Time-course analysis of Streptococcus sanguinis after manganese depletion reveals changes in glycolytic and nucleic acid metabolites

Abstract

Introduction Manganese is important for the endocarditis pathogen Streptococcus sanguinis. Little is known about why manganese is required for virulence or how it impacts the metabolome of streptococci. Objectives We applied untargeted metabolomics to cells and media to understand temporal changes resulting from manganese depletion. Methods EDTA was added to a S. sanguinis manganese-transporter mutant in aerobic fermentor conditions. Cell and media samples were collected pre- and post-EDTA treatment. Metabolomics data were generated using positive and negative modes of data acquisition on an LC–MS/MS system. Data were subjected to statistical processing using MetaboAnalyst and time-course analysis using Short Time series Expression Miner (STEM). Recombinant enzymes were assayed for metal dependence. Results We observed quantitative changes in 534 and 422 metabolites in cells and media, respectively, after EDTA addition. The 173 cellular metabolites identified as significantly different indicated enrichment of purine and pyrimidine metabolism. Further multivariate analysis revealed that the top 15 cellular metabolites belonged primarily to lipids and redox metabolites. The STEM analysis revealed global changes in cells and media in comparable metabolic pathways. Glycolytic intermediates such as fructose-1,6-bisphosphate increased, suggesting that enzymes that utilize them require manganese for activity or expression. Recombinant enzymes were confirmed to utilize manganese in vitro. Nucleosides accumulated, possibly due to a blockage in conversion to nucleobases resulting from manganese-dependent regulation. Conclusion Differential analysis of metabolites revealed the activation of a number of metabolic pathways in response to manganese depletion, many of which are connected to carbon catabolite repression.