RESUMO
Lyme disease is on the rise. Caused by a spirochete Borreliella burgdorferi, it affects an estimated 500,000 people in the United States alone. The antibiotics currently used to treat Lyme disease are broad spectrum, damage the microbiome, and select for resistance in non-target bacteria. We therefore sought to identify a compound acting selectively against B. burgdorferi. A screen of soil micro-organisms revealed a compound highly selective against spirochetes, including B. burgdorferi. Unexpectedly, this compound was determined to be hygromycin A, a known antimicrobial produced by Streptomyces hygroscopicus. Hygromycin A targets the ribosomes and is taken up by B. burgdorferi, explaining its selectivity. Hygromycin A cleared the B. burgdorferi infection in mice, including animals that ingested the compound in a bait, and was less disruptive to the fecal microbiome than clinically relevant antibiotics. This selective antibiotic holds the promise of providing a better therapeutic for Lyme disease and eradicating it in the environment.
Assuntos
Antibacterianos/uso terapêutico , Doença de Lyme/tratamento farmacológico , Animais , Borrelia burgdorferi/efeitos dos fármacos , Calibragem , Cinamatos/química , Cinamatos/farmacologia , Cinamatos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Fezes/microbiologia , Feminino , Células HEK293 , Células Hep G2 , Humanos , Higromicina B/análogos & derivados , Higromicina B/química , Higromicina B/farmacologia , Higromicina B/uso terapêutico , Doença de Lyme/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Microbiota/efeitos dos fármacosRESUMO
Multidrug efflux is one of the major mechanisms of antibiotic resistance identified in clinical isolates of the human pathogen Acinetobacter baumannii. The multiple antibiotic resistance in this species is often enabled by the overproduction of the tripartite efflux pump AdeABC. In this pump, AdeB is the inner membrane transporter from the resistance-nodulation-division (RND) superfamily of proteins, which is responsible for the recognition and efflux of multiple structurally unrelated compounds. Like other RND transporters, AdeB is a trimeric protein with ligand-binding sites located in the large periplasmic domains. Previous structural studies, however, highlighted the uniqueness of AdeB interactions with ligands. Up to three ligand molecules were bound to one protomer of AdeB, mapping its substrate translocation path. In this study, we introduced single and double substitutions in the identified ligand-binding sites of AdeB. Our results show that the mechanism of substrate translocation by AdeB is different from that of other characterized RND transporters and that the functional interactions between the sites are nonadditive. We identified AdeB mutants with both the loss and the gain of antibiotic susceptibility phenotypes, as well as AdeB mutations making A. baumannii cells overproducing such pump variants even more susceptible to multiple antibiotics than efflux-deficient cells. IMPORTANCE Multidrug efflux pumps of the resistance-nodulation-division superfamily of proteins are important contributors to various aspects of bacterial physiology and antibiotic resistance. Studies of the best-characterized model transporter AcrB from Escherichia coli suggested that these transporters operate by a functional rotation mechanism in which various substrates bind to at least two different binding sites. This study suggests that the mechanism of AdeB is distinct and that the binding sites in this transporter are functionally linked.
Assuntos
Acinetobacter baumannii , Proteínas de Escherichia coli , Humanos , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Proteínas de Bactérias/metabolismo , Ligantes , Antibacterianos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Sítios de Ligação , Escherichia coli/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Testes de Sensibilidade Microbiana , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMO
Gram-negative bacteria are notoriously more resistant to antibiotics than Gram-positive bacteria, primarily due to the presence of the outer membrane and a plethora of active efflux pumps. However, the potency of antibiotics also varies dramatically between different Gram-negative pathogens, suggesting major mechanistic differences in how antibiotics penetrate permeability barriers. Two approaches are used broadly to analyze how permeability barriers affect intracellular accumulation of antibiotics. One compares the antibacterial activities of compounds, while the other measures the total intracellular concentrations of compounds in nongrowing cells, with both approaches using strains harboring wild-type or genetically modified efflux systems and permeability barriers. Whether the two assays provide similar mechanistic insights remains unclear. In this study, we analyzed the intracellular accumulation and antibacterial activities of antibiotics representative of major clinical classes in three Gram-negative pathogens of high clinical importance, Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter baumannii. We found that both assays are informative about properties of permeability barriers, but there is no quantitative agreement between the assays. Our results show that the three pathogens differ dramatically in their permeability barriers, with the outer membrane playing the dominant role in E. coli and P. aeruginosa but efflux dominating in A. baumannii. However, even compounds of the same chemotype may use different permeation pathways depending on small chemical modifications. Accordingly, a classification analysis revealed limited conservation of molecular properties that define compound penetration into the three bacteria.
Assuntos
Antibacterianos , Escherichia coli , Antibacterianos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Transporte Biológico , Bactérias Gram-Negativas/metabolismo , Permeabilidade , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/metabolismoRESUMO
An internal collection of commercial and synthetically derived small molecule compounds was screened against several drug-resistant bacterial pathogens. Compound 1, a known N, N-disubstituted 2-aminobenzothiazole, was found to be a potent inhibitor of Staphylococcus aureus and several associated clinically relevant strains of methicillin-resistant S. aureus suggesting a possible novel mechanism of inhibition. It failed to show activity in any of the Gram-negative pathogens it was tested in. Evaluation in Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1, as well as in their respective hyperporinated and efflux pump-deletion mutants revealed that activity in Gram-negative bacteria is diminished because this benzothiazole scaffold is a substrate for bacterial efflux pumps. Several analogs of 1 were synthesized to generate basic structure-activity relationships for the scaffold which highlighted that the N-propyl imidazole moiety was critical for the observed antibacterial activity.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Staphylococcus aureus , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Antibacterianos/química , Relação Estrutura-Atividade , Bactérias , Escherichia coli , Proteínas de BactériasRESUMO
Cell envelope plays a dual role in the life of bacteria by simultaneously protecting it from a hostile environment and facilitating access to beneficial molecules. At the heart of this ability lie the restrictive properties of the cellular membrane augmented by efflux transporters, which preclude intracellular penetration of most molecules except with the help of specialized uptake mediators. Recently, kinetic properties of the cell envelope came into focus driven on one hand by the urgent need in new antibiotics and, on the other hand, by experimental and theoretical advances in studies of transmembrane transport. A notable result from these studies is the development of a kinetic formalism that integrates the Michaelis-Menten behavior of individual transporters with transmembrane diffusion and offers a quantitative basis for the analysis of intracellular penetration of bioactive compounds. This review surveys key experimental and computational approaches to the investigation of transport by individual translocators and in whole cells, summarizes key findings from these studies and outlines implications for antibiotic discovery. Special emphasis is placed on Gram-negative bacteria, whose envelope contains two separate membranes. This feature sets these organisms apart from Gram-positive bacteria and eukaryotic cells by providing them with full benefits of the synergy between slow transmembrane diffusion and active efflux.
Assuntos
Bactérias Gram-Negativas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Membrana Externa Bacteriana/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/efeitos dos fármacos , Humanos , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Proteínas de Membrana Transportadoras/química , Modelos Biológicos , Simulação de Dinâmica MolecularRESUMO
Multidrug resistant (MDR) strains of Acinetobacter baumannii present a serious clinical challenge. The development of antibiotic resistance in this species is enabled by efflux pumps of the Resistance-Nodulation-Division (RND) superfamily of proteins creating an efficient permeability barrier for antibiotics. At least three RND pumps, AdeABC, AdeIJK, and AdeFGH are encoded in the A. baumannii genome and are reported to contribute to antibiotic resistance in clinical isolates. In this study, we analyzed the contributions of AdeABC and AdeIJK in antibiotic resistance and growth physiology of the two MDR strains, AYE and AB5075. We found that not only the two pumps have nonoverlapping substrate specificities, their inactivation leads to specific nonoverlapping changes in gene expression as determined by RNA sequencing and confirmed by gene knockouts and growth phenotypes. Our results suggest that inactivation of AdeIJK elicits broader changes in the abundances of mRNAs and this response is modified in the absence of AdeB. In contrast, inactivation of AdeB leads to a focused cellular response, which is not sensitive to the activity of AdeIJK. We identified additional efflux pumps and transcriptional regulators that contribute to MDR phenotype of clinical A. baumannii isolates.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla , Proteínas de Membrana Transportadoras/metabolismo , Infecções por Acinetobacter/microbiologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Lipídeo A/metabolismo , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Fenótipo , RNA Bacteriano/metabolismo , Análise de Sequência de RNA , Especificidade por SubstratoRESUMO
Transporters belonging to the resistance-nodulation-division (RND) superfamily of proteins are invariably present in the genomes of Gram-negative bacteria and are largely responsible for the intrinsic antibiotic resistance of these organisms. The numbers of genes encoding RND transporters per genome vary from 1 to 16 and correlate with the environmental versatilities of bacterial species. Pseudomonas aeruginosa strain PAO1, a ubiquitous nosocomial pathogen, possesses 12 RND pumps, which are implicated in the development of clinical multidrug resistance and known to contribute to virulence, quorum sensing, and many other physiological functions. In this study, we analyzed how P. aeruginosa's physiology adapts to a lack of RND-mediated efflux activities. A combination of transcriptomics, metabolomics, genetic, and analytical approaches showed that the P. aeruginosa PΔ6 strain, lacking the six best-characterized RND pumps, activates a specific adaptation response that involves significant changes in the abundance and activities of several transport system, quorum sensing, iron acquisition, and lipid A modification pathways. Our results demonstrate that these cells accumulate large quantities of Pseudomonas quinolone signals (PQS), which triggers iron starvation and activation of siderophore biosynthesis and acquisition pathways. The accumulation of iron in turn activates lipid A modification and membrane protection pathways. A transcriptionally regulated RND pump, MuxABC-OpmB, contributes to these transformations by controlling the concentration of coumarins. Our results suggest that these changes reduce the permeability barrier of the outer membrane and are needed to protect the cell envelope of efflux-deficient P. aeruginosa.
Assuntos
Lipídeo A , Pseudomonas aeruginosa , Ferro , Proteínas de Membrana Transportadoras/genética , Pseudomonas aeruginosa/genética , Percepção de QuorumRESUMO
Drug discovery faces a crisis. The industry has used up the "obvious" space in which to find novel drugs for biomedical applications, and productivity is declining. One strategy to combat this is rational approaches to expand the search space without relying on chemical intuition, to avoid rediscovery of similar spaces. In this work, we present proof of concept of an approach to rationally identify a "chemical vocabulary" related to a specific drug activity of interest without employing known rules. We focus on the pressing concern of multidrug resistance in Pseudomonas aeruginosa by searching for submolecules that promote compound entry into this bacterium. By synergizing theory, computation, and experiment, we validate our approach, explain the molecular mechanism behind identified fragments promoting compound entry, and select candidate compounds from an external library that display good permeation ability.
Assuntos
Antibacterianos , Vocabulário , Algoritmos , Antibacterianos/farmacologia , Bactérias Gram-Negativas , Aprendizado de Máquina , Pseudomonas aeruginosaRESUMO
Genetic activation of the bacterial two-component signal transduction system, CpxRA, abolishes the virulence of a number of pathogens in human and murine infection models. Recently, 2,3,4,9-tetrahydro-1H-carbazol-1-amines were shown to activate the CpxRA system by inhibiting the phosphatase activity of CpxA. Herein we report the initial structure-activity relationships of this scaffold by focusing on three approaches 1) A-ring substitution, 2) B-ring deconstruction to provide N-arylated amino acid derivatives, and 3) C-ring elimination to give 2-ethylamino substituted indoles. These studies demonstrate that the A-ring is amenable to functionalization and provides a promising avenue for continued optimization of this chemotype. Further investigations revealed that the C-ring is not necessary for activity, although it likely provides conformational constraint that is beneficial to potency, and that the (R) stereochemistry is required at the primary amine. Simplification of the scaffold through deconstruction of the B-ring led to inactive compounds, highlighting the importance of the indole core. A new lead compound 26 was identified, which manifests a â¼30-fold improvement in CpxA phosphatase inhibition over the initial hit. Comparison of amino and des-amino derivatives in bacterial strains differing in membrane permeability and efflux capabilities demonstrate that the amine is required not only for target engagement but also for permeation and accumulation in Escherichia coli.
Assuntos
Carbazóis/uso terapêutico , Animais , Carbazóis/farmacologia , Humanos , Camundongos , Relação Estrutura-AtividadeRESUMO
Antibiotic-resistant Acinetobacter baumannii causes infections that are extremely difficult to treat. A significant role in these resistance profiles is attributed to multidrug efflux pumps, especially those belonging to the resistance-nodulation-cell division (RND) superfamily of transporters. In this study, we analyzed functions and properties of RND efflux pumps in A. baumannii ATCC 17978. This strain is susceptible to antibiotics and does not contain mutations that are commonly selected upon exposure to high concentrations of antibiotics. We constructed derivatives of ATCC 17978 lacking chromosomally encoded RND pumps and complemented these strains by the plasmid-borne genes. We analyzed the substrate selectivities and efficiencies of the individual pumps in the context of native outer membranes and their hyperporinated variants. Our results show that inactivation of AdeIJK provides the strongest potentiation of antibiotic activities, whereas inactivation of AdeFGH triggers the overexpression of AdeAB. The plasmid-borne overproduction complements the hypersusceptible phenotypes of the efflux deletion mutants to the levels of the parental ATCC 17978. Only a few antibiotics strongly benefitted from the overproduction of efflux pumps and antibacterial activities of some of those depended on the synergistic interaction with the low permeability barrier of the outer membrane. Either overproduction or inactivation of efflux pumps change dramatically the lipidome of ATCC 17978. We conclude that efflux pumps of A. baumannii are tightly integrated into physiology of this bacterium and that clinical levels of antibiotic resistance in A. baumannii isolates are unlikely to be reached solely due to the overproduction of RND efflux pumps.IMPORTANCE RND-type efflux pumps are important contributors in development of clinical antibiotic resistance in A. baumannii However, their specific roles and the extent of contribution to antibiotic resistance remain unclear. We analyzed antibacterial activities of antibiotics in strains with different permeability barriers and found that the role of active efflux in antibiotic resistance of A. baumannii is limited to a few select antibiotics. Our results further show that the impact of efflux pump overproduction on antibiotic susceptibility is significantly lower than the previously reported for clinical isolates. Additional mechanisms of resistance, in particular those that improve the permeability barriers of bacterial cells and act synergistically with active efflux pumps are likely involved in antibiotic resistance of clinical A. baumannii isolates.
Assuntos
Acinetobacter baumannii/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Especificidade por SubstratoRESUMO
The ability Gram-negative pathogens have at adapting and protecting themselves against antibiotics has increasingly become a public health threat. Data-driven models identifying molecular properties that correlate with outer membrane (OM) permeation and growth inhibition while avoiding efflux could guide the discovery of novel classes of antibiotics. Here we evaluate 174 molecular descriptors in 1260 antimicrobial compounds and study their correlations with antibacterial activity in Gram-negative Pseudomonas aeruginosa. The descriptors are derived from traditional approaches quantifying the compounds' intrinsic physicochemical properties, together with, bacterium-specific from ensemble docking of compounds targeting specific MexB binding pockets, and all-atom molecular dynamics simulations in different subregions of the OM model. Using these descriptors and the measured inhibitory concentrations, we design a statistical protocol to identify predictors of OM permeation/inhibition. We find consistent rules across most of our data highlighting the role of the interaction between the compounds and the OM. An implementation of the rules uncovered in our study is shown, and it demonstrates the accuracy of our approach in a set of previously unseen compounds. Our analysis sheds new light on the key properties drug candidates need to effectively permeate/inhibit P. aeruginosa, and opens the gate to similar data-driven studies in other Gram-negative pathogens.
RESUMO
Multidrug-resistant Acinetobacter baumannii is a serious threat pathogen rapidly spreading in clinics and causing a range of complicated human infections. The major contributor to A. baumannii antibiotic resistance is the overproduction of AdeIJK and AdeABC multidrug efflux pumps of the resistance-nodulation-division (RND) superfamily of proteins. The dominant role of efflux in antibiotic resistance and the relatively high permeability of the A. baumannii outer membrane to amphiphilic compounds make this pathogen a promising target for the discovery of clinically relevant efflux pump inhibitors. In this study, we identified 4,6-diaminoquoniline analogs with inhibitory activities against A. baumannii AdeIJK efflux pump and followed up on these compounds with a focused synthetic program to improve the target specificity and to reduce cytotoxicity. We identified several candidates that potentiate antibacterial activities of antibiotics erythromycin, tetracycline, and novobiocin not only in the laboratory antibiotic susceptible strain A. baumannii ATCC17978 but also in multidrug-resistant clinical isolates AB5075 and AYE. The best analogs potentiated the activities of antibiotics in low micromolar concentrations, did not have antibacterial activities on their own, inhibited AdeIJK-mediated efflux of its fluorescent substrate ethidium ion, and had low cytotoxicity in A549 human lung epithelial cells.
Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Antibacterianos , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade Microbiana , Acinetobacter baumannii/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/tratamento farmacológico , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Células A549 , Sinergismo FarmacológicoRESUMO
Multidrug efflux transporters are major contributors to the antibiotic resistance of Acinetobacter baumannii in clinical settings. Previous studies showed that these transporters are tightly integrated into the physiology of A. baumannii and have diverse functions. However, for many of the efflux pumps, such functions remain poorly defined. In this study, we characterized two putative drug efflux pumps, AmfAB and AmfCD (Acinetobacter Major Facilitator), that are homologous to EmrAB-like transporters from Escherichia coli and other Gram-negative bacteria. These pumps comprise the Major Facilitator Superfamily (MFS) transporters AmfB and AmfD and the periplasmic membrane fusion proteins AmfA and AmfC, respectively. We inactivated and overproduced these pumps in the wild-type ATCC 17978 strain and its derivative strains lacking the major efflux pumps from the Resistance-Nodulation-Division (RND) superfamily and characterized antibiotic susceptibilities and growth of the strains under stresses typical during human infections. We found that neither AmfAB nor AmfCD contribute to the antibiotic non-susceptibility phenotypes of A. baumannii. The two pumps, however, are critical for the adaptation and growth of the bacterium under acidic stress, whereas AmfCD also contributes to growth under conditions of low iron, high temperature, and in the presence of bile salts. These functions are dependent on the presence of the RND pumps, the inactivation of which further diminishes A. baumannii survival and growth. Our results suggest that MFS transporters contribute to stress survival by affecting the permeability properties of the A. baumannii cell envelope.
RESUMO
Transporters of the resistance-nodulation-cell division (RND) superfamily of proteins are the dominant multidrug efflux power of Gram-negative bacteria. The major RND efflux pump of Pseudomonas aeruginosa is MexAB-OprM, in which the inner membrane transporter MexB is responsible for the recognition and binding of compounds. The high importance of this pump in clinical antibiotic resistance made it a subject of intense investigations and a promising target for the discovery of efflux pump inhibitors. This study is focused on a series of peptidomimetic compounds developed as effective inhibitors of MexAB-OprM. We performed multi-copy molecular dynamics simulations, machine-learning (ML) analyses, and site-directed mutagenesis of MexB to investigate interactions of MexB with representatives of efflux avoiders, substrates, and inhibitors. The analysis of both direct and water-mediated protein-ligand interactions revealed characteristic patterns for each class, highlighting significant differences between them. We found that efflux avoiders poorly interact with the access binding site of MexB, and inhibition engages amino acid residues that are not directly involved in binding and transport of substrates. In agreement, machine-learning models selected different residues predictive of MexB substrates and inhibitors. The differences in interactions were further validated by site-directed mutagenesis. We conclude that the substrate translocation and inhibition pathways of MexB split at the interface (between the main putative binding sites) and at the deep binding pocket and that interactions outside of the hydrophobic patch contribute to the inhibition of MexB. This molecular-level information could help in the rational design of new inhibitors and antibiotics less susceptible to the efflux mechanism. IMPORTANCE Multidrug transporters recognize and expel from cells a broad range of ligands including their own inhibitors. The difference between the substrate translocation and inhibition routes remains unclear. In this study, machine learning and computational and experimental approaches were used to understand dynamics of MexB interactions with its ligands. Our results show that some ligands engage a certain combination of polar and charged residues in MexB binding sites to be effectively expelled into the exit funnel, whereas others engage aromatic and hydrophobic residues that slow down or hinder the next step in the transporter cycle. These findings suggest that all MexB ligands fit into this substrate-inhibitor spectrum depending on their physico-chemical structures and properties.
Assuntos
Proteínas da Membrana Bacteriana Externa , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Ligantes , Testes de Sensibilidade Microbiana , Proteínas de Membrana Transportadoras/metabolismoRESUMO
Bacterial drug-efflux transporters act synergistically with diffusion barriers of cellular membranes and other resistance mechanisms to protect cells from antibiotics and toxic metabolites. Their critical roles in clinical antibiotic and multidrug resistance are well established. In addition, a large body of evidence has been accumulated in support of their important contributions to bacterial growth and proliferation during infections. However, how these diverse functions of drug transporters are integrated at the level of bacterial cell physiology remains unclear. This opinion briefly summarizes the current understanding of substrate specificities and physiological roles of drug-efflux pumps from Resistance-Nodulation-Division (RND) superfamily of proteins in two ESKAPE pathogens Pseudomonas aeruginosa and Acinetobacter baumannii. Based on the analysis of phenotypic and transcriptomic studies in vitro and in vivo, we propose that RND pumps of Gram-negative bacteria fall into three categories: constitutively expressed, regulated, and silent. These three categories of efflux pumps participate in different physiological programs, which are not involved in the central metabolism and bacterial growth.
Assuntos
Acinetobacter baumannii , Farmacorresistência Bacteriana Múltipla , Acinetobacter baumannii/genética , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMO
The clinical success of linezolid for treating Gram-positive infections paired with the high conservation of bacterial ribosomes predicts that if oxazolidinones were engineered to accumulate in Gram-negative bacteria, then this pharmacological class would find broad utility in eradicating infections. Here, we report an investigative study of a strategically designed library of oxazolidinones to determine the effects of molecular structure on accumulation and biological activity. Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa strains with varying degrees of compromise (in efflux and outer membrane) were used to identify motifs that hinder permeation across the outer membrane and/or enhance efflux susceptibility broadly and specifically between species. The results illustrate that small changes in molecular structure are enough to overcome the efflux and/or permeation issues of this scaffold. Three oxazolidinone analogues (3e, 8d, and 8o) were identified that exhibit activity against all three pathogens assessed, a biological profile not observed for linezolid.
Assuntos
Oxazolidinonas , Oxazolidinonas/farmacologia , Oxazolidinonas/química , Linezolida/farmacologia , Testes de Sensibilidade Microbiana , Antibacterianos/química , Bactérias Gram-Negativas , Escherichia coliRESUMO
Two membrane cell envelopes act as selective permeability barriers in Gram-negative bacteria, protecting cells against antibiotics and other small molecules. Significant efforts are being directed toward understanding how small molecules permeate these barriers. In this study, we developed an approach to analyze the permeation of compounds into Gram-negative bacteria and applied it to Pseudomonas aeruginosa, an important human pathogen notorious for resistance to multiple antibiotics. The approach uses mass spectrometric measurements of accumulation of a library of structurally diverse compounds in four isogenic strains of P. aeruginosa with varied permeability barriers. We further developed a machine learning algorithm that generates a deterministic classification model with minimal synonymity between the descriptors. This model predicted good permeators into P. aeruginosa with an accuracy of 89% and precision above 58%. The good permeators are broadly distributed in the property space and can be mapped to six distinct regions representing diverse chemical scaffolds. We posit that this approach can be used for more detailed mapping of the property space and for rational design of compounds with high Gram-negative permeability.
Assuntos
Bactérias Gram-Negativas , Pseudomonas aeruginosa , Antibacterianos/química , Membrana Celular/metabolismo , Bactérias Gram-Negativas/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Permeabilidade , Pseudomonas aeruginosa/metabolismoRESUMO
Acinetobacter baumannii is a Gram-negative pathogen that has emerged as one of the most highly antibiotic-resistant bacteria worldwide. Multidrug efflux within these highly drug-resistant strains and other opportunistic pathogens is a major cause of failure of drug-based treatments of infectious diseases. The best-characterized multidrug efflux system in A. baumannii is the prevalent Acinetobacterdrug efflux B (AdeB) pump, which is a member of the resistance-nodulation-cell division (RND) superfamily. Here, we report six structures of the trimeric AdeB multidrug efflux pump in the presence of ethidium bromide using single-particle cryoelectron microscopy (cryo-EM). These structures allow us to directly observe various novel conformational states of the AdeB trimer, including the transmembrane region of trimeric AdeB can be associated with form a trimer assembly or dissociated into "dimer plus monomer" and "monomer plus monomer plus monomer" configurations. We also discover that a single AdeB protomer can simultaneously anchor a number of ethidium ligands and that different AdeB protomers can bind ethidium molecules simultaneously. Combined with molecular dynamics (MD) simulations, we reveal a drug transport mechanism that involves multiple multidrug-binding sites and various transient states of the AdeB membrane protein. Our data suggest that each AdeB protomer within the trimer binds and exports drugs independently.IMPORTANCEAcinetobacter baumannii has emerged as one of the most highly antibiotic-resistant Gram-negative pathogens. The prevalent AdeB multidrug efflux pump mediates resistance to a broad spectrum of clinically relevant antimicrobial agents. Here, we report six cryo-EM structures of the trimeric AdeB pump in the presence of ethidium bromide. We discover that a single AdeB protomer can simultaneously anchor a number of ligands, and different AdeB protomers can bind ethidium molecules simultaneously. The results indicate that each AdeB protomer within the trimer recognizes and extrudes drugs independently.
Assuntos
Acinetobacter baumannii/genética , Proteínas de Bactérias/química , Microscopia Crioeletrônica , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Membrana Transportadoras/química , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/ultraestrutura , Antibacterianos/farmacologia , Proteínas de Bactérias/ultraestrutura , Divisão Celular/efeitos dos fármacos , Etídio/farmacologia , Proteínas de Membrana Transportadoras/ultraestruturaRESUMO
The structure of the TriABC inner membrane component of the triclosan/SDS-specific efflux pump from Pseudomonas aeruginosa was determined by cryoelectron microscopy to 4.5 Å resolution. The complete structure of the inner membrane transporter TriC of the resistance-nodulation-division (RND) superfamily was solved, including a partial structure of the fused periplasmic membrane fusion subunits, TriA and TriB. The substrate-free conformation of TriABC represents an intermediate step in efflux complex assembly before the engagement of the outer membrane channel. Structural analysis identified a tunnel network whose constriction impedes substrate efflux, indicating inhibition of TriABC in the unengaged state. Blind docking studies revealed binding to TriC at the same loci by substrates and bulkier non-substrates. Together with functional analyses, we propose that selective substrate translocation involves conformational gating at the tunnel narrowing that, together with conformational ordering of TriA and TriB, creates an engaged state capable of mediating substrate efflux.