The polyamino-isoprenyl potentiator NV716 revives disused antibiotics against Gram-negative bacteria in broth, infected monocytes, or biofilms, by disturbing the barrier effect of their outer membrane (original) (raw)
2022, European journal of medicinal chemistry
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Research in Microbiology, 2018
Antibiotic resistance is a serious threat to public health. Significant efforts are currently directed toward containment of the spread of resistance, finding new therapeutic options concerning resistant human and animal pathogens, and addressing the gaps in the fundamental understanding of mechanisms of resistance. Experimental data and kinetic modeling revealed a major factor in resistance, the synergy between active efflux and the low permeability barrier of the outer membrane, which dramatically reduces the intracellular accumulation of many antibiotics. The structural and mechanistic particularities of trans-envelope efflux pumps amplify the effectiveness of cell envelopes as permeability barriers. An important feature of this synergism is that efflux pumps and the outer membrane barriers are mechanistically independent and select antibiotics based on different physicochemical properties. The synergism amplifies even weak polyspecificity of multidrug efflux pumps and creates a major hurdle in the discovery and development of new therapeutics against Gram-negative pathogens.
PLoS ONE, 2013
Active efflux of antimicrobial agents is a primary mechanism by which bacterial pathogens can become multidrug resistant. The combined use of efflux pump inhibitors (EPIs) with pump substrates is under exploration to overcome efflux-mediated multidrug resistance. Phenylalanine-arginine b-naphthylamide (PAbN) is a well-studied EPI that is routinely combined with fluoroquinolone antibiotics, but few studies have assessed its utility in combination with b-lactam antibiotics. The initial goal of this study was to assess the efficacy of b-lactams in combination with PAbN against the opportunistic pathogen, Pseudomonas aeruginosa. PAbN reduced the minimal inhibitory concentrations (MICs) of several b-lactam antibiotics against P. aeruginosa; however, the susceptibility changes were not due entirely to efflux inhibition. Upon PAbN treatment, intracellular levels of the chromosomally-encoded AmpC b-lactamase that inactivates b-lactam antibiotics were significantly reduced and AmpC levels in supernatants correspondingly increased, potentially due to permeabilization of the outer membrane. PAbN treatment caused a significant increase in uptake of 8-anilino-1-naphthylenesulfonic acid, a fluorescent hydrophobic probe, and sensitized P. aeruginosa to bulky antibiotics (e.g. vancomycin) that are normally incapable of crossing the outer membrane, as well as to detergent-like bile salts. Supplementation of growth media with magnesium to stabilize the outer membrane increased MICs in the presence of PAbN and restored resistance to vancomycin. Thus, PAbN permeabilizes bacterial membranes in a concentration-dependent manner at levels below those typically used in combination studies, and this additional mode of action should be considered when using PAbN as a control for efflux studies.
Physiological Functions of Bacterial “Multidrug” Efflux Pumps
Chemical Reviews, 2021
Bacterial multidrug efflux pumps have come to prominence in human and veterinary pathogenesis, since they help bacteria protect themselves against the antimicrobials used to overcome their infections. However, it is increasingly realised that many, probably most, such pumps have physiological roles that are distinct from protection of bacteria against antimicrobials administered by humans. Here we undertake a broad survey of the proteins involved, allied to detailed examples of their evolution, energetics, structures, chemical recognition and molecular mechanisms, together with the experimental strategies that enable rapid and economical progress in understanding their true physiological roles. Once these roles are established, the knowledge can be harnessed to design more effective drugs, improve existing microbial production of drugs for clinical practice and of feedstocks for commercial exploitation, and even develop more sustainable biological processes that avoid, for example, utilisation of petroleum. TOC graphic Contents 1. Introduction 1.1 Antimicrobial efflux evolved independently many times in bacteria 1.2 The conservation of drug efflux pumps further alludes to a role outside drug resistance 1.3 Drug efflux pumps encoded in a single bacterial strain frequently have overlapping profiles for recognition of antimicrobials 1.4 The regulatory circuits controlling efflux pump expression are often not tuned to resistance functions 1.5 Overview 2. The movement of small molecules across bacterial cell envelopes 2.1 Bacterial cell envelopes 2.1.1 The Gram-positive cell envelope 2.1.2 The Gram-negative cell envelope 2.2 Families and superfamiles of proteins that include multidrug efflux pumps 2.2.1 The ATP binding cassette superfamily 2.2.2 The major facilitator superfamily 2.2.3 The resistance/nodulation/cell division superfamily 2.2.4 Drug/metabolite transporter superfamily 2.2.5 The multidrug and toxic compound extrusion transporter family 2.2.6 The Proteobacterial antimicrobial efflux family 2.2.7 The AbgT family of transport proteins 2.3 Overview 3. The substrate binding regions of multidrug efflux pumps allow functional promiscuity 3.1 Poly-specific binding sites in bacterial transcriptional regulatory proteins 3.2 Binding sites in RND pumps 3.3 Promiscuous binding sites and coupling reactions in drug exporting MFS transporters 3.4 Binding sites in drug exporting ABC superfamily pumps 3.5 Binding sites in SMR family pumps 3.6 Binding sites in MATE family efflux pumps 3.7 Binding sites in recently identified efflux pump families, AbgT and PACE family pumps 3.8 The contribution of tripartite complex components in controlling substrate specificity 3.9 Overview 4. Physiological functions of polyspecific bacterial efflux pumps 4.1 Transport of mammalian host-derived antimicrobial peptides 4.2 Protection against mammalian bile acids/salts and hormones 4.3 Fatty acid export 4.4 Protection against plant derived toxins 4.5 Tolerance towards aromatic hydrocarbons 4.6 Resistance to heavy metals 4.7 Polyamine efflux 4.8 Guanidinium efflux 4.9 Primary metabolite efflux 4.10 pH and salt tolerance 4.11 Protection against oxidative and nitrosative stress 4.12 Cell to cell signalling 4.13 Bacterial biofilm formation 4.14 Secretion of molecules involved in competitive bacterial interactions 4.15 Metal ion acquisition through siderophore efflux 4.16 Necrosignallinga novel, non-efflux related function of efflux pumps 4.17 Overview 5. The discovery and characterisation of novel efflux pumps and their substrates 5.1 Recognition of efflux proteins from bioinformatics 5.2 Transcriptomics identify a novel protein whose expression is responsive to chlorhexidine 5.3 Common features of the novel protein family 5.3.1 Prediction of four transmembrane helices per monomer. 5.3.2 Recurring structural motifs 5.4 Transfer of the target gene from an inconvenient pathogen to a convenient E. coli host for expression and purification of the PACE proteins and investigation of their properties. 5.4.1 The native host organism. 5.4.2 Automated determination of interactions of an individual cloned gene with many biocides 5.4.3 Assays with fluorescent artificial substrates 5.4.4 Direct measurements of efflux activity of AceI in E. coli using radioisotopelabelled chlorhexidine 5.4.5 Conclusions 5.5 Production and purification of membrane transport proteins for direct physical chemistry assays to test binding of potential ligands. 5.5.1 Introduction 5.5.2 Fluorescence changes of endogenous tryptophan residues in the purified AceI protein detect binding of substrates and/or inhibitors 5.5.3 Measurements of circular dichroism and changes in melting curves authenticate and extend identification of substrates and/or inhibitors. 5.5.4 Conclusions 5.6 Is there a natural substrate for transport by the AceI protein? 5.6.1 Introduction 5.6.2 Transport of radioisotope-labelled compounds by E. coli and A. baumannii cells induced for activity of the AceI protein 5.6.2.1 The AceI protein and its E15Q variant expressed in E. coli 5.6.2.2 Diamines and expression of the aceI gene in A. baumannii 5.6.2.3 Toxicity of diamines towards growth of A. baumannii 5.6.2.4 Transport of radiolabelled cadaverine by A. baumannii 5.6.3 Conclusions 5.7 The AceI protein of A. baumannii is a cadaverine/H+ efflux transport protein in vitro 5.7.1 Introduction 5.7.2 Transport of radiolabelled substrates by proteoliposomes 5.7.3 Coupling of transport to an electrochemical gradient of protons 5.7.4 Conclusions 6. Conclusions and future perspectives Biographies Acknowledgements Abbreviations References
mBio
Gram-negative bacteria are notoriously resistant to antibiotics, but the extent of the resistance varies broadly between species. We report that in significant human pathogens Acinetobacter baumannii , Pseudomonas aeruginosa , and Burkholderia spp., the differences in antibiotic resistance are largely defined by their penetration into the cell. For all tested antibiotics, the intracellular penetration was determined by a synergistic relationship between active efflux and the permeability barrier. We found that the outer membrane (OM) and efflux pumps select compounds on the basis of distinct properties and together universally protect bacteria from structurally diverse antibiotics. On the basis of their interactions with the permeability barriers, antibiotics can be divided into four clusters that occupy defined physicochemical spaces. Our results suggest that rules of intracellular penetration are intrinsic to these clusters. The identified specificities in the permeability barrier...
Inhibitors of efflux pumps in Gram-negative bacteria
Trends in Molecular Medicine, 2005
In Gram-negative bacteria, efflux complexes, consisting of an inner-membrane pump, a periplasmic adaptor protein and outer-membrane channel, provide an efficient means for the export of structurally unrelated drugs, causing the multidrug-resistance phenotype. Resistance due to this antibiotic efflux is an increasing problem worldwide. A new molecular challenge is to combat this transport by searching for new molecules to block efflux and thus restore drug susceptibility to resistant clinical strains. Recent data shed new light on the structure and activity of the archetypal efflux pumps AcrAB-TolC and MexAB-OprM. Here, we describe recent insights into the molecular mechanisms of bacterial efflux pumps and their inhibitors. Current progress for the clinical use of efflux-pump inhibitors and new strategies to combat the drug-efflux mechanisms will be discussed.
Bacterial resistance to antibiotics: Active efflux and reduced uptake
Advanced Drug Delivery Reviews, 2005
Antibiotic resistance of bacterial pathogens is a fast emerging global crisis and an understanding of the underlying resistance mechanisms is paramount for design and development of new therapeutic strategies. Permeability barriers for and active efflux of drug molecules are two resistance mechanisms that have been implicated in various infectious outbreaks of antibioticresistant pathogens, suggesting that these mechanisms may be good targets for new drugs. The synergism of reduced uptake and efflux is most evident in the multiplicative action of the outer membrane permeability barrier and active efflux, which results in high-level intrinsic and/or acquired resistance in many clinically important Gram-negative bacteria. This review summarizes the current knowledge of these two important resistance mechanisms and potential strategies to overcome them. Recent advances in understanding the physical structures, function and regulation of efflux systems will facilitate exploitation of pumps as new drug targets. D
Journal of bacteriology, 1997
A major feature of the MexAB-OprM multidrug efflux pump which distinguishes it from the MexCD-OprJ and MexEF-OprN multidrug efflux systems in Pseudomonas aeruginosa is its ability to export a wide variety of beta-lactam antibiotics. Given the periplasmic location of their targets it is feasible that beta-lactams exit the cell via the outer membrane OprM without interaction with MexA and MexB, though the latter appear to be necessary for OprM function. To test this, chimeric MexAB-OprJ and MexCD-OprM efflux pumps were reconstituted in delta mexCD delta oprM and delta mexAB delta oprJ strains, respectively, and the influence of the exchange of outer membrane components on substrate (i.e., beta-lactam) specificity was assessed. Both chimeric pumps were active in antibiotic efflux, as evidenced by their contributions to resistance to a variety of antimicrobial agents, although there was no change in resistance profiles relative to the native pumps, indicating that OprM is not the determ...
Bacterial multidrug efflux pumps: … and some suggestions on how they might work
The Biochemist, 2015
One of the key mechanisms that confers drug resistance in bacteria involves the action of energyconsuming transporters that drive the efflux of toxic compounds from the cell. In the Gramnegative group of bacteria, which have a characteristic system of two lipid membranes, some of these transporters are the engine components of tripartite assemblies that span both membranes and the interstitial periplasm. These assemblies have been the focus of extensive structural and mechanistic studies for decades, and we provide a brief synopsis of the current understanding of how they are organized into functioning machines, how those machines might work in detail and the likely points in the machinery that could be targets for therapeutic intervention.
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