Cytoplasmic membrane thinning observed by interfacial dyes is likely a common effect of bactericidal antibiotics (original) (raw)
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BIO-PROTOCOL
Membrane fluidity is a key parameter of bacterial membranes that undergoes quick adaptation in response to environmental challenges and has recently emerged as an important factor in the antibacterial mechanism of membrane-targeting antibiotics. The specific level of membrane fluidity is not uniform across the bacterial cell membrane. Rather, specialized microdomains associated with different cellular functions can exhibit fluidity values that significantly deviate from the average. Assessing changes in the overall membrane fluidity and formation of membrane microdomains is therefore pivotal to understand both the functional organization of the bacterial cell membrane as well as antibiotic mechanisms. Here we describe how two fluorescent membrane dyes, laurdan and DiIC12, can be employed to assess membrane fluidity in living bacteria. We focus on Bacillus subtilis, since this organism has been relatively well-studied with respect to membrane domains. However, we also describe how these assays can be adapted for other bacteria such as Staphylococcus aureus and Streptococcus pneumoniae.
ACS Infectious Diseases, 2020
The formation of reactive oxygen species (ROS) induced by bactericidal antibiotics has been associated with a common, nonspecific mechanism of cellular death. Herein, we report real-time single-cell fluorescence studies on Escherichia coli stained with a fluorogenic probe for lipid peroxyl radicals showing the generation of this form of ROS when exposed to the minimum inhibitory concentration (MIC) and 10× MIC of the fluoroquinolone antibiotic ciprofloxacin (3 and 30 μM, respectively). Single-cell intensity−time trajectories show an induction period followed by an accelerating phase for cells treated with antibiotic, where initial and maximum intensity achieved following 3.5 h of incubation with antibiotic showed dose-dependent average values. A large fraction of bacteria remains viable after the studies, indicating ROS formation is occurring a priori of cell death. Punctate structures are observed, consistent with membrane blebbing. The addition of a membrane embedding lipid peroxyl radical scavenger, an αtocopherol analogue, to the media increased the MIC of ciprofloxacin. Lipid peroxyl radical formation precedes E. coli cell death and may be invoked in a cascade event including membrane disruption and consequent cell wall permeabilization. Altogether, our work illustrates that lipid peroxidation is caused by ciprofloxacin in E. coli and suppressed by α-tocopherol analogues. Lipid peroxidation may be invoked in a cascade event including membrane disruption and consequent cell wall permeabilization. Our work provides a methodology to assess antibiotic-induced membrane peroxidation at the single-cell level; this methodology provides opportunities to explore the scope and nature of lipid peroxidation in antibiotic-induced cell lethality.
Sensing and Bio-Sensing Research, 2015
Understanding the molecular basis of interactions between antibiotics affecting bacterial cell wall biosynthesis and cellular membranes is important in rational drug design of new drugs to overcome resistance. However, a precise understanding of how bacteriostatic antibiotics effect action often neglects the effect of biophysical forces involved following antibiotic-receptor binding events. We have employed a combination of a label-free binding biosensor (surface plasmon resonance, SPR) and a force biosensor (in-plane stress cantilever), together with model membrane systems to study the complex interplay between glycopeptide antibiotics, their cognate ligands and different model membranes. Bacterial cell wall precursor analogue N-a-Docosanoyl-e-acetyl-Lys-D-Alanine-D-Alanine (doc-KAA) was inserted into lipid layers comprised of zwitterionic or anionic lipids then exposed to either vancomycin or the membrane-anchored glycopeptide antibiotic teicoplanin. Binding affinities and kinetics of the antibiotics to these model membranes were influenced by electrostatic interactions with the different lipid backgrounds, in addition to ligand affinities. In addition, cantilever sensors coated with model membranes showed that planar surface stress changes were induced by glycopeptide antibiotics adsorption and caused compressive surface stress generation in a ligand-dependent manner.
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.
Nanoscale Features of Tunable Bacterial Outer Membrane Models Revealed by Correlative Microscopy
Langmuir
The rise of antibiotic resistance is a growing worldwide human health issue, with major socioeconomic implications. An understanding of the interactions occurring at the bacterial membrane is crucial for the generation of new antibiotics. Supported lipid bilayers (SLBs) made from reconstituted lipid vesicles have been used to mimic these membranes, but their utility has been restricted by the simplistic nature of these systems. A breakthrough in the field has come with the use of outer membrane vesicles derived from Gram-negative bacteria to form SLBs, thus providing a more physiologically relevant system. These complex bilayer systems hold promise but have not yet been fully characterized in terms of their composition, ratio of natural to synthetic components, and membrane protein content. Here, we use correlative atomic force microscopy (AFM) with structured illumination microscopy (SIM) for the accurate mapping of complex lipid bilayers that consist of a synthetic fraction and a fraction of lipids derived from Escherichia coli outer membrane vesicles (OMVs). We exploit the high resolution and molecular specificity that SIM can offer to identify areas of interest in these bilayers and the enhanced resolution that AFM provides to create detailed topography maps of the bilayers. We are thus able to understand the way in which the two different lipid fractions (natural and synthetic) mix within the bilayers, and we can quantify the amount of bacterial membrane incorporated into the bilayer. We prove the system's tunability by generating bilayers made using OMVs engineered to contain a green fluorescent protein (GFP) binding nanobody fused with the porin OmpA. We are able to directly visualize protein−protein interactions between GFP and the nanobody complex. Our work sets the foundation for accurately understanding the composition and properties of OMV-derived SLBs to generate a highresolution platform for investigating bacterial membrane interactions for the development of next-generation antibiotics.
Influence of polymyxins on the structural dynamics of Escherichia coli lipid membranes
Talanta, 2003
Polymyxins are a family of nonribosomic cationic peptide antibiotics highly effective against Gram-negative bacteria. Two members of this family, Polymyxins B and E (PxB, PxE), form molecular vesicle Á/vesicle contacts and promote a selective exchange of phospholipids at very low concentrations in the membrane, a biophysical phenomenon that can be the basis of their antibiotic mode of action. To get more insight into the interaction of these antibiotics with the lipid membrane, their effect on the structural dynamics of bilayers prepared with lipids extracted from the membrane of Escherichia coli was determined using fluorescently labeled phopholipids. Steady-state anisotropy measurements with probes that localize at different positions in the membrane give information on the effects of polymyxins on the mobility of the phospholipids. Results with PxB, PxE, colymycin M and polymyxin B nonapeptide (PxB-NP), a deacylated derivative with no antibiotic properties, are compared. At low peptide concentrations (B/2 mol%) PxB and PxE bind to the membranes superficially, affecting very slightly the ordering of the lipids at the outermost part of the bilayer. Above this concentration, PxB and PxE insert more deeply in the bilayer, increasing lipid order both in the gel and liquid Á/crystal states and modifying phase transitions. Fluorescence experiments with pyrene labeled phospholipids indicate that the increase in lipid packing is accompanied by an enrichment of phospholipids in the bilayers. In contrast, colymycin M and PxB-NP did not modify lipid packing or phase transition, nor did they induce microdomain formation. The possible significance of these results in the antibiotic mode of action of PxB and PxE is discussed. The combination of spectroscopic techniques described here can be useful as part of a general method of screening for new antibiotics that act on the membrane by the same mechanism as polymyxins.
Analytical and Bioanalytical Chemistry, 2010
The phase transition temperatures of several lipidic systems were determined using two different techniques: dynamic light scattering (DLS) and steady-state fluorescence anisotropy, using two fluorescent probes that report different membrane regions (TMA-DPH and DPH). Atomic force microscopy (AFM) was used as a complementary technique to characterize different lipid model systems under study. The systems were chosen due to the increased interest in bacterial membrane studies due to the problem of antibiotic drug resistance. The simpler models studied comprised of mixtures of POPE and POPG lipids, which form a commonly used model system for Escherichia coli membranes. Given the important role of cardiolipin (CL) in natural membranes, a ternary model system, POPE/POPG/CL, was then considered. The results obtained in these mimetic systems were compared with those obtained for the natural systems E. coli polar and total lipid extract. DLS and fluorescence anisotropy are not commonly used to study lipid phase transitions, but it was shown that they can give useful information about the thermotropic behaviors of model systems for bacterial membranes. These two techniques provided very similar results, validating their use as methods to measure phase transitions in lipid model systems. The temperature transitions obtained from these two very different techniques and the AFM results clearly show that cardiolipin is a fundamental component to mimic bacteria membranes. The results suggest that the less commonly used ternary system is a considerably better mimic for natural E. coli membranes than binary lipid mixture.
Stress induced alterations in the outer membrane of Escherichia coli K-12 strain
2019
In the present study, Gram-negative (Escherichia coli K-12) bacterial biomembrane involvement was studied in the presence of modulating factors such as EDTA, Mg+2 ions and EDTA and Mg+2 ions in combination. Also, the release of proteins and their involvement during the transport of β-Lactams namely Ceftriaxone and Cefazolin were studied. The broader applications of Ceftriaxone for Pharmaceutical implications offer greater advantage as compared to pre-existing β-Lactams. Due to the availability of more signal molecules in the membranes there is enhanced toxicity at 5 mM EDTA concentration, and easy entrapment of antibiotics, thus enhanced sensitivity levels. A concentration of 15 mM Mg+2 ions was found to be toxic for E.coli whereas it exhibited luxuriant growth with decreasing Mg+2 ion concentration under antibiotic stress. On the contrary, when 5 mM EDTA is treated in combination with Mg+2, it attributed reduced signals available on the membrane surface therefore, reduced drug sens...
Visualization of Antimicrobial-Induced Bacterial Membrane Disruption with a Bicolor AIEgen
Chemosensors
Gram-negative bacteria are difficult to kill due to their complex cell envelope, including the outer membrane (OM) and cytoplasmic membrane (CM). To monitor the membranolytic action of antimicrobials on Gram-negative bacteria would facilitate the development of effective antimicrobials. In this paper, an aggregation-induced emission luminogen (AIEgen) with microenvironment-sensitive properties was employed to indicate the interaction of antimicrobials with the OM and CM of Gram-negative bacteria. The damaged extent of OM and CM caused by antimicrobials with the change of dosage and incubation time can be visually captured based on the variation of two emission colors of IQ-Cm responding to OM-defective (green) and CM-disruptive bacteria (orange). Meanwhile, the activity assessment of antimicrobials can be easily realized within 1~2 h based on the distinct response of IQ-Cm to live and dead E. coli, which is much faster than the agar plate culture. This probe may shed light on the un...