Winther, A. R. | bioRxiv Science

bioRxiv2025

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

The aminoacyltransferase MurM is an important penicillin resistance determinant in Streptococcus pneumoniae. This enzyme attaches a serine or alanine to the dipeptide side chain of lipid II, resulting in branched muropeptides that can be crosslinked to stem peptides in the peptidoglycan layer by penicillin binding proteins (PBPs). Deletion of murM results in only linear muropeptides, and more importantly a significant reduction in resistance. Highly penicillin resistant pneumococci are known to express low-affinity PBPs, an altered MurM protein, and possess a highly branched cell wall structure. 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 murM nor elevated levels of cell wall branching affected the resistance level. 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 cells 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 remodelling 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 Streptococcus pneumoniae. 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 remodelling mechanisms. These insights could offer new perspectives on why a branched cell wall is important for penicillin resistance in pneumococci.

microbiology↗

bioRxiv2024

Genome-wide analysis of fitness determinants of Staphylococcus aureus during growth in milk

Staphylococcus aureus is a major concern in the dairy industry due to its significance as a pathogen causing bovine mastitis as well as a source of food poisoning. The nutrient-rich milk environment supports bacterial growth, but the specific genetic determinants that facilitate S. aureus proliferation and persistence in milk are poorly understood. In this study, we conducted a genome-wide CRISPR interference sequencing (CRISPRi-seq) screen to identify fitness determinants essential for S. aureus growth and survival in milk. We identified 282 milk-essential genes, including those with key roles in DNA replication, protein synthesis, and metabolism. Comparative analysis with brain heart infusion (BHI) as growth medium, revealed 79 genes with differential fitness, highlighting specific adaptations required for growth in milk. Notably, we found that purine biosynthesis, folate cycle pathways, and metal acquisition were particularly important in this environment. Based on this, we further demonstrate that S. aureus is more sensitive to the folate inhibitors trimethoprim-sulfamethoxazole (TMP-SMX) in milk and identify several genes whose knockdown results in hypersensitivity to TMP-SMX in milk. Additionally, our analysis showed a relatively reduced importance of cell wall components, such as teichoic acids, for S. aureus fitness in milk, which is also reflected in reduced efficiency of antimicrobials targeting teichoic acids. Together, these findings provide new insights into the genetic basis of S. aureus fitness and antibiotic susceptibility in milk, offering directions for novel treatment strategies against bovine mastitis.

microbiology↗