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Staphylococcus aureus is a human pathogen that can cause a wide range of diseases. Although formerly regarded as extracellular pathogen, it has been shown that S. aureus can also be internalized by host cells and persist within these cells. In the present study, we comparatively analyzed survival and physiological adaptation of S. aureus HG001 after internalization by two human lung epithelial cell lines (S9 and A549), and human embryonic kidney cells (HEK 293). Combining enrichment of bacteria from host-pathogen assays by cell sorting and quantitation of the pathogen's proteome by mass spectrometry we characterized S. aureus adaptation during the initial phase between 2.5 h and 6.5 h post-infection. Starting with about 2 × 106 bacteria, roughly 1450 S. aureus proteins, including virulence factors and metabolic enzymes were identified by spectral comparison and classical database searches. Most of the bacterial adaptation reactions, such as decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied. However, differences were noted in central carbon metabolism including regulation of fermentation and threonine degradation. Since these differences coincided with different intracellular growth behavior, complementary profiling of the metabolome of the different non-infected host cell types was performed. This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate. With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and regulatory mutants.
Swine are regarded as promising biomedical models, but the dynamics of theirgastrointestinal microbiome have been much less investigated than that of humans or mice. The aimof this study was to establish an integrated multi-omics protocol to investigate the fecal microbiomeof healthy swine. To this end, a preparation and analysis protocol including integrated samplepreparation for meta-omics analyses of deep-frozen feces was developed. Subsequent data integrationlinked microbiome composition with function, and metabolic activity with protein inventories, i.e.,16S rRNA data and expressed proteins, and identified proteins with corresponding metabolites.16S rRNA gene amplicon and metaproteomics analyses revealed a fecal microbiome dominated byPrevotellaceae,Lactobacillaceae,Lachnospiraceae,RuminococcaceaeandClostridiaceae.Similar microbiomecompositions in feces and colon, but not ileum samples, were observed, showing that feces can serveas minimal-invasive proxy for porcine colon microbiomes. Longitudinal dynamics in composition,e.g., temporal decreased abundance ofLactobacillaceaeandStreptococcaceaeduring the experiment,were not reflected in microbiome function. Instead, metaproteomics and metabolomics showed arather stable functional state, as evident from short-chain fatty acids (SCFA) profiles and associatedmetaproteome functions, pointing towards functional redundancy among microbiome constituents.In conclusion, our pipeline generates congruent data from different omics approaches on the taxonomyand functionality of the intestinal microbiome of swine.