Enterococci are important causative agents of a particularly serious form of bacterial endocarditis, as well as other infections. They are now among the top three nosocomial pathogens in the US. Infections caused by the enterococci are notoriously recalcitrant to antibiotic treatment, and relatively little is known about the genetic and molecular basis for the virulence of this group of bacteria. This proposal represents a multidisciplinary effort of two investigators, an enterococcal geneticist, and medical microbiologist, to elucidate the role of specific cell wall components of the Gram-positive microbe, Enterococcus faecalis in the pathogenesis of endocarditis. The investigators will be especially interested in the Lipoteichoic Acid (LTA) component of the cell wall of these organisms. Purified enterococcal LTA has recently been shown to have some important biological effects, such as induction of cytokine production and enhancement of the activity of protein toxins, which are reminiscent of the activities of the Lipopolysaccharide (endotoxin) component of Gram-negative bacteria. LTA has also been suggested as an important adhesion, but direct proof of its role in bacterial pathogenesis has been hampered by the lack of a genetic system to manipulate LTA expression in isogeneic bacterial strains. The investigators will take advantage of recently isolated cell wall mutants defective in expression of a surface receptor (Enterococcal Binding Substance-EBS) involved in bacterial conjugation, and with structural alterations in LTA, to determine the role of LTA and other cell wall components in Enterococcal virulence. Specific objectives include: 1) Complementation and sequencing analysis of three genetic loci involved in expression of wild-type EBS, and construction of isogeneic strains expressing various combinations of wild-type and mutant EBS loci. 2) Biochemical analysis of the cell wall components of E. faecalis strains constructed in Objective #1, in order to identify the genes directly involved in biosynthesis of LTA and other wall components. 3) Analysis of the pathogenic properties of the strains described above in a rabbit model for endocarditis, and analysis of the ability of purified LTAs from these strains to induce cytokine production in vitro. The phenotypic effects of EBS mutations on expression of two other known enterococcal virulence factors will also be determined.
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