Despite the advance of antimicrobial therapy, Staphylococcus aureus continues to be a major pathogen, both in the community and in hospital settings. The recent emergence of multiple antibiotic resistance, including that of vancomycin, is beginning to pose significant public health problems. A major concern with prior pathogenic studies of S. aureus is that they are largely conducted with cells grown in culture media. As S. aureus can alter its gene expression in response to changing environments, it is our hypothesis that gene expression in vivo likely differs from those in vitro. To address this issue, an E. coli-S. aureus shuttle vector containing a promoterless gfpuv reporter gene preceded by a polylinker region was constructed. Preliminary data indicated that the expression of Green Fluorescent Protein (GFP) from this vector in S. aureus is dependent on the strength of the upstream promoter, thus rendering this vector useful for studying gene expression in vivo. In this proposal, one major goal is to optimize GFP expression of this shuttle vector in a S. aureus host to evaluate virulence gene expression in an acute rabbit endocarditis (IE) model, as well as a chronic guinea pig tissue cage model. By assessing serial GFP expression of selected virulence determinants (derived from in vitro and limited in vivo studies) in target tissues, the temporal, sequential and tissue-targeting steps in the infectious process will be defined, thereby leading to a better understanding of the molecular physiology of infection at the level of gene expression in various tissues. To search for novel genes preferentially expressed in vivo, a shuttle vector library, consisting of random chromosomal fragments, representing possible promoter elements, upstream of an optimized gfp variant (excitation maxima 488nm) reporter gene, will be constructed in S. aureus. The S. aureus plasmid library will be used to infect Swiss mice IP and rabbits with experimental IE. Applying the differential fluorescence induction (DFI) strategy, promoter elements that are preferentially activated in vivo (i.e., green fluorescent colonies) will be sorted by a Fluorescent Activated Cell Sorter. By analyzing these promoters and their respective genes, novel genes that are preferentially expressed in vivo will be identified and characterized. Analysis of these genes and their gene product may possibly lead to new insights into the pathogenesis of S. aureus infections. Our long-term goal is to examine some of these unique gene products for the development of vaccine and novel antimicrobial strategies against S. aureus.
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