Wolan, D. W. | Search bioRxiv

bioRxiv2018

Triflic acid treatment enables LC-MS/MS analysis of insoluble bacterial biomass

The lysis and extraction of soluble bacterial proteins from cells is a common practice for proteomics analyses, but insoluble bacterial biomasses are often left behind. Here, we show that with triflic acid treatment, the insoluble bacterial biomass of Gram- and Gram+ bacteria can be rendered soluble. We use LC-MS/MS shotgun proteomics to show that bacterial proteins in the soluble and insoluble post-lysis fractions differ significantly. Additionally, in the case of Gram- Pseudomonas aeruginosa, triflic acid treatment enables the enrichment of cell envelope-associated proteins. Finally, we apply triflic acid to a human microbiome sample to show that this treatment is robust and enables the identification of a new, complementary subset of proteins from a complex microbial mixture.

systems biology↗

bioRxiv2018

The Genetic Basis for the Cooperative Bioactivation of Plant Lignans by a Human Gut Bacterial Consortium

Plant-derived lignans, consumed daily by most individuals, are inversely associated with breast cancer; however, their bioactivity is only exerted following gut bacterial conversion to enterolignans. Here, we dissect a four-species bacterial consortium sufficient for all four chemical reactions in this pathway. Comparative genomics and heterologous expression experiments identified the first enzyme in the pathway. Transcriptional profiling (RNAseq) independently identified the same gene and linked a single genomic locus to each of the remaining biotransformations. Remarkably, we detected the complete bacterial lignan metabolism pathway in the majority of human gut microbiomes. Together, these results are an important step towards a molecular genetic understanding of the gut bacterial bioactivation of lignans and other plant secondary metabolites to downstream metabolites relevant to human disease.\n\nOne Sentence SummaryBess et al. provide a first step towards elucidating the molecular genetic basis for the cooperative gut bacterial bioactivation of plant lignans, consumed daily by most individuals, to phytoestrogenic enterolignans.

microbiology↗

bioRxiv2017

Metaproteomics of colonic microbiota unveils discrete protein functions among colitic mice and control groups

Metaproteomics can greatly assist established high-throughput sequencing methodologies to provide systems biological insights into the alterations of microbial protein functionalities correlated with disease-associated dysbiosis of the intestinal microbiota. Here, we utilized the well-characterized murine T cell transfer model of colitis to find specific changes within the intestinal luminal proteome associated with inflammation. MS proteomic analysis of colonic samples permitted the identification of {bsim}10,000-12,000 unique peptides that corresponded to 5,610 protein clusters identified across three groups, including the colitic Rag1-/- T cell recipients, isogenic Rag1-/- controls, and wild-type mice. We demonstrate that the colitic mice exhibited a significant increase in Proteobacteria and Verrucomicrobia and show that such alterations in the microbial communities contributed to the enrichment of specific proteins with transcription and translation gene ontology terms. In combination with 16S sequencing, our metaproteomics-based microbiome studies provide a foundation for assessing alterations in intestinal luminal protein functionalities in a robust and well-characterized mouse model of colitis, and set the stage for future studies to further explore the functional mechanisms of altered protein functionalities associated with dysbiosis and inflammation.\n\nStatement of significance of the studyThe commensal gut microbiota is essential to maintaining health and has a primary role in digestion/metabolism, homeostasis, and protection from pathogenic bacteria. High-throughput sequencing has established Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria as the four major bacterial phyla that comprise the ecological makeup of the intestinal microbiota. However, the tremendous inter-/intra-variability in microbial composition across individuals, as well as along the length of the intestinal tract has made it difficult to definitively ascertain specific bacterial species associated with health or as drivers of disease states, including inflammatory bowel disease. In this study, we expanded upon the current metaproteomics techniques and use the robust and highly reproducible murine T cell transfer model of colitis as well as a comprehensive database of mouse, human, plant, and all microbial genomes sequenced to date to elucidate alterations in both host and gut microbial proteins associated with intestinal inflammation. Our results show that host genetics, gut microbiota, and inflammation have tremendous influences on the intestinal luminal proteomic landscape.

bioinformatics↗