uridine-diphosphate-n-acetylmuramic-acid and Staphylococcal-Infections

Lipid II is critical for peptidoglycan synthesis, which is the main component of the bacterial cell wall. Lipid II is a relatively conserved and important part of the cell wall biosynthesis pathway and is targeted by antibiotics such as the lantibiotics, which achieve their function by disrupting the biosynthesis of the cell wall. Given the urgent need for development of novel antibiotics to counter the growing threat of bacterial infection resistance, it is imperative that a thorough molecular-level characterization of the molecules targeted by antibiotics be achieved. To this end, we present a molecular dynamics simulation study of the conformational dynamics of Lipid II within a detailed model of the Staphylococcus aureus cell membrane. We show that Lipid II is able to adopt a range of conformations, even within the packed lipidic environment of the membrane. Our simulations also reveal dimerization of Lipid II mediated by cations. In the presence of the defensin peptide plectasin, the conformational lability of Lipid II allows it to form loose complexes with the protein, via a number of different binding modes.

Topics: Binding Sites; Cell Membrane; Models, Molecular; Molecular Dynamics Simulation; Peptides; Protein Conformation; Staphylococcal Infections; Staphylococcus aureus; Uridine Diphosphate N-Acetylmuramic Acid

We have previously reported on the functional interaction of Lipid II with human alpha-defensins, a class of antimicrobial peptides. Lipid II is an essential precursor for bacterial cell wall biosynthesis and an ideal and validated target for natural antibiotic compounds. Using a combination of structural, functional and in silico analyses, we present here the molecular basis for defensin-Lipid II binding. Based on the complex of Lipid II with Human Neutrophil peptide-1, we could identify and characterize chemically diverse low-molecular weight compounds that mimic the interactions between HNP-1 and Lipid II. Lead compound BAS00127538 was further characterized structurally and functionally; it specifically interacts with the N-acetyl muramic acid moiety and isoprenyl tail of Lipid II, targets cell wall synthesis and was protective in an in vivo model for sepsis. For the first time, we have identified and characterized low molecular weight synthetic compounds that target Lipid II with high specificity and affinity. Optimization of these compounds may allow for their development as novel, next generation therapeutic agents for the treatment of Gram-positive pathogenic infections.

Topics: Anti-Bacterial Agents; Defensins; Drug Delivery Systems; Humans; Indoles; Methicillin-Resistant Staphylococcus aureus; Peptidomimetics; Pyrans; Staphylococcal Infections; Uridine Diphosphate N-Acetylmuramic Acid

The peptidoglycan of Staphylococcus aureus is characterized by a high degree of crosslinking and almost completely lacks free carboxyl groups, due to amidation of the D-glutamic acid in the stem peptide. Amidation of peptidoglycan has been proposed to play a decisive role in polymerization of cell wall building blocks, correlating with the crosslinking of neighboring peptidoglycan stem peptides. Mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin. We identified the enzymes catalyzing the formation of D-glutamine in position 2 of the stem peptide. We provide biochemical evidence that the reaction is catalyzed by a glutamine amidotransferase-like protein and a Mur ligase homologue, encoded by SA1707 and SA1708, respectively. Both proteins, for which we propose the designation GatD and MurT, are required for amidation and appear to form a physically stable bi-enzyme complex. To investigate the reaction in vitro we purified recombinant GatD and MurT His-tag fusion proteins and their potential substrates, i.e. UDP-MurNAc-pentapeptide, as well as the membrane-bound cell wall precursors lipid I, lipid II and lipid II-Gly₅. In vitro amidation occurred with all bactoprenol-bound intermediates, suggesting that in vivo lipid II and/or lipid II-Gly₅ may be substrates for GatD/MurT. Inactivation of the GatD active site abolished lipid II amidation. Both, murT and gatD are organized in an operon and are essential genes of S. aureus. BLAST analysis revealed the presence of homologous transcriptional units in a number of gram-positive pathogens, e.g. Mycobacterium tuberculosis, Streptococcus pneumonia and Clostridium perfringens, all known to have a D-iso-glutamine containing PG. A less negatively charged PG reduces susceptibility towards defensins and may play a general role in innate immune signaling.

Topics: Animals; Bacterial Proteins; Catalysis; Cell Wall; Humans; Immunity, Innate; Multienzyme Complexes; Mutation; Operon; Signal Transduction; Staphylococcal Infections; Staphylococcus aureus; Uridine Diphosphate N-Acetylmuramic Acid

Cell wall thickening is a common feature among daptomycin-resistant Staphylococcus aureus strains. However, the mechanism(s) leading to this phenotype is unknown. We examined a number of cell wall synthesis pathway parameters in an isogenic strain set of S. aureus bloodstream isolates obtained from a patient with recalcitrant endocarditis who failed daptomycin therapy, including the initial daptomycin-susceptible parental strain (strain 616) and two daptomycin-resistant strains (strains 701 and 703) isolated during daptomycin therapy. Transmission electron microscopy demonstrated significantly thicker cell walls in the daptomycin-resistant strains than in the daptomycin-susceptible strain, a finding which was compatible with significant differences in dry cell weight of strain 616 versus strains 701 to 703 (P < 0.05). Results of detailed analysis of cell wall muropeptide composition, the degree of peptide side chain cross-linkage, and the amount of the peptidoglycan precursor, UDP-MurNAc-pentapeptide, were similar in the daptomycin-susceptible and daptomycin-resistant isolates. In contrast, the daptomycin-resistant strains contained less O-acetylated peptidoglycan. Importantly, both daptomycin-resistant strains synthesized significantly more wall teichoic acid (WTA) than the parental strain (P < 0.001). Moreover, the proportion of D-alanylated WTA species was substantially higher in the daptomycin-resistant strains than in the daptomycin-susceptible parental strain (P < 0.05 in comparing strain 616 versus strain 701). The latter phenotypic findings correlated with (i) enhanced tagA and dltA gene expression, respectively, and (ii) an increase in surface positive charge observed in the daptomycin-resistant versus daptomycin-susceptible isolates. Collectively, these data suggest that increases in WTA synthesis and the degree of its D-alanylation may play a major role in the daptomycin-resistant phenotype in some S. aureus strains.

Topics: Alanine; Anti-Bacterial Agents; Bacterial Proteins; Cell Wall; Daptomycin; Drug Resistance, Bacterial; Endocarditis, Bacterial; Humans; Lipoproteins; Microbial Sensitivity Tests; Microscopy, Electron, Transmission; Peptidoglycan; Staphylococcal Infections; Staphylococcus aureus; Teichoic Acids; Uridine Diphosphate N-Acetylmuramic Acid