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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI038897-05
Application #
6149801
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1996-02-01
Project End
2001-01-31
Budget Start
2000-02-01
Budget End
2001-01-31
Support Year
5
Fiscal Year
2000
Total Cost
$255,738
Indirect Cost
Name
University of California Los Angeles
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Winstel, Volker; Missiakas, Dominique; Schneewind, Olaf (2018) Staphylococcus aureus targets the purine salvage pathway to kill phagocytes. Proc Natl Acad Sci U S A 115:6846-6851
Yu, Wenqi; Missiakas, Dominique; Schneewind, Olaf (2018) Septal secretion of protein A in Staphylococcus aureus requires SecA and lipoteichoic acid synthesis. Elife 7:
Bobrovskyy, Maksym; Willing, Stephanie E; Schneewind, Olaf et al. (2018) EssH peptidoglycan hydrolase enables Staphylococcus aureus type VII secretion across the bacterial cell wall envelope. J Bacteriol :
Sun, Yan; Emolo, Carla; Holtfreter, Silva et al. (2018) Staphylococcal protein A contributes to persistent colonization of mice with Staphylococcus aureus. J Bacteriol :
Ohr, Ryan Jay; Anderson, Mark; Shi, Miaomiao et al. (2017) EssD, a Nuclease Effector of the Staphylococcus aureus ESS Pathway. J Bacteriol 199:
Anderson, Mark; Ohr, Ryan Jay; Aly, Khaled A et al. (2017) EssE Promotes Staphylococcus aureus ESS-Dependent Protein Secretion To Modify Host Immune Responses during Infection. J Bacteriol 199:
Nygaard, Tyler K; Kobayashi, Scott D; Freedman, Brett et al. (2016) Interaction of Staphylococci with Human B cells. PLoS One 11:e0164410
Missiakas, Dominique; Schneewind, Olaf (2016) Staphylococcus aureus vaccines: Deviating from the carol. J Exp Med 213:1645-53
Chan, Yvonne G Y; Frankel, Matthew B; Missiakas, Dominique et al. (2016) SagB Glucosaminidase Is a Determinant of Staphylococcus aureus Glycan Chain Length, Antibiotic Susceptibility, and Protein Secretion. J Bacteriol 198:1123-36
Kim, Hwan Keun; Falugi, Fabiana; Missiakas, Dominique M et al. (2016) Peptidoglycan-linked protein A promotes T cell-dependent antibody expansion during Staphylococcus aureus infection. Proc Natl Acad Sci U S A 113:5718-23

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