Project description:Analysis of changes in gene expression in Enterococcus faecalis OG1 delta-EF2638 mutant compared to wild-type OG1 strain. The deletion mutant has a growth defect when grown with aeration The mutant presented in this study is described and characterized in Vesic, D. and Kristich, C.J. 2012. A Rex-family transcriptional repressor influnces H2O2 accumulation by Enterococcus faecalis. (submitted for publication) Microarray analysis was done using RNA isolated from two independent cultures of wild-type Enterococcus faecalis OG1 and two independent cultres of Enterococcus faecalis OG1 delta-EF2638 mutant; each RNA sample was subjected to triplicate hybridization (technical replicates) . Microarrays were custom designed to investigate expression of ORFs in Enterococcus faecalis OG1RF genome. The arrays were designed based on the OG1RF annotation generated with the Rapid Annotation Using Subsystem Technology (RAST) server (Aziz et. al. 2008. BMC Genomics 9:75), as described in Frank et al (2012) Infect. Immun. 80:539. The aim was eighteen probe pairs per ORF, each of which is present in triplicate.
Project description:The role of mucosal invariant T (MAIT) cells in the lung tumor microenvironment re-mains poorly understood, especially in the setting of immune checkpoint inhibitors. We identified intratumoral MAIT cells from paired single cell RNA and TCR sequencing datasets of tumor infil-trating CD3 T cells isolated from non-small cell lung cancer tumors in patients receiving neoadju-vant PD-1 blockade therapy. MAIT cells were subclustered to identify conventional MAIT-associ-ated TCR clonotypes, which were then tested for the recognition of bacteria using a MAIT TCR capture functional assay. Strikingly, surveillance of intratumoral bacteria in lung cancer patients revealed Enterococcus spp. that are not directly recognized by MAIT cells but, nevertheless can synergize with exogenous riboflavin biosynthesis-derived metabolites to induce expression of MR1 by antigen presenting cells (APC, dendritic cells, B cells and mononuclear phagocytes). Enhanced MR1 cell surface expression resulted from enterococcal-mediated perturbation of an endo-lysosomal vacuolar pathway with recycling of early endosomal MR1 to the APC cytoplasmic membrane. Riboflavin auxotrophic Enterococcus spp may therefore exercise their beneficial im-munomodulatory functions upon immune checkpoint blockade treatment, at least in part, by pro-moting intratumoral MR1 expression and innate-like T cell activation. Our results indicate that composition of the intratumoral microbiome during immune checkpoint inhibitor treatment has the potential to impact the function of human intratumoral MAIT cells.
Project description:Gene content in various Enterococcus faecalis strains compared to E. faecalis V583. Strains have been compared to the V583 strain by comparative genomic hybridization using genome-wide PCR-based microarrays representing the V583 genome. Genes have been deemed "present" or "divergent" in the various strains.
