Deficiency of RgpG Causes Major Defects in Cell Division and Biofilm Formation, and Deficiency of LytR-CpsA-Psr Family Proteins Leads to Accumulation of Cell Wall Antigens in Culture Medium by Streptococcus mutans

Abstract

Streptococcus mutans is known to possess rhamnose-glucose polysaccharide (RGP), a major cell wall antigen. S. mutans strains deficient in rgpG, encoding the first enzyme of the RGP biosynthesis pathway, were constructed by allelic exchange. The rgpG deficiency had no effect on growth rate but caused major defects in cell division and altered cell morphology. Unlike the coccoid wild type, the rgpG mutant existed primarily in chains of swollen, "squarish" dividing cells. Deficiency of rgpG also causes significant reduction in biofilm formation (P < 0.01). Double and triple mutants with deficiency in brpA and/or psr, genes coding for the LytR-CpsA-Psr family proteins BrpA and Psr, which were previously shown to play important roles in cell envelope biogenesis, were constructed using the rgpG mutant. There were no major differences in growth rates between the wild-type strain and the rgpG brpA and rgpG psr double mutants, but the growth rate of the rgpG brpA psr triple mutant was reduced drastically (P < 0.001). Under transmission electron microscopy, both double mutants resembled the rgpG mutant, while the triple mutant existed as giant cells with multiple asymmetric septa. When analyzed by immunoblotting, the rgpG mutant displayed major reductions in cell wall antigens compared to the wild type, while little or no signal was detected with the double and triple mutants and the brpA and psr single mutants. These results suggest that RgpG in S. mutans plays a critical role in cell division and biofilm formation and that BrpA and Psr may be responsible for attachment of cell wall antigens to the cell envelope.IMPORTANCEStreptococcus mutans, a major etiological agent of human dental caries, produces rhamnose-glucose polysaccharide (RGP) as the major cell wall antigen. This study provides direct evidence that deficiency of RgpG, the first enzyme of the RGP biosynthesis pathway, caused major defects in cell division and morphology and reduced biofilm formation by S. mutans, indicative of a significant role of RGP in cell division and biofilm formation in S. mutans These results are novel not only in S. mutans, but also other streptococci that produce RGP. This study also shows that the LytR-CpsA-Psr family proteins BrpA and Psr in S. mutans are involved in attachment of RGP and probably other cell wall glycopolymers to the peptidoglycan. In addition, the results also suggest that BrpA and Psr may play a direct role in cell division and biofilm formation in S. mutans This study reveals new potential targets to develop anticaries therapeutics.

Keywords: BrpA; LCP proteins; Psr; Streptococcus mutans; TEM analysis; biofilm formation; cell division; cell wall antigens; dental caries; rhamnose-glucose polysaccharides.

FIG 1

Growth study. The S. mutans wild-type strain (UA159), its rgpG (TW322 and TW393), brpA (TW14D), psr (TW251), rgpG brpA (TW340), rgpG psr (TW341), and rgpG brpA psr (TW343) mutants, and the rgpG complement strain (TW393C) were grown in BHI (A and C), semidefined biofilm medium (A) with glucose (BMG) and sucrose (BMS) as the carbohydrate sources, and BHI adjusted to pH 7.0 and 6.0 (B). The optical density at 600 nm (OD₆₀₀) of the cultures was monitored continuously using Bioscreen C with a sterile mineral oil overlay. The data presented here are representative of more than three separate experiments.

FIG 2

TEM analysis. The S. mutans wild-type strain (UA159) (A) and its rgpG (TW322) (B), rgpG brpA (TW340) (C), rgpG psr (TW341) (D), and rgpG brpA psr (TW343) (E) mutants were grown in BHI until the mid-exponential phase (OD₆₀₀ of ≅0.4). The images presented here were taken at a magnification of 10,000×, and scale bars are 500 nm. Panel F shows inserts of the magnified cell envelope of the different strains with the triple mutant TW343 showing a significantly thicker peptidoglycan (pg) layer and a thinner/fainter cytoplasmic membrane (cm).

FIG 3

Biofilm formation. The S. mutans wild-type strain (UA159), its rgpG mutants (TW322 for the 96-well plate model and TW393 for HA discs), and the rgpG complement strain (TW393C) were grown in BM supplemented with glucose (BMG), sucrose (BMS), and glucose plus sucrose (BMGS). Biofilms were grown in 96-well plates (A) and HA discs vertically placed in 12-well plates (B and C) for 24 and 48 h. By the end of the experiments, biofilms on 96-well plates were stained with crystal violet and analyzed using a spectrophotometer (A); biofilms on HA discs were fixed overnight and processed for SEM analysis (B), or stained using the LIVE/DEAD fluorescent staining kit and examined by confocal microscopy (C). The results presented in panel A are averages (±standard deviation [SD] shown by error bars) of biofilms after 24 and 48 h from more than three independent experiments. * and # indicate statistically significant difference at P < 0.001 and P < 0.05, respectively, compared to the wild type. Panel B shows representative SEM images (at 5,000×) of 24-h biofilms grown in BM plus glucose and sucrose. Panel C shows representative compressed xyz, xz, and yz confocal microscopic images (512 by 512) of biofilms grown in BM with glucose and sucrose.

FIG 3

Biofilm formation. The S. mutans wild-type strain (UA159), its rgpG mutants (TW322 for the 96-well plate model and TW393 for HA discs), and the rgpG complement strain (TW393C) were grown in BM supplemented with glucose (BMG), sucrose (BMS), and glucose plus sucrose (BMGS). Biofilms were grown in 96-well plates (A) and HA discs vertically placed in 12-well plates (B and C) for 24 and 48 h. By the end of the experiments, biofilms on 96-well plates were stained with crystal violet and analyzed using a spectrophotometer (A); biofilms on HA discs were fixed overnight and processed for SEM analysis (B), or stained using the LIVE/DEAD fluorescent staining kit and examined by confocal microscopy (C). The results presented in panel A are averages (±standard deviation [SD] shown by error bars) of biofilms after 24 and 48 h from more than three independent experiments. * and # indicate statistically significant difference at P < 0.001 and P < 0.05, respectively, compared to the wild type. Panel B shows representative SEM images (at 5,000×) of 24-h biofilms grown in BM plus glucose and sucrose. Panel C shows representative compressed xyz, xz, and yz confocal microscopic images (512 by 512) of biofilms grown in BM with glucose and sucrose.

FIG 4

Biofilm formation. The S. mutans wild-type strain (UA159) and its rgpG (TW322), brpA (TW14D), psr (TW251), rpgG brpA (TW340), rgpG psr (TW341), and rgpG brpA psr (TW343) mutants were grown in BM containing glucose, sucrose, and glucose plus sucrose. Biofilms were grown in 96-well plates and measured using a spectrophotometer following crystal violet staining. ANOVA and Tukey's pairwise comparison were used to analyze the differences between different strains. The data presented here represent the average (±SD shown by error bars) from more than three separate sets of experiments with cultures grown in BM plus glucose and sucrose. #, P < 0.001 versus UA159 and TW343 and <0.05 versus TW14D, TW251, TW340, and TW341; *, P < 0.001 versus UA159 and TW343 and <0.05 versus TW322; **, P < 0.001 versus all others.

FIG 5

Immunoblot analysis. Cell wall antigens were isolated from murein sacculi of the S. mutans wild-type strain (UA159), its rgpG (TW322), rpgG brpA (TW340), rgpG psr (TW341), and rgpG brpA psr (TW343) mutants, and the rgpG complement strain (TW322C) and were probed with whole-cell antiserum adsorbed with UA159 or TW343. The bar graph shows results when probed with antiserum adsorbed with the triple mutant, TW343, and expressed as mean of the percentage of signal intensities relative to UA159 (±SD represented in numbers above the individual bars) from two separate experiments. *, significant difference at P < 0.001 compared to the wild type. Images show the slot blots of cell wall antigens (a) and cell-free culture medium preparations (b) probed with the TW343 adsorbed antiserum. Samples were loaded as indicated in the bar graph above. The two images presented in panels a and b were taken from the same immunoblots with samples loaded at different rows.