Conjugal transfer of R68.45 and FP5 between Pseudomonas aeruginosa strains in a freshwater environment

Recent concern over the release of genetically engineered organisms has resulted in a need for information about the potential for gene transfer in the environment. In this study, the conjugal transfer in Pseudomonas aeruginosa of the plasmids R68.45 and FP5 was demonstrated in the freshwater environment of Fort Loudoun Resevoir, Knoxville, Tenn. When genetically well defined plasmid donor and recipient strains were introduced into test chambers suspended in Fort Loudoun Lake, transfer of both plasmids was observed. Conjugation occurred in both the presence and absence of the natural microbial community. The number of transconjugants recovered was lower when the natural community was present. Transfer of the broad-host-range plasmid R68.45 to organisms other than the introduced recipient was not observed in these chambers but was observed in laboratory simulations when an organism isolated from lakewater was used as the recipient strain. Although the plasmids transferred in laboratory studies were genetically and physically stable, a significant number of transconjugants recovered from the field trials contained deletions and other genetic rearrangements, suggesting that factors which increase gene instability are operating in the environment. The potential for conjugal transfer of genetic material must be considered in evaluating the release of any genetically engineered microorganism into a freshwater environment.Peer reviewedMicrobiology and Molecular Genetic

Gene-diet interactions associated with complex trait variation in an advanced intercross outbred mouse line

Phenotypic variation of quantitative traits is orchestrated by a complex interplay between the environment (e.g. diet) and genetics. However, the impact of gene-environment interactions on phenotypic traits mostly remains elusive. To address this, we feed 1154 mice of an autoimmunity-prone intercross line (AIL) three different diets. We find that diet substantially contributes to the variability of complex traits and unmasks additional genetic susceptibility quantitative trait loci (QTL). By performing whole-genome sequencing of the AIL founder strains, we resolve these QTLs to few or single candidate genes. To address whether diet can also modulate genetic predisposition towards a given trait, we set NZM2410/J mice on similar dietary regimens as AIL mice. Our data suggest that diet modifies genetic susceptibility to lupus and shifts intestinal bacterial and fungal community composition, which precedes clinical disease manifestation. Collectively, our study underlines the importance of including environmental factors in genetic association studies

Mitochondrial gene polymorphism is associated with gut microbial communities in mice

Gut microbial communities are key mediators of health and disease and have the capacity to drive the pathogenesis of diverse complex diseases including metabolic and chronic inflammatory diseases as well as aging. Host genetics is also a major determinant of disease phenotypes, whereby two different genomes play a role, the nuclear (nDNA)-and mitochondrial genome (mtDNA). We investigated the impact of mutations in mtDNA on the gut microbiota using conplastic mouse strains exhibiting distinct mutations in their mtDNA on an identical nDNA. Each of three strain tested harbors a distinct gut microbiota, ranging from differences at the phylum-to operational taxonomic units level. The C57BL/6J-mt FVB/NJ strain, carrying a mutation in the mitochondrial ATP8 synthase gene, exhibits higher Firmicutes abundance than Bacteroidetes, indicating a possible indicative for metabolic dysfunctions. In line with this, the C57BL/6J-mt FVB/NJ displays a variety of different phenotypes, including increased susceptibility to metabolic-related and inflammatory disorders. Furthermore, we discuss the cross-talk between mitochondrial genome/mitochondria and commensal microbiota in relation to clinical phenotypes. In summary, we demonstrate that mutations in mtDNA lead to significant differences in the composition of gut microbial communities in mice. Such differences may facilitate the emergence of metabolic disease and therefore constitute potential therapeutic targets. © 2017 The Author(s)

Complex interactions between gut microbiota and the mitochondria contribute to the pathogenesis of autoimmune skin diseases

Autoimmune diseases are thought to be caused by complex interactions of genetic and environmental factors. Shifts in gut microbial communities and the reduction in their diversity are found in a variety of diseases, including autoimmune diseases. Bullous pemphigoid is the most common autoimmune blistering skin disease (AIBD), which is mediated by autoantibodies targeting type XVII collagen. To the best of our knowledge, no experimental studies have been conducted to elucidate the impact of gut microbiota in AIBD. Therefore, we first induced a well-established experimental epidermolysis bullosa acquisita (EBA) in germ-free mice. Germfree C57BL/6Tac mice, which received pathogenic IgG against type VII collagen exhibited significantly milder disease severity compared to the conventionally housed C57BL/6Tac mice (padj. T), compared to wild-type C57BL/6J (B6) mice. In the human equivalent gene, MT-ATP8, polymorphisms are associated with bullous pemphigoid in a German cohort. In line with this human study, B6-mtFVB mice demonstrated significantly less skin inflammation than B6 mice when experimental EBA was induced (P < 0.05). To further confirm a potential interaction of the gut microbiota and AIBD, shotgun metagenomics of cecum content, RNA-seq of cecum epithelium samples, and untargeted metabolomics of liver samples of B6-mtFVB and B6 mice were performed. Integrated analysis of shotgun metagenomics and untargeted metabolomics analysis is currently being conducted to identify the gut microbiome derived metabolites that may modulate host immune cell response. Additionally, we evaluated the immune cell metabolism in CD4 + T cells, a pivotal player in the pathogenesis of skin inflammation in the experimental EBA. CD4 + T cells from B6-mtFVB mice demonstrated a higher ratio of glycolysis to oxidative phosphorylation than those from B6 mice (P = 0.0008). This change may further modulate CD4 + T cell function, as we observed less cellular proliferation capacity upon immunological stress induced by anti-CD3 and anti-CD28 antibodies (P < 0.05) and less proinflammatory cytokine IL-17 production in CD4 + T cells from B6-mtFVB mice than B6 mice (P < 0.01). We also evaluated gut permeability via gene expression of tight junction proteins (Claudin-2, Claudin-15, Occludin and ZO-1). Expression levels of these genes were reduced in small intestine, cecum and colon samples of B6-mtFVB compared to those of B6 mice. Our findings show 1) experimental evidence demonstrating the importance of the gut microbiota in the pathogenesis of AIBD, and that 2) a single polymorphism in the mitochondrial genome impacts the composition of gut microbiota, host metabolites and immune cell metabolism, which further modulate disease severity in autoimmune skin inflammation in mice. To explore the link between gut microbiota and immune cell interaction, we plan to identify gut microbiota-derived metabolites and evaluate their modulatory effect on immune cells contributing to autoimmune skin inflammation in vitro and in germ-free B6-mtFVB and B6 mice which are currently being generated

Design and synthesis of nature-inspired chromenopyrroles as potential modulators of mitochondrial metabolism

Chromenopyrrole derivatives with multiple stereocenters and variable ring fusion pattern are found in many natural products and biologically appealing molecules. By employing a build/couple/pair strategy, we have recently reported on the discovery of a serendipitous cascade to access a diverse collection of chromenopyrroles. This protocol features a one-pot cascade that includes the generation of azomethine ylide and intramolecular [3 + 2]-cycloaddition. Phenotypic screening of the developed pilot library enabled the identification of chemical probes that efficiently suppress mitochondrial membrane potential, elevate reactive oxygen species content, and deplete ATP content in a hepatoma cell line (Hepa1-6), without affecting the proliferation of T- or B-cells. This selective targeting represents a new approach for the treatment of cancer. [Figure not available: see fulltext.