Peptidoglycan Cross-Linking Preferences of <i>Staphylococcus aureus</i> Penicillin-Binding Proteins Have Implications for Treating MRSA Infections

Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) infections are a global public health problem. MRSA strains have acquired a non-native penicillin-binding protein called PBP2a that cross-links peptidoglycan when the native <i>S. aureus</i> PBPs are inhibited by β-lactams. It has been proposed that the native <i>S. aureus</i> PBPs can use cell wall precursors having different glycine branch lengths (penta-, tri-, or monoglycine), while PBP2a can only cross-link peptidoglycan strands bearing a complete pentaglycine branch. This hypothesis has never been tested because the necessary substrates have not been available. Here, we compared the ability of PBP2a and two native <i>S. aureus</i> transpeptidases to cross-link peptidoglycan strands bearing different glycine branches. We show that purified PBP2a can cross-link glycan strands bearing penta- and triglycine, but not monoglycine, and experiments in cells provide support for these findings. Because PBP2a cannot cross-link peptidoglycan containing monoglycine, this study implicates the enzyme (FemA) that extends the monoglycine branch to triglycine on Lipid II as an ideal target for small molecules that restore sensitivity of MRSA to β-lactams

Detection of Lipid-Linked Peptidoglycan Precursors by Exploiting an Unexpected Transpeptidase Reaction

Penicillin-binding proteins (PBPs) are involved in the synthesis and remodeling of bacterial peptidoglycan (PG). <i>Staphylococcus aureus</i> expresses four PBPs. Genetic studies in <i>S. aureus</i> have implicated PBP4 in the formation of highly cross-linked PG, but biochemical studies have not reached a consensus on its primary enzymatic activity. Using synthetic Lipid II, we show here that PBP4 preferentially acts as a transpeptidase (TP) <i>in vitro</i>. Moreover, it is the PBP primarily responsible for incorporating exogenous d-amino acids into cellular PG, implying that it also has TP activity <i>in vivo</i>. Notably, PBP4 efficiently exchanges d-amino acids not only into PG polymers but also into the PG monomers Lipid I and Lipid II. This is the first demonstration that any TP domain of a PBP can activate the PG monomer building blocks. Exploiting the promiscuous TP activity of PBP4, we developed a simple, highly sensitive assay to detect cellular pools of lipid-linked PG precursors, which are of notoriously low abundance. This method, which addresses a longstanding problem, is useful for assessing how genetic and pharmacological perturbations affect precursor levels, and may facilitate studies to elucidate antibiotic mechanism of action

The Mechanism of Action of Lysobactin

Lysobactin, also known as katanosin B, is a potent antibiotic with in vivo efficacy against Staphylococcus aureus and Streptococcus pneumoniae. It was previously shown to inhibit peptidoglycan (PG) biosynthesis, but its molecular mechanism of action has not been established. Using enzyme inhibition assays, we show that lysobactin forms 1:1 complexes with Lipid I, Lipid II, and Lipid II_A^WTA, substrates in the PG and wall teichoic acid (WTA) biosynthetic pathways. Therefore, lysobactin, like ramoplanin and teixobactin, recognizes the reducing end of lipid-linked cell wall precursors. We show that despite its ability to bind precursors from different pathways, lysobactin’s cellular mechanism of killing is due exclusively to Lipid II binding, which causes septal defects and catastrophic cell envelope damage

Silver Nanowire-Induced Sensitivity Enhancement of Optical Oxygen Sensors Based on AgNWs–Palladium Octaethylporphine–Poly(methyl methacrylate) Microfiber Mats Prepared by Electrospinning

Sensitivity enhancement of optical oxygen sensors is crucial for the characterization of nearly anoxic systems and oxygen quantification in trace amounts. In this work, for the first time we presented the introduction of silver nanowires (AgNWs) as a sensitivity booster for optical oxygen sensors based on AgNWs–palladium octaethylporphine–poly­(methyl methacrylate) (AgNWs@PdOEP–PMMA) microfiber mats prepared by electrospinning. Herein, a series of sensing microfiber mats with different loading ratios of high aspect ratio AgNWs were fabricated, and the corresponding sensitivity enhancement was systematically investigated. With increasing incorporated ratios, the AgNWs@PdOEP–PMMA-sensing microfiber mats exhibited a swift response (approx. 1.8 s) and a dramatic sensitivity enhancement (by 243% for the range of oxygen concentration 0−10% and 235% for the range of oxygen concentration 0–100%) when compared to the pure PdOEP–PMMA microfiber mat. Additionally, the as-prepared sensing films were experimentally confirmed to be highly photostable and reproducible. The advantages of AgNW-induced sensitivity enhancement could be useful for the rational design and realization of revolutionary highly sensitive sensors and expected to be readily applicable to many other high-performance gas sensor devices

Multifunctional PHPMA-Derived Polymer for Ratiometric pH Sensing, Fluorescence Imaging, and Magnetic Resonance Imaging

In this paper, we report synthesis and characterization of a novel multimodality (MRI/fluorescence) probe for pH sensing and imaging. A multifunctional polymer was derived from poly­(<i>N</i>-(2-hydroxypropyl)­methacrylamide) (PHPMA) and integrated with a naphthalimide-based-ratiometric fluorescence probe and a gadolinium–1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid complex (Gd–DOTA complex). The polymer was characterized using UV–vis absorption spectrophotometry, fluorescence spectrofluorophotometry, magnetic resonance imaging (MRI), and confocal microscopy for optical and MRI-based pH sensing and cellular imaging. In vitro labeling of macrophage J774 and esophageal CP-A cell lines shows the polymer’s ability to be internalized in the cells. The transverse relaxation time (<i>T</i>₂) of the polymer was observed to be pH-dependent, whereas the spin-lattice relaxation time (<i>T</i>₁) was not. The pH probe in the polymer shows a strong fluorescence-based ratiometric pH response with emission window changes, exhibiting blue emission under acidic conditions and green emission under basic conditions, respectively. This study provides new materials with multimodalities for pH sensing and imaging

Broad-Spectrum Antimicrobial Supramolecular Assemblies with Distinctive Size and Shape

With the increased prevalence of antibiotic-resistant infections, there is an urgent need for innovative antimicrobial treatments. One such area being actively explored is the use of self-assembling cationic polymers. This relatively new class of materials was inspired by biologically pervasive cationic host defense peptides. The antimicrobial action of both the synthetic polymers and naturally occurring peptides is believed to be complemented by their three-dimensional structure. In an effort to evaluate shape effects on antimicrobial materials, triblock polymers were polymerized from an assembly directing terephthalamide-bisurea core. Simple changes to this core, such as the addition of a methylene spacer, served to direct self-assembly into distinct morphologiesspheres and rods. Computational modeling also demonstrated how subtle core changes could directly alter urea stacking motifs manifesting in unique multidirectional hydrogen-bond networks despite the vast majority of material consisting of poly(lactide) (interior block) and cationic polycarbonates (exterior block). Upon testing the spherical and rod-like morphologies for antimicrobial properties, it was found that both possessed broad-spectrum activity (Gram-negative and Gram-positive bacteria as well as fungi) with minimal hemolysis, although only the rod-like assemblies were effective against Candida albicans