Attenuation of penicillin resistance in a peptidoglycan O‐acetyl transferase mutant of Streptococcus pneumoniae

Abstract

SummaryThe level of penicillin resistance in clinical isolates of Streptococcus pneumoniae depends not only on the reduced affinity of penicillin binding proteins (PBPs) but also on the functioning of enzymes that modify the stem peptide structure of cell wall precursors. We used mariner mutagenesis in search of additional genetic determinants that may further attenuate the level of penicillin resistance in the bacteria. A mariner mutant of the highly penicillinresistant S. pneumoniae strain Pen6 showed reduction of the penicillin minimum inhibitory concentration (MIC) from 6 to 0.75 mg ml -1 . Decrease in penicillin MIC was also observed upon introduction of the mutation (named provisionally adr, for attenuator of drug resistance) into representatives of major epidemic clones of penicillin-resistant pneumococci. Attenuation of resistance levels was specific for b-lactams. The adr mutant has retained unchanged (low affinity) PBPs, unaltered murM gene and unchanged cell wall stem peptide composition, but the mutant became hypersensitive to exogenous lysozyme and complementation experiments showed that both phenotypes -reduced resistance and lysozyme sensitivity -were linked to the defective adr gene. DNA sequence comparison and chemical analysis of the cell wall identified adr as the structural gene of the pneumococcal peptidoglycan O-acetylase.

O-Acetylation of Peptidoglycan in Gram-negative Bacteria

“…As first discovered in Staphylococcus aureus (15) and later found in Streptococcus pneumoniae (16), a single integral membrane enzyme, OatA, would appear to both translocate acetate from the cytoplasm and then transfer it to PG, but this has not been demonstrated biochemically. Indeed, despite considerable effort by ourselves and others, including those investigating the Grampositive bacteria (15,16), an in vitro kinetic assay has yet to be developed for any PG O-acetyltransferase and thus no quantitative data have been reported. This is understandable given the complexities that are associated with accommodating in vitro an integral membrane protein acting in concert with a peripheral membrane protein for the modification of a totally insoluble substrate.…”

“…This would require that a source of acetate from within the cytoplasm be transported across the cytoplasmic membrane prior to being attached to the newly ligated muramoyl residues in PG. An O-acetyltransferase (OatA) that serves these functions has been discovered in some Gram-positive bacteria (15,16), but neither a homolog nor a paralog have been found in Gramnegative bacteria (2,3).…”

SpxB Regulates O-Acetylation-dependent Resistance of Lactococcus lactis Peptidoglycan to Hydrolysis

“…As expected, spxB itself was among the genes showing markedly increased expression in VES3910, thus confirming that multicopy cloning leads to its overexpression (20-fold). Among the up-regulated genes, only yvhB had a predicted function that could be directly related to the cell wall and consequently to the lysozyme-resistant phenotype; the encoded protein showed 30% identity to OatA, the PG O-acetylase of S. aureus (19), and 23% identity to its pneumococcal ortholog Adr (20). On the basis of this observation and enzyme activity assays detailed below, we assigned yvhB the name oatA.…”

“…The gene oatA (encoding PG O-acetyltransferase) was identified in Staphylococcus aureus; an oatA mutant showed increased sensitivity to lysozyme (19). The analogous mutant was later isolated in S. pneumoniae and named adr for its attenuation of the drug (penicillin) resistance phenotype (20). The concerted action of PG O-acetyltransferases and O-acetylpeptidoglycan esterases was proposed to participate in the control of PG degradation (21), although the genetic mechanisms of such regulation remain unknown.…”

Nod2 sensing of lysozyme-digested peptidoglycan promotes macrophage recruitment and clearance of S. pneumoniae colonization in mice

“…Because of the abundance of lysozyme on host surfaces, many mucosal pathogens have acquired resistance to lysozyme-mediated degradation by modifying residues of their peptidoglycan backbone (26). S. pneumoniae uses two distinct peptidoglycan modifications to prevent lysozyme digestion; deacetylation of GlcNAc by PgdA (27,28) and O-acetylation of N-acetylmuramic acid by Adr (29). A mutant strain lacking both of these modifications (pgdA -adr -) was hypersensitive to hydrolysis by recombinant human lysozyme, and clearance was accelerated (30).…”