Molecular characterization of the dextran-binding lectin B gene dblB of Streptococcus criceti in Streptococcus mutans strain GS-5 with mutations in both gbpC and spaP genes.

Haruki Tamura,Arisa Yamada,Hirohisa Kato

doi:10.1266/ggs.89.41

Abstract

Streptococcus mutans, a cariogenic agent, has a glucan-binding protein gene, gbpC, and S. criceti possesses four gbpC homologs, including dblA and dblB, as does S. sobrinus. The S. criceti dblB gene encodes a 1,717-amino-acid protein having two repetitive alanine-rich and proline-rich regions and an LPXTG motif, which is recognized by the sortase SrtA, near the C terminus. Reverse transcription-PCR analysis indicated no cotranscription of the dblA and dblB genes of S. criceti. As we could not obtain a dblB mutant of S. criceti, the dblB gene was characterized in S. mutans strain GS-5, which has genetic mutations in both gbpC and spaP genes and shows an inability to agglutinate triggered by dextran. A dextran-induced agglutination assay showed that S. mutans cells carrying dblB agglutinated in the presence of dextran. A hydrophobicity assay showed that the cells containing dblB were hydrophobic. A biofilm formation assay showed that the dblB gene was associated with biofilm formation by cells cultivated in brain heart infusion broth supplemented with glucose and maltose, but not sucrose. Nucleotide sequence analysis of the S. criceti strains studied revealed a frameshift mutation in the srtA gene encoding sortase, but intact dblA and dblB genes were found in dextran-induced agglutination-negative strains, whereas intact dblA, dblB and srtA genes were found in dextran-induced agglutination-positive strains. These results suggest the cell-surface localization of dblA and dblB gene products by SrtA and the responsibility of dblB for dextran-induced agglutination, cell-surface hydrophobicity and biofilm formation in S. criceti.

Highlights

Streptococcus mutans is a cariogenic agent in humans and possesses cell-surface proteins such as glucan-binding protein C (GbpC) (Sato et al, 1997) and SpaP, which contain an LPXTG motif recognized by sortase SrtA for anchorage to the cell wall (Igarashi, 2004)
Reverse transcription-PCR (RT-PCR) analysis of dblA and dblB genes Nucleotide sequences of the dblA and dblB genes of S. criceti HS-6T were retrieved from DDBJ/EMBL/GenBank databases
As dblA and dblB genes are arranged in tandem (Fig. 1A), their expression was examined by RT-PCR

Summary

Introduction

Streptococcus mutans is a cariogenic agent in humans (for review, see Colby and Russell, 1997) and possesses cell-surface proteins such as glucan-binding protein C (GbpC) (Sato et al, 1997) and SpaP ( known as antigen I/II, PAc, P1 and antigen B) (for review, see Brady et al, 2010), which contain an LPXTG motif recognized by sortase SrtA for anchorage to the cell wall (Igarashi, 2004). GbpC is responsible for dextran-dependent agglutination in S. mutans grown under stress conditions such as at a subinhibitory concentration of tetracycline, 4% ethanol, or 42°C incubation (Sato et al, 1997). S. mutans GS-5 fails to agglutinate in the presence of dextran owing to a nonsense mutation in the gbpC gene (Sato et al, 2002). A frameshift mutation in the spaP gene has been found in one strain of GS-5 (Murakami et al, 1997), but intact spaP has been identified in other strains of GS-5 (Sato et al, 2002; Biswas and Biswas, 2012)

Methods

Results

Conclusion