Trigger factor in Streptococcus mutans is involved in stress tolerance, competence development, and biofilm formation - PubMed (original) (raw)

Trigger factor in Streptococcus mutans is involved in stress tolerance, competence development, and biofilm formation

Zezhang T Wen et al. Infect Immun. 2005 Jan.

Abstract

Trigger factor is a ribosome-associated peptidyl-prolyl cis/trans isomerase that is highly conserved in most bacteria. A gene, designated ropA, encoding an apparent trigger factor homologue, was identified in Streptococcus mutans, the primary etiological agent of human dental caries. Inactivation of ropA had no major impact on growth rate in planktonic cultures under the conditions tested, although the RopA-deficient mutant formed long chains in broth. Deficiency of RopA decreased tolerance to acid killing and to oxidative stresses induced by hydrogen peroxide and paraquat, and it reduced transformation efficiency about 200-fold. Addition of synthetic competence-stimulating peptide to the culture medium enhanced transformability of both the mutant and wild-type strains, although the ropA strain did not attain levels of competence observed for the parent. Loss of RopA decreased the capacity of S. mutans to form biofilms by over 80% when cultivated in glucose, but it increased biofilm formation by over 50% when sucrose was provided as the carbohydrate source. Western blot analysis revealed that the expression of glucosyltransferases B and D was lower in the RopA-deficient mutant. These results suggest that RopA is a key regulator of acid and oxidative stress tolerance, genetic competence, and biofilm formation, all critical virulence properties of S. mutans.

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Figures

FIG. 1.

Acid killing and hydrogen peroxide challenge assays. For acid tolerance, all S. mutans strains were grown in BHI until mid-exponential phase (OD600 ≅ 0.3), harvested by centrifugation, washed once with 0.1 M glycine buffer, pH 7.0, and then subjected to acid killing at pH 2.8 for 45 min. For hydrogen peroxide challenge, the mid-exponential-phase cultures were incubated in buffer containing 0.2% (vol/vol) hydrogen peroxide for 2 h. The surviving cells were plated on BHI agar plates in triplicate, and results are expressed as survival rate over time. See text for more details. Data presented are means ± standard deviations (error bars) of three independent experiments. *, P < 0.01.

FIG. 2.

Transformation efficiency. S. mutans strains were evaluated for their ability to transform foreign DNA (plasmid) with or without inclusion of the synthetic CSP (500 ng μl−1). The bar graph represents the average of at least three independent experiments, with standard deviations denoted by the error bars. *, P < 0.01.

FIG. 3.

SEM analysis of biofilms. The 24-h biofilms of S. mutans strains UA159 (panels 1 and 3) and TW90 (panels 2 and 4) were grown on HA disks that were deposited in 24-well cell culture clusters in semidefined BM medium with glucose (panels 1 and 2) or sucrose (panels 3 and 4) as the carbohydrate source. Magnification in all images, ×1,000.

FIG. 4.

Western blot analysis. Whole-cell extracts (20 μg of total protein) of UA159 (lanes 1), TW90A (lanes 2), and TW90 (lane 3) were separated by SDS-10% PAGE. The separated proteins were then blotted to a polyvinylidene difluoride membrane and probed with anti-GtfB (A) and anti-GtfD (B) polyclonal antibodies.

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Trigger factor in Streptococcus mutans is involved in stress tolerance, competence development, and biofilm formation - PubMed (original) (raw)