences are frequently copresent with bacterial infection at multiple sites. Discerning mechanisms of bacterially induced viral reactivation would explain the molecular basis of polymicrobial infections. We hypothesized that bacterial end-products from oral and sexually transmitted pathogens and bacterial components initiate viral reactivation from latency and augment viral pathogenesis. Latently infected cell lines such as BCBL-1 (Kaposi's Sarcoma Associated Herpesvirus (KSHV)) and B958 (Epstein Barr Virus (EBV)) were incubated in vitro with crude spent media from oral bacteria. Cells were then assayed for promoter activation or state of infection by viral gene expression and Gardella gel analysis. To investigate mechanisms of reactivation, Histone Deacetylase (HDAC) inhibition potential and Protein Kinase C (PKC) activity were measured. Following incubation with crude spent media from bacteria, viral immediate early promoters were activated such as ORF -50 (KSHV), ICP0 (Herpes Simplex Virus (HSV), and BRLF1 (EBV). The KSHV early gene, Pan promoter, was upregulated and linear genomes were detected. HDAC inhibition activity as well as kinase activity increased significantly following pathogen spent media treatment from Porphyromas gingivalis, Fusobacterium nucleatum, and Staphylococcus aureus. Interestingly, distinct oral bacterial pathogens such as F.nucleatum, P.gingivalis, Prevotella intermedius, and S.aureus differentially reactivated EBV, KSHV and HSV. Using crude spent media from STD pathogens, Trichomonas vaginalis and Neisseria gonorrhea, we demonstrated that they decreased HDAC activity and activated immediate early viral promoters such as ORF 50 (KSHV), and ICP0 (HSV). The ability of both oral and STD bacteria to decrease HDAC activity leads to decreases in innate immune responses. Using gene expression studies and promoter based assays it was demonstrated that induction of gene transcription of Interferon Regulatory factor 3 (IRF 3) and interferon alpha mediated induction of interferon stimulated gene transcription decreased subsequent to treatment with bacterial spent media from P.gingivalis, F.nucleatum, and S.aureus. Bacterial spent media dose dependently decreased activation of interferon beta promoter. Chromatin immunoprecipitation (ChIP) analysis detected decreased binding of RNA polymerase II on the IFN[beta] promoter following bacterial spent media treatment. Following viral challenge, there was decreased secretion of IFN[beta] from fibrosarcoma cell line, 2fTGH, in the presence of bacterial spent media. Interestingly, the presence of bacterial LPS in the spent media did not affect the ability of metabolites to decrease innate antiviral immune responses. Spent media treatment upregulated the expression of NLRX1, an antagonist of the innate mitochondrial antiviral signaling (MAVS) response. Collectively, these data demonstrate that bacteria in a polymicrobial environment may potentiate viral reactivation and enhance viral pathogenesis