Project description:Enterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants.
Project description:We examined fecal proteins from normal subjects (n=6), patients with inflammatory bowel disease (n=8), patients with colorectal adenoma (n=8), and patients with colorectal cancer (n=14) in the hope of identifying novel fecal protein markers that could be used to identify colorectal diseases.
Project description:Patients with metastatic pancreatic ductal adenocarcinoma (PDAC) survive longer if disease spreads to the lung but not the liver. We generated overlapping, multi-omic datasets to identify molecular and cellular features that distinguish patients whose disease develops liver metastasis (liver cohort) from those whose disease develops lung metastasis without liver metastases (lung cohort). Lung cohort patients survived longer than liver cohort patients, despite sharing the same tumor subtype. We developed a pORG gene set enriched in liver cohort versus lung cohort primary tumors. We identified ongoing replication stress (RS) response pathways in high pORG/liver cohort tumors, while low pORG/lung cohort tumors had greater densities of lymphocytes and shared T cell clonal responses. Our study demonstrates that liver-avid PDAC is associated with tolerance to ongoing RS, limited tumor immunity, and less favorable outcomes; whereas low RS, lung-avid/liver-averse tumors are associated with active tumor immunity that may account for favorable outcomes.
Project description:In Staphylococcus aureus, the role of the GGDEF domain containing protein GdpS remains poorly understood. Previous studies reported that gdpS mutant strains had decreased biofilm formation due to changes in icaADBC expression that were independent of cyclic-di-GMP levels. We deleted gdpS in three unrelated S. aureus isolates, and analyzed the resultant mutants for alterations in biofilm formation, metabolism and transcription. Dynamic imaging during biofilm development showed that GdpS inhibited early biofilm formation in only two out of the three strains examined, without affecting bacterial survival. However, quantification of biofilm formation using crystal violet staining revealed that inactivation of gdpS affected biofilm formation in all three studied strains. Extraction of metabolites from S. aureus cells confirmed the absence of cyclic-di-GMP, suggesting that biofilm formation in this species differs from that in other Gram-positive organisms. In addition, targeted mutagenesis demonstrated that the GGDEF domain was not required for GdpS activity. Transcriptomic analysis revealed that the vast majority of GGDEF-regulated genes were involved in virulence, metabolism, cell wall biogenesis and eDNA release. Finally, expression of lrgAB or deletion of cidABC in a strain lacking gdpS confirmed the role of GdpS on regulation of eDNA production that occurred without an increase in cell autolysis. In summary, S. aureus GdpS contributes to cell-to-cell interactions during early biofilm formation by influencing expression of lrgAB and cidABC mediated eDNA release. We conclude that GdpS acts as a negative regulator of eDNA release. Three strains UAMS-1, SA113 and SA564 were used in this study to compare wt with gdpS mutant after 5 hours of growth in static conditions (biofilm formation).
Project description:In Staphylococcus aureus, the role of the GGDEF domain containing protein GdpS remains poorly understood. Previous studies reported that gdpS mutant strains had decreased biofilm formation due to changes in icaADBC expression that were independent of cyclic-di-GMP levels. We deleted gdpS in three unrelated S. aureus isolates, and analyzed the resultant mutants for alterations in biofilm formation, metabolism and transcription. Dynamic imaging during biofilm development showed that GdpS inhibited early biofilm formation in only two out of the three strains examined, without affecting bacterial survival. However, quantification of biofilm formation using crystal violet staining revealed that inactivation of gdpS affected biofilm formation in all three studied strains. Extraction of metabolites from S. aureus cells confirmed the absence of cyclic-di-GMP, suggesting that biofilm formation in this species differs from that in other Gram-positive organisms. In addition, targeted mutagenesis demonstrated that the GGDEF domain was not required for GdpS activity. Transcriptomic analysis revealed that the vast majority of GGDEF-regulated genes were involved in virulence, metabolism, cell wall biogenesis and eDNA release. Finally, expression of lrgAB or deletion of cidABC in a strain lacking gdpS confirmed the role of GdpS on regulation of eDNA production that occurred without an increase in cell autolysis. In summary, S. aureus GdpS contributes to cell-to-cell interactions during early biofilm formation by influencing expression of lrgAB and cidABC mediated eDNA release. We conclude that GdpS acts as a negative regulator of eDNA release.
Project description:3'-end RNA sequencing of inflamed and non-inflamed resected intestial tissue from IBD patients, as well as from non-tumor intestinal tissue resected from patients with colorectal cancer