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 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.

Uncovering the activities, biological roles, and regulation of bacterial cell wall hydrolases and tailoring enzymes

“…The clear preference of WTA ligases for modifying nascent peptidoglycan is consistent with their membrane-anchored location. S. aureus OatA is also anchored in the membrane and may also display a preference for uncross-linked peptidoglycan, although detailed studies of its substrate preferences that could help elucidate when it acts have not been done (127). The studies described here exemplify how using defined substrates that mimic peptidoglycan intermediates can provide temporal information on cell wall assembly.…”

“…The Gram-negative O-acetyltransferase PatB can only acetylate substrates that are at least 3 sugars in length (125,126). The S. aureus O-acetyltransferase OatA prefers to acetylate uncross-linked glycan strands containing intact stem pentapeptides as opposed to those with trimmed peptides (127). Whether OatA can transfer acetyl groups to cross-linked peptidoglycan was not tested.…”

Environment Shapes the Accessible Daptomycin Resistance Mechanisms inEnterococcus faecium

“…Seven different oatA mutations were observed between Bioreactor Population 1 and Population 2, six of which resulted in a truncation of the catalytic C-terminal domain (predicted residues 460-628). OatA catalyzes the acetylation of the C-6 hydroxyl group of N -acetylmuramic acid (MurNAc), which contributes to lysozyme resistance and is linked to increased pathogenesis in many species (45–47). Here, two different mutations were identified in end-point isolates: E480* and E460* (Table 2).…”

Genetic Determinants Enabling Medium-Dependent Adaptation to Nafcillin in Methicillin-Resistant Staphylococcus aureus

“…Mutations in mecA were all located in the active site of PBP2a (87), suggesting an alteration in the target for nafcillin, and thus enabling transpeptidase activity to proceed. Mutations affecting synthesis and acquisition of branched-chain amino acids, as well as biosynthesis of peptidoglycan and its precursors potentially suggest a reorganization of metabolic activity more representative of host infection (71,81,88). Importantly, mutations in oatA have been previously shown to have significant impact on S. aureus interaction with the host, potentially allowing enhanced intracellular survival to escape from largely extracellularly acting antibiotics like beta-lactams.…”