Serotype 5 and 8 capsular polysaccharides are the two most common capsule serotypes produced from clinical isolates of Staphylococcusaureus. Strains of both of these capsule types have very thin capsules under laboratory conditions which are referred to as microcapsules. Previous studies, including some by this applicant, have examined capsule genes of the heavily encapsulated type 1 staphylococci. Detailed analyses of the capsule genes of the type 5 and type 8 microcapsules are not available. The proposed study will examine the structure of the genes required for production of the type 8 capsule, their regulation of expression, and their inferred products. These studies will complement the work that Dr. Lee has already done on type 1 capsule genes and studies underway by others on the type 5 capsule genes. Dr. Lee has already cloned overlapping fragments of 30 kb of contiguous DNA unique to the type 8 strain. The genes required for type 8 capsule expression were found in fragments covering about 12 kb of this region. These findings have put him ahead of the group at Harvard studying the type 5 capsule genes. The genes in this 12 kb region will be cloned and sequenced.The genetic organization of this locus will be investigated by northern blot and complementation studies. Transcription start sites and ends will be revealed by S1 mapping. Gene function will be investigated using capsule production and export as readout systems. The role of the type 8 capsulein virulence will be investigated using a mouse lethality model and an in vitro phagocytosis assay. It is anticipated that these studies will lead to better strategies for protection against this human pathogen.
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