Summary Staphylocuccus aureus is a significant human pathogen that causes a wide range of diseases, some of which are serious and difficult to treat. Current treatment methods are largely limited to antibiotic therapy. However, S. aureus is capable of forming biofilm on implants or tissues rendering the bacteria resistance to antibiotics and to host immune defense system without acqiuring genetic resistance traits. Thus, biofilm formation often leads to treament failures. Biofilms are bacterial communities consist of multiple layers of bacterial cells encased within an extracellular matrix that keeps the bacteria within the biofilm structure. To date, staphylococcal biofilm matrix has been shown to consist of polysaccharides, proteins and extracellular DNA but the composition is not been fully defined. Understanding biofilm matrix composition is therefore critical to unveil biofilm formaiton mechanism and provide potential targets for therapeutic or diagnostic development. Diverse biofilm matrix proteins have been reported but only very few species of matrix polysaccharide have been identified. We have shown that mutations in an previously uncharacterized gene cluster of putative polysaccharide sythesis genes in the S. aureus genome reduced biofilm formation. These results indicate that the gene cluster is involved in the production of a novel polysaccharide, which contributes to S. aureus virulence by promoting biofilm formation. Thus, the goal of this application is to verify and study this novel polysaccharide. To this end, we will molecularly characterize the gene cluster and biochemically identify the novel polysaccharide in Aim 1 and study the role of this polysacharide in biofilm formation and in virulence in an implant bone infection model in Aim 2.