Role of P. gingivalis PtpA Tyrosine Phosphatase in Exopolysaccharide Production

Objectives: The PtpA tyrosine phosphatase of P. gingivalis in involved in regulation heterotypic P. gingivalis-S. gordonii biofilm development, however the physiological pathways controlled by this enzyme have not been determined. The objective of this study was to examine the role of PtpA in production of exopolysaccharide and in expression of exopolysaccharide genes by P. gingivalis. Methods: P. gingivalis strain 33277, along with a ptpA mutant, a ptpA mutant complemented in trans with the wild type ptpA allele, and a ptpA mutant complemented with a catalytic domain mutant of ptpA were labeled with Syto-17 and deposited in multi-well plates. Exopolysaccharide was stained with FITC-Concanavalin A and FITC-WGA. Exopolysaccharide production was calculated by the ratio of FITC:Syto-17 fluorescence. mRNA levels of exopolysaccharide related genes were measured by quantitative RT-PCR. Results: Loss of PtpA resulted in a 2.5 fold increase in exopolysaccharide production (p<0.01). Similarly complementation of the PtpA mutant with PtpA containing a C10S substitution in the catalytic domain resulted in an increase in exopolysaccharide (p<0.01). In contrast, complementation of the mutant with the wild type ptpA allele restored exopolysaccharide to wild type levels. Loss of PtpA or of its catalytic activity caused differential regulation of several genes involved in polysaccharide production. Upregulated (p<0.01) in the absence of PtpA were: PG0436, a putative capsular polysaccharide transport protein; rfbD a putative dTDP-4-dehydrorhamnose 3,5-epimerase, involved in the biosynthesis and degradation of surface polysaccharides; porS and porR, involved in polysaccharide biosynthesis and surface retention; and wbbL a putative rhamnosyltransferase present in a locus that also contains several glucosyltransferases. Conclusions: The PtpA tyrosine phosphatase of P. gingivalis downregulated exopolysaccharide production through transcriptional regulation of several genes involved in biosynthesis and transport. Supported by NIDCR DE12505 and T32DE007200