Staphylococcus aureus is an opportunistic bacterial pathogen responsible for a diverse spectrum of human and animal diseases. Although few S. aureus isolates are highly encapsulated, ~90% of strains are microencapsulated, i.e., they produce uronic acid-containing extracellular polysaccharides that can be detected biochemically or serologically in bacterial cell extracts. Three are ll capsular serotypes of S. aureus; types 5 and 8 predominate, comprising ~22% and ~53% of isolates examined. The biologic relevance of type 5 and 8 microcapsules is unknown since microencapsulated strains are isolated from both pathologic and commensal sources. To determine whether type 5 and 8 microcapsules promote staphylococcal virulence, isogenic, capsule-negative mutants will be created from prototype strains of S. aureus by transposon mutagenesis. The relative virulence of the parent strains and the unencapsulated mutants will be examined using well-established mouse models of staphylococcal infection. Furthermore, the antiphagocytic properties of the type 5 and 8 microcapsules will be examined using an in vitro opsonophagocytic killing assay. Quantitative comparisons will be made of the levels of complement and specific antibodies needed to opsonize the isogenic strains for phagocytosis. Mutants negative for type 8 capsule expression will be used to identify genes responsible for synthesis of the S. aureus capsule. This will be accomplished by cloning DNA from the type 8 capsule-negative mutant into Escherichia coli and selecting for the antibiotic-resistance marker of the inserted transposon. DNA sequences flanking the transposon (presumably encoding proteins involved in capsule expression) will be used to probe cosmid libraries constructed from a type 8 strain. E. coli bearing cosmid clones that hybridize to the flanking sequences should contain large segments of DNA involved in S. aureus capsule expression. The cloned genes will be used to restore capsule expression in mutant S. aureus strains and to probe genomic digests from the ll S. aureus capsule serotypes. Tn5 mutagenesis and complementation studies will identify gene clusters on the cloned DNA fragments that are essential for capsule expression. These analyses will further our understanding of the genetic mechanisms underlying capsule expression and regulation in S. aureus.
