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Title: In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA)

Abstract

The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA) represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain. We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groupsmore » from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens.« less

Authors:
 [1];  [1];  [2]; ORCiD logo [3]; ORCiD logo [4]; ORCiD logo [5]; ORCiD logo [5]; ORCiD logo [5];  [2]; ORCiD logo [1]
  1. Univ. of Guelph, ON (Canada)
  2. Univ. of Toronto, ON (Canada); The Hospital for Sick Children, Toronto, ON (Canada)
  3. Univ. of Birmingham (United Kingdom)
  4. Brookhaven National Lab. (BNL), Upton, NY (United States)
  5. Univ. of Warwick, Coventry (United Kingdom)
Publication Date:
Research Org.:
Brookhaven National Lab. (BNL), Upton, NY (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1425067
Report Number(s):
BNL-203203-2018-JAAM
Journal ID: ISSN 1553-7374
Grant/Contract Number:  
SC0012704
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
PLoS Pathogens
Additional Journal Information:
Journal Volume: 13; Journal Issue: 10; Journal ID: ISSN 1553-7374
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; Bacterial cell wall; Peptidoglycan; O-Acetylation; O-Acetyltransferase; X-Ray crystallography; Mechanism of action

Citation Formats

Sychantha, David, Jones, Carys S., Little, Dustin J., Moynihan, Patrick J., Robinson, Howard, Galley, Nicola F., Roper, David I., Dowson, Christopher G., Howell, P. Lynne, and Clarke, Anthony J. In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA). United States: N. p., 2017. Web. doi:10.1371/journal.ppat.1006667.
Sychantha, David, Jones, Carys S., Little, Dustin J., Moynihan, Patrick J., Robinson, Howard, Galley, Nicola F., Roper, David I., Dowson, Christopher G., Howell, P. Lynne, & Clarke, Anthony J. In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA). United States. doi:10.1371/journal.ppat.1006667.
Sychantha, David, Jones, Carys S., Little, Dustin J., Moynihan, Patrick J., Robinson, Howard, Galley, Nicola F., Roper, David I., Dowson, Christopher G., Howell, P. Lynne, and Clarke, Anthony J. Fri . "In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA)". United States. doi:10.1371/journal.ppat.1006667. https://www.osti.gov/servlets/purl/1425067.
@article{osti_1425067,
title = {In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA)},
author = {Sychantha, David and Jones, Carys S. and Little, Dustin J. and Moynihan, Patrick J. and Robinson, Howard and Galley, Nicola F. and Roper, David I. and Dowson, Christopher G. and Howell, P. Lynne and Clarke, Anthony J.},
abstractNote = {The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA) represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain. We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens.},
doi = {10.1371/journal.ppat.1006667},
journal = {PLoS Pathogens},
issn = {1553-7374},
number = 10,
volume = 13,
place = {United States},
year = {2017},
month = {10}
}

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    Works referencing / citing this record:

    Peptidoglycan O-Acetylation as a Virulence Factor: Its Effect on Lysozyme in the Innate Immune System
    journal, July 2019


    Peptidoglycan O-Acetylation as a Virulence Factor: Its Effect on Lysozyme in the Innate Immune System
    journal, July 2019