H-083. Effect of covR and vicK Gene Inactivation in the Expression of Virulence Genes in Strains of Streptococcus mutans

R. N. Stipp1, R. B. Gonçalves1, J. F. Höfling1, D. J. Smith2, R. O. Mattos-Graner1;
1Piracicaba Dent. Sch. – FOP/UNICAMP, Piracicaba, SP, BRAZIL, 2The Forsyth Inst., Boston, MA.

Background: Expression of genes encoding glucosyltransferases B, C (gtfB, gtfC) and glucan-binding protein B (gbpB) by S. mutans (SM) strains is associated with particular capacities of strains to accumulate into biofilms. Genes encoding these virulence proteins appear to be controlled by global two-component transduction systems (TCS), Vic and Cov. Whether these systems work similarly among strains that differ with regard to their virulence pattern expression is unclear. Objective: The aim of this study was to compare the effect of inactivation of the Vic and Cov genes on the expression of gtfB/C and gbpB among different SM strains. Methods: Nonpolar deletion mutations of vicK or covR were performed by PCR ligation mutagenesis in three SM strains, UA159, 20A3 and 5ST1. covR and vicK mutants (MT) and the respective wild type (WT) strains were grown in BHI (37°C;10%CO2;5h) to absorbances (A550nm) of 0.3; cells and culture fluids were then collected. RNAs extracted from cell samples were analyzed in semi-quantitative RT-PCR assays for transcript levels of gtfB, gtfC and gbpB. Amounts of cell-associated and secreted GbpB were also analyzed in western blot assays probed with specific GbpB anti-serum. Results: Inactivation of covR resulted in a 20 to 30% increase in levels of gtfB and gtfC transcripts (p<0.05, ANOVA) in all the strains analyzed, while the vicK deletion resulted in 10 to 20% decrease in the transcription of gtfB, gtfC and gbpB (p<0.05, ANOVA). Forty to 60% reduction in levels of GbpB were observed among the three vicK- mutants in comparison with the respective WT strains (p<0.05, ANOVA). covR mutants also produced 60 to 250% more GbpB than WT strains (p<0.01 ANOVA). Conclusions: Data indicate that Vic and Cov systems play roles in the expression of virulence genes among the strains analyzed, in that gtfB, gtfC and gbpB are under the negative control of covR, while the same genes are positively regulated by the vicK TCS. Supported by FAPESP 02/07156-1, 06/55933-8 and NIH-Fogarty TW-06324.