Streptococcus mutans Extracellular DNA Is Upregulated during Growth in Biofilms, Actively Released via Membrane Vesicles, and Influenced by Components of the Protein Secretion Machinery

Abstract

f Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5-and 24-h biofilms was increased by >3-and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA.

Influence of Growth Stages and Nutrient Media on Production of Membrane Vesicles of Clostridium perfringens Type A and their Molecular Characterization

“…The present observations on trend of protein concentration was similar to the observations reported by Brown et al, (2014) Liao et al, (2014) reported the presence of DNA in the extracellular vesicles of gram-positive bacteria.…”

Immunoactive Clostridial Membrane Vesicle Production Is Regulated by a Sporulation Factor

“…Previous studies have revealed that MV release is conserved in Gram-negative cells and that MVs are associated with virulence factor delivery, protein secretion, cell-cell communication, protection from phage infection, and other biological processes (2,8). Although Gram-positive bacteria have no outer membrane and are covered by a rigid, thick cell wall, MV production was observed in many Gram-positive bacteria, such as Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes, Streptococcus mutans, S. pneumoniae, Mycobacteria spp., and Lactobacillus rhamnosus (14)(15)(16)(17)(18)(19)(20)(21). In addition, Grampositive bacterial MVs have been reported to deliver virulence factors to host cells to stimulate an immune response in recipient cells and to facilitate biofilm formation by extracellular DNA release via the MVs (17,22,23), suggesting that functional MV release is also conserved in Gram-positive bacteria.…”

“…Although Gram-positive bacteria have no outer membrane and are covered by a rigid, thick cell wall, MV production was observed in many Gram-positive bacteria, such as Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes, Streptococcus mutans, S. pneumoniae, Mycobacteria spp., and Lactobacillus rhamnosus (14)(15)(16)(17)(18)(19)(20)(21). In addition, Grampositive bacterial MVs have been reported to deliver virulence factors to host cells to stimulate an immune response in recipient cells and to facilitate biofilm formation by extracellular DNA release via the MVs (17,22,23), suggesting that functional MV release is also conserved in Gram-positive bacteria. In Gram-negative bacteria, MV production is stimulated by accumulation of the unnecessary proteins in the periplasm, charge repulsion, and the SOS response (8,24).…”

“…In Mycobacterium tuberculosis, virR mutant strains overproduce MVs; thus, VirR proteins inhibit vesiculogenesis (26). Mutation of srtA, which encodes the enzyme involved in anchoring cell surface proteins, affects protein composition in MVs of S. mutans but not the amount of MVs (17). However, the regulatory factor activating MV production and release is not known in Gram-positive bacteria.…”

Type III Secretion System Translocon Component EseB Forms Filaments on and Mediates Autoaggregation of and Biofilm Formation by Edwardsiella tarda

“…Filamentous appendages are also observed in other bacteria, for example, outer membrane vesicles (OMVs) on Myxococcus xanthus (42), nanofibers on Streptococcus mutans (43), and rigid nanowires on Shewanella oneidensis (44). Rigid nanowires, which are quite similar to E. tarda EseB filaments in morphology and distribution, are electrically conductive in direct response to electron acceptor limitation (44).…”