Isolation and Characterization of Extracellular Membrane Vesicles From Streptococcus mutans

Objectives: Streptococcus mutans produces extracellular membrane vesicles (MV’s) that carry protein and eDNA. Protein content is altered in a deletion mutant lacking Sortase A. In Bacillus subtilis MV’s are disrupted by the lipoprotein surfactin and accumulate in a Δsfp mutant. The current objective is to characterize MV’s from S. mutans WT, ΔsrtA, and Δsfp strains, compare thier protein and lipid content, and evaluate MV contribution to S. mutans biofilm development.
Methods: MV’s were prepared from overnight cultures of S. mutans WT (NG8 and UA159) and corresponding mutant strains grown in chemically defined medium. Cell free supernatants were concentrated ~200-fold in an Amicon^TM stirred cell with a 100 kDa cutoff PL membrane. MV’s were harvested by centrifugation at 100,000xg and isolated on an OptiPrep^TM density gradient.
Results: MV’s were visualized by transmission electron microscopy. Nanosight analysis demonstrated final yields of ~6.0x10⁹-1.7x10¹¹ MV particles per liter of culture supernatant with mean diameters of 96-182 nm. Dynamic light scattering revealed a poly-disperse population and zeta potential measurement indicated a net negative charge. Western blot analysis revealed membrane proteins YidC1 and YidC2 and other members of the protein translocation machinery including Ffh and SecA. Other prominent proteins were glucan binding protein B, AtlA, and the secreted protein Smu63c, an apparent negative regulator of biofilm cell density and genetic competence. Cell wall anchored adhesins P1 (AgI/II) and WapA were present in low or trace quantities. The active processed form of AtlA was absent in MV’s from the ΔsrtA mutant. Addition of MV’s from WT UA159 consistently increased biofilm formation of that strain, especially in the presence of sucrose.
Conclusions: S. mutans produces extracellular MV’s that contribute to biofilm formation. The effect of MV’s from mutant strains on biofilm formation is being evaluated as is the ability of MV’s from WT S. mutans to rescue biofilm defects of ΔsrtA and ΔatlA mutants. Clinical isolates were identified that produce 5-6 fold more MV’s than S. mutans UA159 and are being evaluated for genomic alterations to explain this phenotype. Mass spectrometry proteomic and lipidomic analyses of MVs and corresponding cytoplasmic membrane preparations are underway.