PGA exopolysaccharide in biofilm formation and pathogenicity of Aggregatibacter a

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DESCRIPTION (provided by applicant): Aggregatibacter actinomycetemcomitans (Aa) and several pathogenic bacteria attach to abiotic surfaces and produce exopolysaccharide that immobilize the bacterial cells on these colonized surfaces. The exopolysaccharide produced by Aa is a homopolymer of ¿-1,6-linked N-acetyl-D-glucosamine (GlcNAc) units. Aa produces this exopolysaccharide (PGA) utilizing a four-gene operon homologous to the pga of Escherichia coli, hms of Yersinia pestis, and ica of Staphylococcus epidermidis. In Aa, the operon pgaABCD carries a deacetylase enzyme (EC 3.5.1.41) encoded by pgaB. Our Preliminary Data show that the enzyme PgaB has the ability to remove acetyl groups from GlcNAc of PGA. More intriguingly, we discovered that PgaB detaches preformed biofilms and inhibits biofilm formation of Aa, Actinobacillus pleuropneumoniae and S. epidermidis when added to the growth medium. We have previously shown that an enzyme dispersin B (DspB) also from Aa prevents the surface attachment of several Gram negative as well as Gram-positive bacterial species through depolymerization of PGA. Unlike DspB, the newly discovered enzyme PgaB is a deacetylase of GlcNAc residues in PGA and through deacetylation alters the charge state of PGA (enrichment of positive charges). This enzyme activity suggests that the mechanism of detachment/inhibition might be through the increased repulsive interactions on the PGA polymer. Importantly, the role of deacetylation in biofilm removal or formation is understudied in these organisms, especially in Aa. We will use PgaB from Aa as a model system to understand these processes. Our long-range goal is to understand the role of PGA in Aa pathogenicity and how the deacetylase activity of PgaB is central to this role. We will use both in vitro as well as in vivo models to tet the overall hypothesis that PGA plays a critical role in Aa virulence. Specifically, the following aims will be studied: Specific Aim 1. Demonstrate that PGA deacetylating ability of the enzyme PgaB is critical for its antibiofilm activity. Specific Aim 2a. Demonstrate that the status and degree of acetylation of PGA contributes to the biofilm formation in Aa. Specific Aim 2b. Demonstrate that the exopolysaccharide PGA contributes to Aa pathogenicity.