Despite the advance of newer antimicrobial therapy, S. aureus continues to be a major pathogen in both community-acquired and nosocomial infections. One reason is that S. aureus is very adept in developing resistance to multiple antibiotics. Unfortunately, there is very little experimental evidence to justify a direct vaccine strategy at present. An alternative approach will be to target the genetic control apparatus of virulence determinants in an attempt to develop new strategies to control serious S. aureus infections. We have identified by transposon mutagenesis a locus on the S. aureus chromosome, designated sar, that is involved in the regulation of several extracellular proteins some of which are involved in pathogenesis. Recent studies have provided some insights into a possible mechanism by which exoprotein synthesis is controlled by sar. In particular, we have found that a functioning sar locus is required for the nominal transcription of RNAII and RNAIII (P2 and P3), the agr regulatory molecules. As agr is a pleiotropic regulator of exoprotein synthesis, we hypothesize that the sar locus may regulate exoprotein transcription via the control of agr. To test this hypothesis, the sar locus will be cloned and sequenced. Transcriptional analysis of ORF(s) coupled with hybridization studies with selected sar probes will allow us to discern the extent of individual transcripts and the corresponding gene products within the locus. Whether the sar locus represents a novel regulatory system will be determined by sequence analysis. By constructing a series of deleted mutations within the sar locus, we will be able to determine the minimum genetic requirement of this locus necessary for the normal agr function. To evaluate if the gene product(s) of sar behave as DNA binding protein(s), some of these proteins will be purified by affinity chromatography from the cell lysate of an expression vector. The purified protein(s) will be used to examine specific sar protein - agr promoter interactions by both gel shift and footprinting assays. The results of these studies will furnish important information on the mechanism of exoprotein control by the sar locus. This knowledge is indispensable for the design of synthetic analogs to interfere with the expression of virulence genes controlled by the sar locus in the future.
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