application abstract): Human infections caused by Gram-positive bacteria often present a serious therapeutic challenge due to the appearance of antibiotic-resistant strains. Of particular concern are Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis, Gram-positive organisms that are by far the most common cause of bacterial infections in American hospitals. These nosocomial pathogens have developed resistance mechanisms to all known antibiotic regimens and the development of novel targets for antimicrobial therapy is urgently needed. Surface proteins of Gram-positive organisms fulfill many important functions during the pathogenesis of human infections. This proposal describes a novel anchor mechanism for surface proteins in Staphylococcus aureus that may serve as a target for antibacterial therapy. Staphylococcal surface proteins harbor a C-terminal sorting signal that is proteolytically cleaved between the threonine (T) and the glycine (G) of its LPXTG sequence motif. The carboxyl threonine is subsequently amide linked to the free amino group of the peptidoglycan crossbridge, thus anchoring the C-terminal end of the polypeptide chain to the staphylococcal cell wall. The elements of this anchor mechanism, the LPXTG motif in the sorting signal and the free amino group in the peptidoglycan crossbridge, are conserved in many different Gram positive species, suggesting that surface protein anchoring occurs by a universal mechanism. To determine whether or not this anchor mechanism is universal in Gram-positive bacteria, Dr. Schneewind will investigate surface protein anchoring in two other Gram-positive organisms: E. faecalis and L. monocytogenes. This proposal describes experiments that investigate the sorting signals, their proteolytic cleavage at the LPXTG motif and the chemical linkage between surface proteins and the peptidoglycan of E. faecalis and L. monocytogenes. Furthermore, he will characterize the peptido glycan substrate required to anchor surface proteins to the cell wall of Gram-positive bacteria. In vitro assays that allow the specific measurement of surface protein anchoring will be established and employed to isolate the catalytic machinery of S. aureus responsible for this reaction. These in vitro assays will also be used to screen for substances that may inhibit surface protein anchoring at the LPXTG motif and thereby disrupt the pathogenesis of infections caused by Gram- positive bacteria.
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