Lipidomic and proteomic evaluation of extracellular membrane vesicles from Streptococcus mutans wild‐type, ΔsrtA and Δsfp strains

Abstract

BackgroundExtracellular membrane vesicles (EMVs) have long been studied in Gram‐negative bacteria. It is more recently established that Gram‐positive bacteria are capable of producing EMVs despite their thick cell wall. Our group showed that the cariogenic bacterium Streptococcus mutans produces EMVs that carry protein and eDNA. EMV protein content is altered in a deletion mutant lacking the transpeptidase, Sortase A. In Bacillus subtilis, EMVs are disrupted by the lipoprotein surfactin and accumulate in a Δsfp mutant. Our current objective is to isolate EMVs from wild‐type, ΔsrtA and Δsfp strains in order to compare their respective lipidomic and proteomic profiles, as well as to compare each strain's EMVs with corresponding cytoplasmic membrane preparations.MethodsEMVs were isolated from four separate replicates of each strain grown in Terleckyj's defined media (TDM). Filtered culture supernatants were concentrated ~50 fold, and EMVs were harvested by high‐speed centrifugation and density gradient isolation. Cytoplasmic membranes were isolated from protoplasts derived from bacterial cells from the same cultures. Lipids were analyzed by capillary‐liquid chromatography mass spectrometry (Cap‐LC‐MS). Proteins were analyzed by Nano‐Liquid chromatography tandem mass spectrometry (Nano‐LC/MS/MS). MV size, concentration, and polydisperity were assessed by transmission electron microscopy, nanoparticle tracking analysis, and dynamic light scattering.ResultsEMVs from the ΔsrtA mutant were enlarged compared to the other strains. Lipid composition of EMV membranes consisted primarily of glycerophospholipids, with some glycerolipids and sphingolipids. Glycerophosphoinsitol (PI) was underrepresented in both mutant strains compared to the WT. Glycerophosphocholine (PC) was overrepresented in the Δsfp mutant compared to the other two strains. Cardiolipin (CL) was overrepresented in EMVs from the WT strain compared to its corresponding cytoplasmic membrane preparations. Proteomics analyses are underway.ConclusionsLipidomic and proteomic analyses will provide information regarding S. mutans vesiculogenesis and potential functional differences in EMVs produced by WT and mutant strains.Support or Funding InformationNIH R21DE025348 NIH R01DE008007This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.