SUMMARY Diet, genetics, and the gut microbiome are determinants of metabolic status, in part through production of metabolites by the gut microbiota. To understand the mechanisms linking these factors, we performed LC-MS-based metabolomic analysis of cecal contents and plasma from C57BL/6J, 129S1/SvImJ, and 129S6/SvEvTac mice on chow or a high-fat diet (HFD) and HFD-treated with vancomycin or metronidazole. Prediction of the functional metagenome of gut bacteria by PICRUSt analysis of 16S sequences revealed dramatic differences in microbial metabolism. Cecal and plasma metabolites showed multifold differences reflecting the combined and integrated effects of diet, antibiotics, host background, and the gut microbiome. Eighteen plasma metabolites correlated positively or negatively with host insulin resistance across strains and diets. Over 1,000 still-unidentified metabolite peaks were also highly regulated by diet, antibiotics, and genetic background. Thus, diet, host genetics, and the gut microbiota interact to create distinct responses in plasma metabolites, which can contribute to regulation of metabolism and insulin resistance.

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Outer membrane vesicles (OMVs) produced by Gram-negative bacteria provide an interesting research material for defining cellenvelope proteins without experimental cell disruption. OMVs are also promising immunogenic platforms and may play important roles in bacterial survival and pathogenesis. We used in-solution trypsin digestion coupled to mass spectrometry to identify 90 proteins present in OMVs of Vibrio cholerae when grown under conditions that activate the TCP pilus virulence regulatory protein (ToxT) virulence regulon. The ToxT expression profile and potential contribution to virulence of these proteins were assessed using ToxT and in vivo RNA-seq, Tn-seq, and cholera stool proteomic and other genome-wide data sets. Thirteen OMV-associated proteins appear to be essential for cell growth, and therefore may represent antibacterial drug targets. Another 12 nonessential OMV proteins, including DegP protease, were required for intestinal colonization in rabbits. Comparative proteomics of a degP mutant revealed the importance of DegP in the incorporation of nine proteins into OMVs, including ones involved in biofilm matrix formation and various substrates of the type II secretion system. Taken together, these results suggest that DegP plays an important role in determining the content of OMVs and also affects phenotypes such as intestinal colonization, proper function of the type II secretion system, and formation of biofilm matrix.HtrA family | in-solution digestion | biofilm formation | CTXϕ phage

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