Affinities and in-plane stress forces between glycopeptide antibiotics and biomimetic bacterial membranes (original) (raw)
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Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection
Journal of Visualized Experiments, 2013
The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity . The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions.
Surface mediated cooperative interactions of drugs enhance mechanical forces for antibiotic action
Scientific Reports, 2017
The alarming increase of pathogenic bacteria that are resistant to multiple antibiotics is now recognized as a major health issue fuelling demand for new drugs. Bacterial resistance is often caused by molecular changes at the bacterial surface, which alter the nature of specific drug-target interactions. Here, we identify a novel mechanism by which drug-target interactions in resistant bacteria can be enhanced. We examined the surface forces generated by four antibiotics; vancomycin, ristomycin, chloroeremomycin and oritavancin against drug-susceptible and drug-resistant targets on a cantilever and demonstrated significant differences in mechanical response when drug-resistant targets are challenged with different antibiotics although no significant differences were observed when using susceptible targets. Remarkably, the binding affinity for oritavancin against drug-resistant targets (70 nM) was found to be 11,000 times stronger than for vancomycin (800 μM), a powerful antibiotic u...
Surface-stress sensors for rapid and ultrasensitive detection of active free drugs in human serum
Nature Nanotechnology, 2014
There is a growing appreciation that mechanical signals can be as important as chemical and electrical signals in biology. To include such signals in a systems biology description for understanding pathobiology and developing therapies, quantitative experiments on how solution-phase and surface chemistry together produce biologically relevant mechanical signals are needed. Because of the appearance of drug-resistant hospital 'superbugs', there is currently great interest in the destruction of bacteria by bound drug-target complexes that stress bacterial cell membranes. Here, we use nanomechanical cantilevers as surface-stress sensors, together with equilibrium theory, to describe quantitatively the mechanical response of a surface receptor to different antibiotics in the presence of competing ligands in solution. The antibiotics examined are the standard, Food and Drug Administration-approved drug of last resort, vancomycin, and the yet-to-be approved oritavancin, which shows promise for controlling vancomycin-resistant infections. The work reveals variations among strong and weak competing ligands, such as proteins in human serum, that determine dosages in drug therapies. The findings further enhance our understanding of the biophysical mode of action of the antibiotics and will help develop better treatments, including choice of drugs as well as dosages, against pathogens.
Journal of Electroanalytical Chemistry, 2018
Lipopeptides are known to show bactericidal activity and due to their simple structure, ease of design, and low cost of implementation, they are often considered potent replacement for many traditional antibiotics. Another important advantage of lipopeptides is related to the fact that their preferential target is a cell membrane. Hence their action is less specific than conventional antibiotics, which means that development of drug resistance by pathogens is less probable in such case. In this paper we have utilized electrochemical methods and in situ atomic force microscopy to evaluate the mode of action of novel lipopeptide C 15 H 31 CO-DPhe-Dab-Dab-Leu-NH 2 on planar lipid bilayer. The latter was composed of phosphatidylethanolamines and phosphatidylglycerols extracts from E. coli. Therefore it can be considered as a simplified model of inner membrane of Gram negative bacteria. We have found that lipopeptide-lipid interactions strongly affect molecular organization of PE/PG bilayer, which is reflected by increased disorder and subsequent perforation of the film. Importantly, fluid domains were identified as preferential sites for insertion of lipopeptide molecules, which tend to accumulate within the membrane. However, above certain threshold ratio the membrane becomes swollen and strongly destabilized. This results in membrane rupture and large mixed lipopeptide-lipid aggregates departure from electrode surface. Based on experimental observations, the mechanism of C 16-fXXL action on bacterial-like model membrane is proposed.
Chemistry - A European Journal, 2003
A novel synthesized watersoluble variant of lipid II (LII) was used to evaluate the noncovalent interactions between a number of glycopeptide antibiotics and their receptor by bioaffinity electrospray ionization mass spectrometry (ESI-MS). The water-soluble variant of lipid II is an improved design, compared to the traditionally used tripeptide N,N'-diacetyl-l-lysyl-d-alanyl-d-alanine (KAA), of the target molecule on the bacterial cell wall. A representative group of glycopeptide antibiotics was selected for this study to evaluate the validity of the novel cell-wall-mimicking target LII. Structure ± function relationships of various glycopeptide antibiotics were investigated by means of 1) bioaffinity mass spectrometry to evaluate solution-phase molecular interactions with both LII and KAA, 2) fluorescence leakage experiments to study the interactions with the membrane-embedded lipid II, and 3) minimum inhibitory concentrations against the indicator strain Micrococcus flavus. Our results with the novel LII molecule reveal that some antibiotics interact differently with KAA and LII. Additionally, our data cast doubt on the hypothesis that antibiotic selfdimerization assists in the invivo efficacy. Finally, the water-soluble lipid II proved to be a better model of the bacterial cell wall.
Journal of Structural Biology, 2006
We report here on an in situ atomic force microscopy study of the interaction of indolicidin, a tryptophan-rich antimicrobial peptide, with phase-segregated zwitterionic DOPC/DSPC supported planar bilayers. By varying the peptide concentration and bilayer composition through the inclusion of anionic lipids (DOPG or DSPG), we found that indolicidin interacts with these model membranes in one of two concentration-dependent manners. At low peptide concentrations, indolicidin forms an amorphous layer on the Xuid domains when these domains contain anionic lipids. At high peptide concentrations, indolicidin appears to initiate a lowering of the gel-phase domains independent of the presence of an anionic lipid. Similar studies performed using membrane-raft mimetic bilayers comprising 30 mol% cholesterol/1:1 DOPC/egg sphingomyelin revealed that indolicidin does not form a carpet-like layer on the zwitterionic DOPC domains at low peptide concentrations and does not induce membrane lowering of the liquid-ordered sphingomyelin/cholesterol-rich domains at high peptide concentration. Simultaneous AFM-confocal microscopy imaging did however reveal that indolicidin preferentially inserts into the Xuid-phase DOPC domains. These data suggest that the indolicidin-membrane association is inXuenced greatly by speciWc electrostatic interactions, lipid Xuidity, and peptide concentration. These insights provide a glimpse into the mechanism of the membrane selectivity of antibacterial peptides and suggest a powerful correlated approach for characterizing peptide-membrane interactions.
Organic & Biomolecular Chemistry, 2010
Functionalised thiols presenting peptides found in the peptidoglycan of vancomycin-sensitive and-resistant bacteria were synthesised and used to form self-assembled monolayers (SAMs) on gold surfaces. This model bacterial cell-wall surface mimic was used to study binding interactions with vancomycin. Force spectroscopy, using the atomic force microscope (AFM), was used to investigate the specific rupture of interfacial vancomycin dimer complexes formed between pairs of vancomycin molecules bound to peptide-coated AFM probe and substrate surfaces. Clear adhesive contacts were observed between the vancomycin-sensitive peptide surfaces when vancomycin was present in solution, and the adhesion force demonstrated a clear dependence on antibiotic concentration.
Antibiotic-induced modifications of the stiffness of bacterial membranes
Journal of Microbiological Methods, 2013
In the latest years the importance of high resolution analysis of the microbial cell surface has been increasingly recognized. Indeed, in order to better understand bacterial physiology and achieve rapid diagnostic and treatment techniques, a thorough investigation of the surface modifications induced on bacteria by different environmental conditions or drugs is essential. Several instruments are nowadays available to observe at high resolution specific properties of microscopic samples. Among these, AFM can routinely study single cells in physiological conditions, measuring the mechanical properties of their membrane at a nanometric scale (force volume). Such analyses, coupled with high resolution investigation of their morphological properties, are increasingly used to characterize the state of single cells. In this work we exploit such technique to characterize bacterial systems. We have performed an analysis of the mechanical properties of bacteria (Escherichia coli) exposed to different conditions. Such measurements were performed on living bacteria, by changing in real-time the liquid environment: standard phosphate buffered saline, antibiotic (ampicillin) in PBS and growth medium. In particular we have focused on the determination of the membrane stiffness modifications induced by these solutions, in particular between stationary and replicating phases and what is the effect of the antibiotic on the bacterial structure.
bioRxiv (Cold Spring Harbor Laboratory), 2023
One way to mitigate the ongoing antimicrobial resistance crisis is to discover and develop new classes of antibiotics. As all antibiotics at some point needs to either cross or interact with the bacterial membrane, there is a need for representative models of bacterial membranes and efficient methods to characterize the interactions to novel antimicrobials-both to generate new knowledge and to screen compound libraries. Since the bacterial cell envelope is a complex assembly of lipids, lipopolysaccharides, membrane proteins and other components, constructing realistic synthetic liposome-based models of the membrane is both difficult and expensive. We here propose to let the bacteria do the hard work for us. Outer membrane vesicles (OMVs) are naturally secreted by Gram-negative bacteria, playing a role in communication between bacteria, as virulence factors, molecular transport or being a part of the antimicrobial resistance mechanism. OMVs consist of the bacterial outer membrane and thus inherit many components and properties of the native outer cell envelope. In this work we have isolated and characterized OMVs from E. coli mutant strains and clinical isolates of the ESKAPE members Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. The OMVs were shown to be representative models for the bacterial membrane in terms of lipid composition with strain specific variations. The OMVs were further used to probe the interactions between OMV and antimicrobial peptides (AMPs) as model compounds by Surface Plasmon Resonance (SPR) and provide proof-of-principle that OMVs can be used as an easily accessible and highly realistic model for the bacterial surface in interaction studies. This further enables direct monitoring of the effect of induction by antibiotics, or the response to host-pathogen interactions.