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

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.

FIG 1

FE-SEM analysis of the eDNA nanofibers. S. mutans UA159 was grown in BM-glucose on hydroxylapatite (A and C to F) and silicon (B) discs for 4 (A and B) and 24 (C to F) h. Panels A to D show a network of eDNA nanofibers connecting cells to substratum (A and B) and cells to cells (C and D); panels E and F show eDNA from cell lysis that was concentrated around broken cells (indicated by asterisks) and often floating with open ends. The inset in panel A shows a close-up of the area indicated by an arrow. Images in panels A and B were taken at 10,000×, C and E at 20,000×, and D and F at 50,000× magnification.

FIG 2

Effect of DNase I on biofilm formation. S. mutans biofilms were grown in FMC with glucose (18 mM) and sucrose (2 mM) as the carbohydrate sources on hydroxylapatite discs vertically deposited in 24-well plates with inclusion of DNase I (DNase) for 5 and 24 h, respectively. Controls received the heat-inactivated enzyme (iDNase). Data presented here represent the averages (± standard deviations) from more than three independent sets of experiments, with an asterisk indicating statistical difference at P < 0.001 compared to the controls.

FIG 3

S. mutans adhesion to apatitic surfaces in presence and absence of eDNA. sHA, saliva-coated hydroxyapatite; gsHA, glucan formed on saliva-coated hydroxyapatite. An asterisk indicates that there is a statistical difference for the samples with eDNA compared to controls without eDNA (P < 0.05).

FIG 4

eDNA production by S. mutans. S. mutans UA159 was grown planktonically in biofilm medium plus glucose (20 mM) in 5-ml Falcon tubes (solid bars) or in biofilms on hydroxylapatite discs vertically deposited in 24-well plates (hatched bars) for 5 and 24 h. Data presented here represent the averages (± standard deviations) from more than 3 independent experiments, with statistic differences at P < 0.01 (#) and P < 0.05 (*) compared to the planktonic counterparts.

FIG 5

EM analysis of S. mutans membrane vesicles. S. mutans UA159 (A) and NG8 (B to D) were grown in BHI broth (B to D) and in biofilm medium on hydroxylapatite discs (A) overnight. FE-SEM (A) and TEM (B) analysis show small blebs on the cell surfaces. Panels C and D show EM images of vesicular structures in cell-free supernatants of NG8 following negative staining with 1% uranyl acetate. Similar vesicles were also seen with UA159 (not shown). Images were taken at magnifications of 50,000× (A), 30,000× (B), 50,000× (C), and 25,000× (D).

FIG 6

Sortase A deficiency on eDNA production. (A) For quantitative analysis, S. mutans UA159, the sortase A-deficient mutant SAB102, and its complement strain, SJ271, which carries a wild-type copy of srtA in a multicopy shuttle vector, pDL271, were grown in the chemically defined medium FMC with glucose (20 mM) for 5-h cultures (−5) or in FMC with glucose (18 mM) plus sucrose (2 mM) for 24-h (−24) and for biofilm (−B) cultures. Biofilms were grown on HA discs that were vertically deposited in wells of 24-well plates. The eDNA in the cell-free supernatant was further normalized and expressed relative to levels for wild-type UA159. Data presented represent averages (± standard deviations) from more than 3 independent experiments, with * and ** indicating statistical difference at P < 0.01 and P < 0.001, respectively, compared to UA159 under the same conditions. Similar results were also obtained during growth in BM. (B) FE-SEM analysis of wild-type S. mutans (UA159) and the SrtA-deficient mutant (SAB102) biofilms on HA discs during growth in BM-glucose. Compared to the wild type, the SrtA-deficient mutant SAB102 consistently displayed few or no eDNA nanofibers in biofilms grown for 24 h. Images were taken at a magnification of 20,000×.

FIG 7

SDS-PAGE (A) and Western blot (B) analysis. S. mutans UA159, its AtlA-deficient mutant (630NP), NG8, and its SrtA-deficient mutant (PC339) were grown in FMC broth overnight, and membrane vesicles were prepared from cell-free supernatants by ultracentrifugation. For controls, a set of overnight cultures was killed by incubating at 60°C for 45 min, washed in sterile PBS, resuspended in FMC, and then allowed to incubate at 37°C overnight. For SDS-PAGE analysis, a 10% gel was used. For Western blotting, antibodies against P1, glucan-binding proteins GbpB and GbpC, and pooled glucosyltransferases (Gtf) were used as probes. Relative to the wild type, NG8, the SrtA-deficient mutant had significantly less P1, GbpB, GbpC, and Gtf, which are known to be surface-associated proteins. Equal volumes of membrane vesicle preparations were loaded on the gel.

FIG 8

eDNA production by S. mutans mutants. S. mutans wild-type NG8 and its derivatives, AH374 (yidC1), AH329 (ffh), AH307 (ftsY), AH378 (yidC2), and AH312 (scRNA), which are deficient in genes (as specified) that encode components of the protein translocation system, during growth in regular FMC with glucose for 5 h. All data presented here are averages (± standard deviations) from three independent experiments and were further normalized to the wild type, with * and ** indicating differences at significance levels of P < 0.05 and P < 0.01, respectively.