Inactivation of the spxA1 or spxA2 gene of Streptococcus mutans decreases virulence in the rat caries model

Abstract

In oral biofilms, the major environmental challenges encountered by Streptococcus mutans are acid and oxidative stresses. Previously, we showed that the transcriptional regulators SpxA1 and SpxA2 are involved in general stress survival of S. mutans with SpxA1 playing a primary role in activation of antioxidant and detoxification strategies whereas SpxA2 serves as a back up activator of oxidative stress genes. We have also found that spxA1 mutant strains (∆spxA1 and ∆spxA1∆spxA2) are outcompeted by peroxigenic oral streptococci in vitro and have impaired abilities to colonize the teeth of rats fed a highly cariogenic diet. Here, we show that the Spx proteins can also exert regulatory roles in the expression of additional virulence attributes of S. mutans. Competence activation is significantly impaired in Δspx strains and the production of mutacin IV and V is virtually abolished in ΔspxA1 strains. Unexpectedly, the ∆spxA2 strain showed increased production of glucans from sucrose, without affecting the total amount of bacteria within biofilms when compared with the parent strain. By using the rat caries model, we showed that the capacity of the ΔspxA1 and ΔspxA2 strains to cause caries on smooth tooth surfaces is significantly impaired. The ∆spxA2 strain also formed fewer lesions on sulcal surfaces. This report reveals that global regulation via Spx contributes to the cariogenic potential of S. mutans and highlights that animal models are essential in the characterization of bacterial traits implicated in virulence.

Keywords: Streptococcus mutans; Spx; dental caries; oxidative stress.

Fig. 1

Competence of S. mutans UA159 and its derivatives. Transformation efficiency of (A, B) parent UA159 and ΔspxA strains and (C, D) UA159 harboring the nisin-inducible pMSP3535 (control) or pMSP3535-Spx plasmids (Spx-overexpressing strains). The integration plasmid pMC340A (donor DNA) was added to cells in early-logarithmic phase (OD₆₀₀ of 0.15) with or without the addition of CSP or nisin. Cultures were incubated at 37°C in 5% CO₂ until entering stationary phase, serially diluted and plated in BHI for total CFU and in BHI containing kanamycin for transformants. (A) pMC340A only (B) pMC340A + CSP, (C) pMC340A only, and (D) pMC340A + nisin. The results represent the mean and standard deviations of at least three independent experiments. Asterisks indicate differences were statistically significant (P ≤0.05) when compared to UA159 (A, B), or UA159 harboring the empty pMSP3535 vector (C, D).

Fig. 2

Mutacin production by S. mutans UA159 and its derivatives. Cultures of S. mutans were grown in BHI to OD₆₀₀ of 0.3 and 15 μl of each culture spotted on BHI plates followed by incubation at 37°C in 5% CO₂. After 24 h incubation, plates were exposed to UV light for 20 min and then overlayed with 5 ml soft BHI agar containing 500 μl of overnight cultures of S. gordonii or L. lactis. Plates were incubated for additional 48 h and the zone of inhibition recorded.

Fig. 3

Biofilm fomation and biomass by S. mutans UA159 and its derivatives. (A) Biofilm formation on microtiter plates. Cultures were grown in LMW containing 1% sucrose for 24 h. Biofilm formation was normalized by total growth to exclude apparent differences due to the growth abilities of each strain (*, P ≤ 0.05). (B) The imaging of EPS and bacteria within intact 3D biofilms was conducted using a method based on incorporation of fluorescently labeled dextran (Alexa Fluor 647, red) by Gtfs during glucan synthesis. Bacterial cells were labeled with SYTO9 (green). The images shown are representative of n = 15. The confocal images were analyzed by AMIRA and COMSTAT (*, P ≤ 0.05).

Fig. 4

Total cultivable microflora (A) and S. mutans (B) recovered from left jaws of animals five weeks after infection with UA159, ΔspxA1 or ΔspxA2 strains. (C) Proportion (%) of S. mutans per total flora. The results represent the mean and standard deviations of one experiment (n=10). Asterisks indicate differences were statistically significant (P ≤0.05) when compared to UA159 * P ≤ 0.05 (ANOVA).