Project description:Mucin hypersecretion, a hallmark of chronic respiratory diseases (CRD), creates a complex microenvironment that reshapes host immunity and microbial behavior. However, its impact on bacteriophage therapy remains poorly understood. Here, we demonstrate that, despite reducing Pseudomonas aeruginosa internalization, mucin increases bacterial-induced cytotoxicity and inflammation in airway epithelial cells, while driving CRD-like transcriptional changes, including hypoxia and stress responses. Mucin selectively downregulates virulence factors without impairing bacterial growth. P. aeruginosa-infecting bacteriophage DMS3vir retained full lytic activity in mucin-rich conditions and, in synergy with mucin, enhanced epithelial cells protection against cytotoxicity. DMS3vir also reduced IL-8 gene expression without triggering antiviral responses. Furthermore, mucin shaped phage-resistant P. aeruginosa phenotypes, altering pigmentation, pigmentation, pyocyanin production, and motility. These changes influenced virulence trade-offs. These findings uncover the dual role of mucins as modulators of infection and sensitizers to phage protection, paving the way for optimized, mucosa-adapted phage therapies in chronic lung diseases.
Project description:As a human tumor virus, EBV is present as a latent infection in its associated malignancies where genetic and epigenetic changes have been shown to impede cellular differentiation and viral reactivation. One such change is increased levels of the Wnt signaling effector, lymphoid enhancer binding factor 1 (LEF1) following EBV epithelial infection. In silico analysis of EBV type 1 and 2 genomes identified over 20 Wnt-response elements suggesting that LEF1 may directly bind the EBV genome and regulate the viral life cycle. Using CUT&RUN-seq, LEF1 was shown to bind the latent EBV genome at various sites encoding viral lytic products that included the immediate early transactivator BZLF1 and viral primase BSLF1 genes. SiRNA depletion of specific LEF1 isoforms revealed that the alternative-promoter derived isoform with an N-terminal truncation (∆N LEF1) transcriptionally repressed lytic genes associated with LEF1 binding. Furthermore, forced expression of the ∆N LEF1 isoform antagonized EBV reactivation from latency. The LEF1 mediated repression requires histone deacetylase activity through either recruitment or a direct intrinsic histone deacetylase activity. SiRNA depletion of LEF1 resulted in increased histone 3 lysine 9 and lysine 27 acetylation at LEF1 binding sites and across the EBV genome. These results support a novel role for LEF1 in maintaining EBV latency and restriction viral reactivation via repressive chromatin remodeling of critical lytic cycle factors
Project description:Chromatin-organizing factors, like CTCF and cohesins, have been implicated in the control of complex viral regulatory programs. We investigated the role of CTCF and cohesin in the control of the latent to lytic switch for Kaposi's Sarcoma-Associated Herpesvirus (KSHV). We found that cohesin subunits, but not CTCF, were required for the repression of KSHV immediate early gene transcription. Depletion of cohesin subunits Rad21, SMC1, or SMC3 resulted in lytic cycle gene transcription and viral DNA replication. In contrast, depletion of CTCF failed to induce lytic transcription or DNA replication. ChiP-Seq analysis revealed that cohesins and CTCF bound to several sites within the immediate early control regions for ORF50 and more distal 5' sites that also regulate the divergently transcribed ORF45-46-47 gene cluster. Rad21 depletion led to a robust increase in ORF45 and ORF47 transcripts, with similar kinetics to that observed with chemical induction by sodium butyrate. During latency, the chromatin between the ORF45 and ORF50 transcription start sites was enriched in histone H3K4me3 with elevated H3K9ac at the ORF45 promoter and elevated H3K27me3 at the ORF50 promoter. A paused form of RNA pol II was loosely associated with the ORF45 promoter region during latency, but was converted to an active elongating form upon reactivation induced by Rad21 depletion. Butyrate-induced transcription of ORF45 and ORF47 was resistant to cyclohexamide, suggesting that these genes have immediate early features similar to ORF50. Butyrate-treatment caused the rapid dissociation of cohesins and loss of CTCF binding at the immediate early gene locus, suggesting that cohesins may be a direct target of butyrate-mediated lytic induction. Our findings implicate cohesins as a major repressor of KSHV lytic gene activation, and function coordinately with CTCF to regulate the switch between latent and lytic gene activity. Study of chromatin-organizing factors, like CTCF and cohesins.