STD NMR spectroscopy: a case study of fosfomycin binding interactions in living bacterial cells

Abstract

O experimento de RMN STD (saturation transfer difference) foi empregado com sucesso na observação das interações de ligação entre fosfomicina e cepas bacterianas resistentes e não resistentes à fosfomicina, diretamente em suspensões celulares vivas sem necessidade de marcação isotópica do ligante ou receptor.A saturation transfer difference (STD) NMR experiment was successfully employed to observe the binding interactions of fosfomycin resistant and non-resistant bacterial strains using living cell suspensions, without the need for isotopic labelling of the ligand or receptor. Keywords: STD NMR in living bacterial cells, membrane-bound proteins, ligand-target interactions, fosfomycin, liposomes IntroductionSaturation transfer difference (STD) is a 1 H NMR technique widely used to investigate ligand (small molecules) and macromolecular (proteins and peptides, 1 carbohydrates, 2 lipids 3 and nucleic acids 4 ) interactions. This tool (a STD experiment) is appropriate to probe biological binding events at the molecular level 5 and is based on the nuclear Overhauser effect (nOe) transfer from the macromolecule to the ligand. It consists of applying a selective radio frequency pulse to the macromolecule at a resonance where no ligand signals are present. The magnetization is transferred to the entire macromolecule via intra-molecular spin diffusion and then this saturation is transferred intermolecularly to bound ligands and detected in the free-ligand solution. The ligand's hydrogen most tightly bound to the macromolecule will receive the most intense magnetization-transfer and the amplitude of these signals will change accordingly to the nOe effects. 5,6 Therefore, the degree of nOe effects reflects the proximity of these protons to the macromolecule, allowing direct observation of the ligand moiety involved in the macromolecule-ligand interaction. Among the vast literature covering biological interactions observed by STD NMR spectroscopy there are few examples in which the detection of those binding processes occurs directly in whole living cells. 7 The information obtained in such investigation is rather important, especially when studying ligand-membrane-bound protein interactions as most biologically relevant proteins are membrane-bound 8 and are often difficult to deal with as they lose their structures and functionality when removed from their natural membrane environment.Herein we show the use of STD NMR to obtain information about direct drug transport into a cell of living bacterial cell suspensions. As an example we used the well-known fosfomycin uptake by cells and the relationship between fosfomycin resistance in bacterial strains.9 Fosfomycin or phosphonomycin, [(1R,2S)-1,2-epoxypropylphosphonic acid)] is a broad spectrum antibiotic against Gram-negative and Grampositive bacteria and has become the first choice for the treatment of certain infections, especially those caused by cephalosporin and penicillin-resistant Streptococcus pneumoniae and, methicillin-and vancomycin-resistant Staphylococcus aureus...

Ex vivo penetration of fosfomycin into healthy and Lawsonia intracellularis‐colonized swine intestinal mucosa

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NMR and molecular recognition. The application of ligand-based NMR methods to monitor molecular interactions

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