The effect of MurM and a branched cell wall structure on penicillin resistance in <i>Streptococcus pneumoniae</i>

The aminoacyltransferase MurM is an important penicillin resistance determinant in <i>Streptococcus pneumoniae</i>. This enzyme attaches a serine or alanine to the side chain of lysine, the third residue of the pentapeptide of lipid II, resulting in branched muropeptides that can be crosslinked to stem peptides in peptidoglycan by penicillin binding proteins (PBPs). Deletion of <i>murM</i> results in only linear muropeptides, and more importantly, a significant reduction in resistance. Highly penicillin-resistant pneumococci express low-affinity PBPs, an altered MurM protein, and possess a highly branched cell wall. It has therefore been hypothesized that MurM, and thus branched muropeptides, are essential for resistance because they are better substrates for low-affinity PBPs. In this study, we found that neither the version of <i>murM</i> nor elevated levels of cell wall branching affected resistance levels. To further support this, we investigated whether branched muropeptide substrates compete better than linear versions with penicillin at the active site of low-affinity PBPs and quantified changes to the stem peptide composition of the resistant Pen6 strain in response to subinhibitory concentrations of penicillin. We found that the level of cell wall branching decreased during penicillin exposure. Together, our results do not support the idea that elevated levels of branched muropeptides (more active MurM) are important for either the function of low-affinity PBPs or the cell's response to penicillin. Nevertheless, since a functional MurM enzyme is important for resistance, we speculate that it might indirectly influence other functions related to cell wall synthesis and remodeling needed for a resistant phenotype.IMPORTANCEA fundamental understanding of the mechanisms behind antibiotic resistance is needed to find strategies to extend the clinical relevance of existing drugs. This study explores the relationship between cell wall composition and penicillin resistance in <i>Streptococcus pneumoniae</i>. Here, we confirm that branched peptide crosslinks in the cell wall are crucial for resistance but found no correlation between elevated branching levels and resistance. Our data suggest that the function of low-affinity penicillin binding proteins is not influenced by the lack of branched cell wall precursors. Instead, a branched cell wall might contribute to resistance via other cell wall biosynthesis and remodeling mechanisms. These insights could offer new perspectives on why a branched cell wall is important for penicillin resistance in pneumococci.