ABSTRACT
Modern medicine is founded on the discovery of penicillin and subsequent small molecules that inhibit bacterial peptidoglycan (PG) and cell wall synthesis. However, the discovery of new chemically and mechanistically distinct classes of PG inhibitors has become exceedingly rare, prompting speculation that intracellular enzymes involved in PG precursor synthesis are not 'druggable' targets. Here, we describe a ß-lactam potentiation screen to identify small molecules that augment the activity of ß-lactams against methicillin-resistant Staphylococcus aureus (MRSA) and mechanistically characterize a compound resulting from this screen, which we have named murgocil. We provide extensive genetic, biochemical, and structural modeling data demonstrating both in vitro and in whole cells that murgocil specifically inhibits the intracellular membrane-associated glycosyltransferase, MurG, which synthesizes the lipid II PG substrate that penicillin binding proteins (PBPs) polymerize and cross-link into the cell wall. Further, we demonstrate that the chemical synergy and cidality achieved between murgocil and the ß-lactam imipenem is mediated through MurG dependent localization of PBP2 to the division septum. Collectively, these data validate our approach to rationally identify new target-specific bioactive ß-lactam potentiation agents and demonstrate that murgocil now serves as a highly selective and potent chemical probe to assist our understanding of PG biosynthesis and cell wall biogenesis across Staphylococcal species.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Outer Membrane Proteins/antagonists & inhibitors , N-Acetylglucosaminyltransferases/antagonists & inhibitors , Peptidoglycan Glycosyltransferase/metabolism , Pyrazoles/pharmacology , Staphylococcus aureus/drug effects , Sterols/pharmacology , Computer Simulation , Drug Resistance, Bacterial , Enzyme Inhibitors/pharmacology , Humans , Microscopy, Fluorescence , Models, Molecular , Pyrazoles/chemistry , Staphylococcus aureus/enzymology , Sterols/chemistryABSTRACT
PURPOSE: The aim of this work was to develop an easy, manageable, and precise analytic tool to describe the tightness of cell layers by a molecular weight ladder. METHODS: Dextrans were labeled by reductive amination with fluorescent 8-aminopyrene-1,3,6-trisulfonate (APTS). This mixture, including the internal standard diazepam, was used for transport studies in Transwell models using Caco-2, ECV304, and PBMEC/C1-2 cell lines. Samples were analyzed by fluorimetry, capillary electrophoresis, and reverse-phase high-performance liquid chromatography. RESULTS: Following this approach, a logarithm correlation of R2 = 0.8958 between transepithelial electrical resistance (TEER) and APTS-dextran permeability was shown. In addition, a TEER-dependent permeability pattern could be observed including each single fraction from free APTS, APTS-glucose up to APTS-dextran consisting of 35 glucose units. The TEER-independent permeability coefficients of diazepam and confocal laser scanning microscopy images confirmed the paracellular transport of APTS-dextran. CONCLUSIONS: All in all, the developed APTS-dextran ladder is a useful tool to characterize cell layer tightness and especially to describe paracellular transport ways and the extent of leakiness of cell layers (for blood-brain barrier or intestinal studies) over time--applying a wide array from smaller to larger molecules at the same time to refine TEER, sucrose, or Evans blue measurements.
Subject(s)
Blood-Brain Barrier/metabolism , Capillary Permeability , Cell Membrane Permeability , Dextrans/metabolism , Endothelial Cells/metabolism , Fluorescent Dyes/metabolism , Intestinal Mucosa/metabolism , Pyrenes/metabolism , Animals , Caco-2 Cells , Dextrans/analysis , Dextrans/chemical synthesis , Dextrans/chemistry , Diazepam/metabolism , Diffusion , Electric Impedance , Electrophoresis, Capillary , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescein-5-isothiocyanate/analysis , Fluorescein-5-isothiocyanate/chemistry , Fluorescein-5-isothiocyanate/metabolism , Fluorescent Dyes/analysis , Fluorescent Dyes/chemical synthesis , Humans , Microscopy, Confocal , Molecular Weight , Pyrenes/analysis , Pyrenes/chemical synthesis , Time FactorsABSTRACT
The aim of this work was the development of an easy manageable analytic system for describing tightness of cell layers in a molecular size dependent manner, which is more precise than currently used ones. Dextrans were labeled by reductive amination with fluorescent 1-aminopyrene-3,6,8-trisulfonate (APTS). This mixture, including internal standard diazepam, was used for transport studies, which were accomplished with an established transwell blood-brain barrier model culturing an immortalized porcine brain microvascular endothelial cell line (PBMEC/C1-2). Samples were analyzed by fluorescence measurements, capillary electrophoresis and RP-LC. Following this approach, a permeability pattern could be achieved including each single fraction from APTS, APTS-glucose to APTS-dextran consisting of 31 glucose units. Permeability coefficients were calculated and ranged from 16.38+/-3.79 microm/min for APTS to 6.07+/-1.23 microm/min for the APTS-dextran with 31 glucose units (diazepam: 67.97+/-7.32 microm/min). All in all, the developed APTS-dextran ladder is an useful tool to characterize cell layer tightness--especially to describe paracellular transport ways and leakiness status of the blood-brain barrier over time--applying a wide range from smaller to larger molecules at the same time in order to refine, e.g. TEER, sucrose or Evans blue measurements.
