Streptococcus mutans possesses three distinct glucosyltransferases (GTFs) and at least three different nonenzymatic glucan binding proteins (GBPs). The precise contributions of each GTF and GBP to plaque development are unknown. Recently we showed that the loss of a glucan-binding protein (GbpA), accomplished through allelic replacement, dramatically altered the architecture of the biofilm formed by cultures of S. mutans. Based on these data we engineered strains with inactivation in genes encoding other extracellular proteins. The loss of GbpC, FruA, or P1 also resulted in changes in the biofilm architecture. To explain these observations we posit that the loss of an extracellular protein may result in specific physical and/or biochemical changes to the organism, or to its immediate environment that affect the structure of the mature biofilm formed by that organism.
The Specific Aims of this application are designed to test these possibilities. Since work with the GbpA has progressed the furthest, investigations of specific GbpA properties are also included in the Aims.
Aim 1) Examine the physical properties of the knockout strains including hydrophobicity, surface charge, and their interactions with cations such as calcium which are important in the development of a biofilm.
Aim 2) Use a flow chamber to examine the events associated with the initial attachment of the knockout strains to the substratum and correlate the results with the structure of the mature biofilms.
Aim 3) Examine how the ratio of GbpA to glucan synthesis correlates with the structure of the biofilm.
Aim 4) Delete or replace the amino terminal domain (non glucan-binding domain) of GbpA and examine how this change affects the phenotypic properties of Streptococcus mutans compared to the wild-type and GbpA knockout strains.
Aim 5) Express phenotypic properties of formation.
Aim 6) Utilize architecture influence gene GbpA in the heterologous host Streptococcus gordonii and examine the organism in the context of sucrose-dependent adhesion and biofilm microarrays to determine how the loss of extracellular proteins and biofilm expression.
