Related Experiment Videos
Construction and characterization of isogenic mutants of Streptococcus mutans deficient in major surface protein
S F Lee1, A Progulske-Fox, G W Erdos
1Department of Oral Biology, University of Florida, Gainesville 32610.
Abstract:
The gene (spaP) coding for the Streptococcus mutans major surface protein antigen P1 (or I/II) has been cloned into Escherichia coli (S. F. Lee, A. Progulske-Fox, and A. S. Bleiweis, Infect. Immun. 56:2114-2119, 1988). In the present study, this gene has been disrupted in vitro by insertional inactivation with pVA981, which carries a Tcr marker, and transformed into S. mutans NG8 (serotype c) by electroporation. Upon homologous recombination, the defective spaP was integrated into the genome as demonstrated by Southern hybridization analysis. One Tcr mutant, designated 834, selected by its nonreactivity with anti-P1 monoclonal antibodies, was found to lack the cell surface fuzzy layer which was clearly present on the parent cells. Analysis of extracellular fluids, sodium dodecyl sulfate-solubilized membranes, and cytoplasmic fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that 834 had protein profiles identical to the parent. However, a 185-kilodalton protein which reacts with anti-P1 antibodies was missing from the wall of 834, suggesting that spaP has been specifically inactivated. This mutant displayed levels of glucosyltransferase and fructosyltransferase activities similar to those of the parent. It was much less hydrophobic than the parent. S. mutans NG8 aggregated readily in the presence of clarified whole saliva or a high-molecular-weight salivary agglutinin. This strain also adhered to agglutinin-coated hydroxyapatite. The P1-negative mutants, however, did not display these two properties, suggesting that P1 may play a role in saliva-mediated aggregation and adherence.
Insights
Streptococcus mutans major surface protein antigen P1 (spaP) gene inactivation resulted in a mutant lacking the cell surface fuzzy layer. This P1-negative mutant showed reduced saliva-mediated aggregation and adherence, suggesting P1’s role in these processes.
Area of Science:
- Microbiology
- Molecular Biology
- Oral Health Research
Background:
- Streptococcus mutans is a primary agent of dental caries.
- The major surface protein antigen P1 (spaP) is crucial for S. mutans virulence.
- Understanding spaP's function is key to developing anti-caries strategies.
Purpose of the Study:
- To investigate the role of the spaP gene in Streptococcus mutans.
- To create and characterize a spaP-deficient mutant of S. mutans.
- To determine the involvement of P1 in bacterial aggregation and adherence.
Main Methods:
- In vitro gene disruption of spaP using insertional inactivation with pVA981.
- Electroporation of the disrupted gene into S. mutans NG8.
- Southern hybridization to confirm homologous recombination.
- Phenotypic analysis of the mutant, including surface structure, protein profiles, enzyme activity, hydrophobicity, aggregation, and adherence assays.
Main Results:
- A tetracycline-resistant (Tcr) mutant (834) lacking the cell surface fuzzy layer was generated.
- The mutant 834 was confirmed to be deficient in the 185-kilodalton P1 antigen.
- Mutant 834 exhibited similar glucosyltransferase and fructosyltransferase activities but reduced hydrophobicity compared to the parent strain.
- P1-negative mutants failed to aggregate in saliva and adhere to agglutinin-coated hydroxyapatite.
Conclusions:
- The spaP gene is essential for the production of the P1 antigen in Streptococcus mutans.
- The P1 antigen plays a significant role in saliva-mediated aggregation and adherence of S. mutans.
- Targeting P1 could be a potential strategy for preventing dental caries.