RESUMEN
ß-lactam antibiotics inhibit bacterial cell wall assembly and, under classical microbiological culture conditions that are generally hypotonic, induce explosive cell death. Here, we show that under more physiological, osmoprotective conditions, for various Gram-positive bacteria, lysis is delayed or abolished, apparently because inhibition of class A penicillin-binding protein leads to a block in autolytic activity. Although these cells still then die by other mechanisms, exogenous lytic enzymes, such as lysozyme, can rescue viability by enabling the escape of cell wall-deficient "L-form" bacteria. This protective L-form conversion was also observed in macrophages and in an animal model, presumably due to the production of host lytic activities, including lysozyme. Our results demonstrate the potential for L-form switching in the host environment and highlight the unexpected effects of innate immune effectors, such as lysozyme, on antibiotic activity. Unlike previously described dormant persisters, L-forms can continue to proliferate in the presence of antibiotic.
Asunto(s)
Antibacterianos/farmacología , Formas L/efectos de los fármacos , Muramidasa/metabolismo , beta-Lactamas/farmacología , Animales , Bacillus subtilis/efectos de los fármacos , Bacteriólisis/efectos de los fármacos , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Hidrolasas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Viabilidad Microbiana/efectos de los fármacos , Osmorregulación/efectos de los fármacos , Penicilina G/farmacología , Proteínas de Unión a las Penicilinas , Peptidoglicano/metabolismo , Profagos/efectos de los fármacos , Células RAW 264.7RESUMEN
Antibiotic-resistant Enterobacterales pose a major threat to healthcare systems worldwide, necessitating the development of novel strategies to fight such hard-to-kill bacteria. One potential approach is to develop molecules that force bacteria to hyper-activate prodrug antibiotics, thus rendering them more effective. In the present work, we aimed to obtain proof-of-concept data to support that small molecules targeting transcriptional regulators can potentiate the antibiotic activity of the prodrug metronidazole (MTZ) against Escherichia coli under aerobic conditions. By screening a chemical library of small molecules, a series of structurally related molecules were identified that had little inherent antibiotic activity but showed substantial activity in combination with ineffective concentrations of MTZ. Transcriptome analyses, functional genetics, thermal shift assays, and electrophoretic mobility shift assays were then used to demonstrate that these MTZ boosters target the transcriptional repressor MarR, resulting in the upregulation of the marRAB operon and its downstream MarA regulon. The associated upregulation of the flavin-containing nitroreductase, NfsA, was then shown to be critical for the booster-mediated potentiation of MTZ antibiotic activity. Transcriptomic studies, biochemical assays, and electron paramagnetic resonance measurements were then used to show that under aerobic conditions, NfsA catalyzed 1-electron reduction of MTZ to the MTZ radical anion which in turn induced lethal DNA damage in E. coli. This work reports the first example of prodrug boosting in Enterobacterales by transcriptional modulators and highlights that MTZ antibiotic activity can be chemically induced under anaerobic growth conditions.
Asunto(s)
Antibacterianos , Proteínas de Escherichia coli , Escherichia coli , Metronidazol , Nitrorreductasas , Proteínas Represoras , Nitrorreductasas/metabolismo , Nitrorreductasas/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Escherichia coli/genética , Metronidazol/farmacología , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Antibacterianos/farmacología , Antibacterianos/química , Aerobiosis , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Methicillin-resistant Staphylococcus aureus, or MRSA, is one of the major causative agents of hospital-acquired infections worldwide. Novel antimicrobial strategies efficient against antibiotic-resistant strains are necessary and not only against S. aureus. Among those, strategies that aim at blocking or dismantling proteins involved in the acquisition of essential nutrients, helping the bacteria to colonize the host, are intensively studied. A major route for S. aureus to acquire iron from the host organism is the Isd (iron surface determinant) system. In particular, the hemoglobin receptors IsdH and IsdB located on the surface of the bacterium are necessary to acquire the heme moiety containing iron, making them a plausible antibacterial target. Herein, we obtained an antibody of camelid origin that blocked heme acquisition. We determined that the antibody recognized the heme-binding pocket of both IsdH and IsdB with nanomolar order affinity through its second and third complementary-determining regions. The mechanism explaining the inhibition of acquisition of heme in vitro could be described as a competitive process in which the complementary-determining region 3 from the antibody blocked the acquisition of heme by the bacterial receptor. Moreover, this antibody markedly reduced the growth of three different pathogenic strains of MRSA. Collectively, our results highlight a mechanism for inhibiting nutrient uptake as an antibacterial strategy against MRSA.
Asunto(s)
Anticuerpos Antibacterianos , Staphylococcus aureus Resistente a Meticilina , Receptores de Superficie Celular , Anticuerpos de Dominio Único , Humanos , Antibacterianos/farmacología , Hemo/metabolismo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/uso terapéutico , Anticuerpos de Dominio Único/biosíntesis , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/farmacología , Infecciones Estafilocócicas/tratamiento farmacológico , Antígenos Bacterianos/inmunología , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Camélidos del Nuevo Mundo , Animales , Unión Proteica/efectos de los fármacos , Modelos Moleculares , Simulación de Dinámica MolecularRESUMEN
The relative lack of marine venom could be attributed to the difficulty in dealing with venomous marine animals. Moreover, the venom of marine animals consists of various bioactive molecules, many of which are proteins with unique properties. In this study, we investigated the potential toxic proteins of jellyfish collected for ligand screening to understand the mechanism of the toxic effects of jellyfish. Since taxonomic identification is problematic due to the lack of proper keys, we conducted morphological and molecular mitochondrial DNA sequencing from COI and ITS regions. The venom extract from nematocysts found along the bell margins was used for protein characterization using SDS-gel electrophoresis and nano-liquid chromatography-tandem mass spectrometry. Ligand screening for the most potent toxin and antibacterial and cytotoxicity assays were carried out. The phylogenetic tree showed distinct clustering from other Catostylus sp. The proteomic analysis revealed venom with many bioactive proteins. Only 13 venom proteins were identified with molecular weights ranging from 4318 to 184,923 Da, exhibiting the venom's complexity. The overall toxin protein composition of Catostylus sp. venom was dominated by potassium channel toxin alpha-KTx. Molecular docking of toxin alpha-KTx 1.13 revealed high specificity towards the human voltage-gated potassium channel Kv3 with a high fitness score and a minimum energy barrier of -17.9 kcal/mol. Disc diffusion and cytotoxicity assays revealed potent antibacterial activity against Klebsiella pneumoniae with no cytotoxicity. Further studies on detailed characterization and therapeutic potentials are warranted.
Asunto(s)
Antibacterianos , Venenos de Cnidarios , Simulación del Acoplamiento Molecular , Péptidos , Animales , Antibacterianos/farmacología , Antibacterianos/química , Humanos , Venenos de Cnidarios/farmacología , Venenos de Cnidarios/química , Péptidos/farmacología , Péptidos/química , Escifozoos , Ligandos , Filogenia , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Proteómica/métodosRESUMEN
The ring-forming AAA+ hexamer ClpC1 associates with the peptidase ClpP1P2 to form a central ATP-driven protease in Mycobacterium tuberculosis (Mtb). ClpC1 is essential for Mtb viability and has been identified as the target of antibacterial peptides like CyclomarinA (CymA) that exhibit strong toxicity toward Mtb. The mechanistic actions of these drugs are poorly understood. Here, we dissected how ClpC1 activity is controlled and how this control is deregulated by CymA. We show that ClpC1 exists in diverse activity states correlating with its assembly. The basal activity of ClpC1 is low, as it predominantly exists in an inactive nonhexameric resting state. We show that CymA stimulates ClpC1 activity by promoting formation of supercomplexes composed of multiple ClpC1 hexameric rings, enhancing ClpC1-ClpP1P2 degradation activity toward various substrates. Both the ClpC1 resting state and the CymA-induced alternative assembly state rely on interactions between the ClpC1 coiled-coil middle domains (MDs). Accordingly, we found that mutation of the conserved aromatic F444 residue located at the MD tip blocks MD interactions and prevents assembly into higher order complexes, thereby leading to constitutive ClpC1 hexamer formation. We demonstrate that this assembly state exhibits the highest ATPase and proteolytic activities, yet its heterologous expression in Escherichia coli is toxic, indicating that the formation of such a state must be tightly controlled. Taken together, these findings define the basis of control of ClpC1 activity and show how ClpC1 overactivation by an antibacterial drug generates toxicity.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Endopeptidasa Clp/metabolismo , Proteínas de Choque Térmico/metabolismo , Mycobacterium tuberculosis , Oligopéptidos/farmacología , Proteínas Bacterianas/química , Endopeptidasa Clp/química , Endopeptidasas/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico/química , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/metabolismo , Péptido Hidrolasas/metabolismo , Péptidos/metabolismoRESUMEN
RNA polymerase (RNAP) binding protein A (RbpA) is essential for mycobacterial viability and regulates transcription initiation by increasing the stability of the RNAP-promoter open complex (RPo). RbpA consists of four domains: an N-terminal tail (NTT), a core domain (CD), a basic linker, and a sigma interaction domain. We have previously shown that truncation of the RbpA NTT and CD increases RPo stabilization by RbpA, implying that these domains inhibit this activity of RbpA. Previously published structural studies showed that the NTT and CD are positioned near multiple RNAP-σA holoenzyme functional domains and predict that the RbpA NTT contributes specific amino acids to the binding site of the antibiotic fidaxomicin (Fdx), which inhibits the formation of the RPo complex. Furthermore, deletion of the NTT results in decreased Mycobacterium smegmatis sensitivity to Fdx, but whether this is caused by a loss in Fdx binding is unknown. We generated a panel of rbpA mutants and found that the RbpA NTT residues predicted to directly interact with Fdx are partially responsible for RbpA-dependent Fdx activity in vitro, while multiple additional RbpA domains contribute to Fdx activity in vivo. Specifically, our results suggest that the RPo-stabilizing activity of RbpA decreases Fdx activity in vivo. In support of the association between RPo stability and Fdx activity, we find that another factor that promotes RPo stability in bacteria, CarD, also impacts to Fdx sensitivity. Our findings highlight how RbpA and other factors may influence RNAP dynamics to affect Fdx sensitivity.
Asunto(s)
Fidaxomicina , Mycobacterium smegmatis , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Fidaxomicina/farmacología , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/genética , Regiones Promotoras Genéticas , Factor sigma/metabolismoRESUMEN
Second messenger molecules play important roles in the responses to various stimuli that can determine a cell's fate under stress conditions. Here, we report that lethal concentrations of aminoglycoside antibiotics result in the production of a dinucleotide alarmone metabolite-diadenosine tetraphosphate (Ap4A), which promotes bacterial cell killing by this class of antibiotics. We show that the treatment of Escherichia coli with lethal concentrations of kanamycin (Kan) dramatically increases the production of Ap4A. This elevation of Ap4A is dependent on the production of a hydroxyl radical and involves the induction of the Ap4A synthetase lysyl-tRNA synthetase (LysU). Ectopic alteration of intracellular Ap4A concentration via the elimination of the Ap4A phosphatase diadenosine tetraphosphatase (ApaH) and the overexpression of LysU causes over a 5,000-fold increase in bacterial killing by aminoglycosides. This increased susceptibility to aminoglycosides correlates with bacterial membrane disruption. Our findings provide a role for the alarmone Ap4A and suggest that blocking Ap4A degradation or increasing its synthesis might constitute an approach to enhance aminoglycoside killing potency by broadening their therapeutic index and thereby allowing lower nontoxic dosages of these antibiotics to be used in the treatment of multidrug-resistant infections.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Antibacterianos/farmacología , Proteínas Portadoras/metabolismo , Fosfatos de Dinucleósidos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Kanamicina/farmacología , Ácido Anhídrido Hidrolasas/genética , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Proteínas Portadoras/genética , Fosfatos de Dinucleósidos/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMEN
An important mechanism of resistance to ß-lactam antibiotics is via their ß-lactamase-catalyzed hydrolysis. Recent work has shown that, in addition to the established hydrolysis products, the reaction of the class D nucleophilic serine ß-lactamases (SBLs) with carbapenems also produces ß-lactones. We report studies on the factors determining ß-lactone formation by class D SBLs. We show that variations in hydrophobic residues at the active site of class D SBLs (i.e. Trp105, Val120, and Leu158, using OXA-48 numbering) impact on the relative levels of ß-lactones and hydrolysis products formed. Some variants, i.e. the OXA-48 V120L and OXA-23 V128L variants, catalyze increased ß-lactone formation compared with the WT enzymes. The results of kinetic and product studies reveal that variations of residues other than those directly involved in catalysis, including those arising from clinically observed mutations, can alter the reaction outcome of class D SBL catalysis. NMR studies show that some class D SBL variants catalyze formation of ß-lactones from all clinically relevant carbapenems regardless of the presence or absence of a 1ß-methyl substituent. Analysis of reported crystal structures for carbapenem-derived acyl-enzyme complexes reveals preferred conformations for hydrolysis and ß-lactone formation. The observation of increased ß-lactone formation by class D SBL variants, including the clinically observed carbapenemase OXA-48 V120L, supports the proposal that class D SBL-catalyzed rearrangement of ß-lactams to ß-lactones is important as a resistance mechanism.
Asunto(s)
Proteínas Bacterianas/metabolismo , Lactonas/metabolismo , beta-Lactamasas/metabolismo , Acinetobacter baumannii/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Farmacorresistencia Microbiana , Hidrólisis , Cinética , Lactonas/química , Espectroscopía de Resonancia Magnética , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , beta-Lactamasas/química , beta-Lactamasas/genéticaRESUMEN
The octapeptins are lipopeptide antibiotics that are structurally similar to polymyxins yet retain activity against polymyxin-resistant Gram-negative pathogens, suggesting they might be used to treat recalcitrant infections. However, the basis of their unique activity is unclear because of the difficulty in generating high-resolution experimental data of the interaction of antimicrobial peptides with lipid membranes. To elucidate these structure-activity relationships, we employed all-atom molecular dynamics simulations with umbrella sampling to investigate the conformational and energetic landscape of octapeptins interacting with bacterial outer membrane (OM). Specifically, we examined the interaction of octapeptin C4 and FADDI-115, lacking a single hydroxyl group compared with octapeptin C4, with the lipid A-phosphoethanolamine modified OM of Acinetobacter baumannii Octapeptin C4 and FADDI-115 both penetrated into the OM hydrophobic center but experienced different conformational transitions from an unfolded to a folded state that was highly dependent on the structural flexibility of their respective N-terminal fatty acyl groups. The additional hydroxyl group present in the fatty acyl group of octapeptin C4 resulted in the molecule becoming trapped in a semifolded state, leading to a higher free energy barrier for OM penetration. The free energy barrier for the translocation through the OM hydrophobic layer was â¼72 kcal/mol for octapeptin C4 and 62 kcal/mol for FADDI-115. Our results help to explain the lower antimicrobial activity previously observed for octapeptin C4 compared with FADDI-115 and more broadly improve our understanding of the structure-function relationships of octapeptins. These findings may facilitate the discovery of next-generation octapeptins against polymyxin-resistant Gram-negative 'superbugs.'
Asunto(s)
Acinetobacter baumannii/química , Membrana Celular/química , Lipopéptidos/química , Simulación de Dinámica Molecular , Relación Estructura-ActividadRESUMEN
Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major human pathogen, and current treatment options to combat this disease are under threat because of the emergence of multidrug-resistant and extensively drug-resistant tuberculosis. High-throughput whole-cell screening of an extensive compound library has recently identified a piperidinol-containing molecule, PIPD1, as a potent lead compound against M. tuberculosis Herein, we show that PIPD1 and related analogs exert in vitro bactericidal activity against the M. tuberculosis strain mc26230 and also against a panel of multidrug-resistant and extensively drug-resistant clinical isolates of M. tuberculosis, suggesting that PIPD1's mode of action differs from those of most first- and second-line anti-tubercular drugs. Selection and DNA sequencing of PIPD1-resistant mycobacterial mutants revealed the presence of single-nucleotide polymorphisms in mmpL3, encoding an inner membrane-associated mycolic acid flippase in M. tuberculosis Results from functional assays with spheroplasts derived from a M. smegmatis strain lacking the endogenous mmpL3 gene but harboring the M. tuberculosis mmpL3 homolog indicated that PIPD1 inhibits the MmpL3-driven translocation of trehalose monomycolate across the inner membrane without altering the proton motive force. Using a predictive structural model of MmpL3 from M. tuberculosis, docking studies revealed a PIPD1-binding cavity recently found to accommodate different inhibitors in M. smegmatis MmpL3. In conclusion, our findings have uncovered bactericidal activity of a new chemical scaffold. Its anti-tubercular activity is mediated by direct inhibition of the flippase activity of MmpL3 rather than by inhibition of the inner membrane proton motive force, significantly advancing our understanding of MmpL3-targeted inhibition in mycobacteria.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Ácidos Micólicos/metabolismo , Piperidinas/farmacología , Antituberculosos/química , Proteínas Bacterianas/metabolismo , Transporte Biológico/efectos de los fármacos , Factores Cordón/metabolismo , Humanos , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Piperidinas/química , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Aminoacyl-tRNA synthetases (ARSs) are universal enzymes that catalyze the attachment of amino acids to the 3' ends of their cognate tRNAs. The resulting aminoacylated tRNAs are escorted to the ribosome where they enter protein synthesis. By specifically matching amino acids to defined anticodon sequences in tRNAs, ARSs are essential to the physical interpretation of the genetic code. In addition to their canonical role in protein synthesis, ARSs are also involved in RNA splicing, transcriptional regulation, translation, and other aspects of cellular homeostasis. Likewise, aminoacylated tRNAs serve as amino acid donors for biosynthetic processes distinct from protein synthesis, including lipid modification and antibiotic biosynthesis. Thanks to the wealth of details on ARS structures and functions and the growing appreciation of their additional roles regulating cellular homeostasis, opportunities for the development of clinically useful ARS inhibitors are emerging to manage microbial and parasite infections. Exploitation of these opportunities has been stimulated by the discovery of new inhibitor frameworks, the use of semi-synthetic approaches combining chemistry and genome engineering, and more powerful techniques for identifying leads from the screening of large chemical libraries. Here, we review the inhibition of ARSs by small molecules, including the various families of natural products, as well as inhibitors developed by either rational design or high-throughput screening as antibiotics and anti-parasitic therapeutics.
Asunto(s)
Aminoacil-ARNt Sintetasas , Antibacterianos , Antiparasitarios , Inhibidores Enzimáticos , Infecciones , Enfermedades Parasitarias , Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Animales , Antibacterianos/química , Antibacterianos/uso terapéutico , Antiparasitarios/química , Antiparasitarios/uso terapéutico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Humanos , Infecciones/tratamiento farmacológico , Infecciones/enzimología , Infecciones/genética , Infecciones/patología , Enfermedades Parasitarias/tratamiento farmacológico , Enfermedades Parasitarias/enzimología , Enfermedades Parasitarias/genética , Empalme del ARN/efectos de los fármacos , ARN de Transferencia/genética , ARN de Transferencia/metabolismoRESUMEN
Resistance of Neisseria gonorrhoeae to extended-spectrum cephalosporins (ESCs) has become a major threat to human health. The primary mechanism by which N. gonorrhoeae becomes resistant to ESCs is by acquiring a mosaic penA allele, encoding penicillin-binding protein 2 (PBP2) variants containing up to 62 mutations compared with WT, of which a subset contribute to resistance. To interpret molecular mechanisms underpinning cephalosporin resistance, it is necessary to know how PBP2 is acylated by ESCs. Here, we report the crystal structures of the transpeptidase domain of WT PBP2 in complex with cefixime and ceftriaxone, along with structures of PBP2 in the apo form and with a phosphate ion bound in the active site at resolutions of 1-7-1.9 Å. These structures reveal that acylation of PBP2 by ESCs is accompanied by rotation of the Thr-498 side chain in the KTG motif to contact the cephalosporin carboxylate, twisting of the ß3 strand to form the oxyanion hole, and rolling of the ß3-ß4 loop toward the active site. Recognition of the cephalosporin carboxylate appears to be the key trigger for formation of an acylation-competent state of PBP2. The structures also begin to explain the impact of mutations implicated in ESC resistance. In particular, a G545S mutation may hinder twisting of ß3 because its side chain hydroxyl forms a hydrogen bond with Thr-498. Overall, our data suggest that acylation is initiated by conformational changes elicited or trapped by binding of ESCs and that these movements are restricted by mutations associated with resistance against ESCs.
Asunto(s)
D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/genética , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/metabolismo , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/ultraestructura , Acilación , Alelos , Sitios de Unión/efectos de los fármacos , Dominio Catalítico , Cefixima/farmacología , Ceftriaxona/farmacología , Resistencia a las Cefalosporinas , Cefalosporinas/farmacología , Gonorrea/genética , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismo , Proteínas de Unión a las Penicilinas/químicaRESUMEN
The final steps of cell-wall biosynthesis in bacteria are carried out by penicillin-binding proteins (PBPs), whose transpeptidase domains form the cross-links in peptidoglycan chains that define the bacterial cell wall. These enzymes are the targets of ß-lactam antibiotics, as their inhibition reduces the structural integrity of the cell wall. Bacterial resistance to antibiotics is a rapidly growing concern; however, the structural underpinnings of PBP-derived antibiotic resistance are poorly understood. PBP4 and PBP5 are low-affinity, class B transpeptidases that confer antibiotic resistance to Enterococcus faecalis and Enterococcus faecium, respectively. Here, we report the crystal structures of PBP4 (1.8 Å) and PBP5 (2.7 Å) in their apo and acyl-enzyme complexes with the ß-lactams benzylpenicillin, imipenem, and ceftaroline. We found that, although these three ß-lactams adopt geometries similar to those observed in other class B PBP structures, there are small, but significant, differences that likely decrease antibiotic efficacy. Further, we also discovered that the N-terminal domain extensions in this class of PBPs undergo large rigid-body rotations without impacting the structure of the catalytic transpeptidase domain. Together, our findings are defining the subtle functional and structural differences in the Enterococcus PBPs that allow them to support transpeptidase activity while also conferring bacterial resistance to antibiotics that function as substrate mimics.
Asunto(s)
Proteínas Bacterianas/química , Enterococcus faecalis/metabolismo , Enterococcus faecium/metabolismo , Proteínas de Unión a las Penicilinas/química , Isoformas de Proteínas/química , Resistencia betalactámica , Acilación , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Carbapenémicos/farmacología , Dominio Catalítico , Cefalosporinas/farmacología , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecium/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/aislamiento & purificación , Proteínas de Unión a las Penicilinas/metabolismo , Penicilinas/metabolismo , Conformación Proteica , Dominios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Resistencia betalactámica/genéticaRESUMEN
Mycobacterium tuberculosis can remain dormant in the host, an ability that explains the failure of many current tuberculosis treatments. Recently, the natural products cyclomarin, ecumicin, and lassomycin have been shown to efficiently kill Mycobacterium tuberculosis persisters. Their target is the N-terminal domain of the hexameric AAA+ ATPase ClpC1, which recognizes, unfolds, and translocates protein substrates, such as proteins containing phosphorylated arginine residues, to the ClpP1P2 protease for degradation. Surprisingly, these antibiotics do not inhibit ClpC1 ATPase activity, and how they cause cell death is still unclear. Here, using NMR and small-angle X-ray scattering, we demonstrate that arginine-phosphate binding to the ClpC1 N-terminal domain induces millisecond dynamics. We show that these dynamics are caused by conformational changes and do not result from unfolding or oligomerization of this domain. Cyclomarin binding to this domain specifically blocked these N-terminal dynamics. On the basis of these results, we propose a mechanism of action involving cyclomarin-induced restriction of ClpC1 dynamics, which modulates the chaperone enzymatic activity leading eventually to cell death.
Asunto(s)
Antibacterianos/farmacología , Arginina/análogos & derivados , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Oligopéptidos/farmacología , Tuberculosis/tratamiento farmacológico , Arginina/farmacología , Muerte Celular , Cristalografía por Rayos X , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Transporte Iónico , Compuestos Organofosforados/farmacología , Fosforilación , Conformación Proteica , Dominios Proteicos , Tuberculosis/metabolismo , Tuberculosis/microbiologíaRESUMEN
Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel ß-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected ß-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many ß-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of ß-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target ß-barrel proteins and the integrity of the Gram-negative OM.
Asunto(s)
Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Proteínas de Escherichia coli/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Péptidos/farmacología , Peptidomiméticos/farmacología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMEN
Allicin (diallyl thiosulfinate) from garlic is a highly potent natural antimicrobial substance. It inhibits growth of a variety of microorganisms, among them antibiotic-resistant strains. However, the precise mode of action of allicin is unknown. Here, we show that growth inhibition of Escherichia coli during allicin exposure coincides with a depletion of the glutathione pool and S-allylmercapto modification of proteins, resulting in overall decreased total sulfhydryl levels. This is accompanied by the induction of the oxidative and heat stress response. We identified and quantified the allicin-induced modification S-allylmercaptocysteine for a set of cytoplasmic proteins by using a combination of label-free mass spectrometry and differential isotope-coded affinity tag labeling of reduced and oxidized thiol residues. Activity of isocitrate lyase AceA, an S-allylmercapto-modified candidate protein, is largely inhibited by allicin treatment in vivo Allicin-induced protein modifications trigger protein aggregation, which largely stabilizes RpoH and thereby induces the heat stress response. At sublethal concentrations, the heat stress response is crucial to overcome allicin stress. Our results indicate that the mode of action of allicin is a combination of a decrease of glutathione levels, unfolding stress, and inactivation of crucial metabolic enzymes through S-allylmercapto modification of cysteines.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Extractos Vegetales/farmacología , Compuestos de Sulfhidrilo/metabolismo , Ácidos Sulfínicos/farmacología , Cisteína/metabolismo , Disulfuros , Escherichia coli/metabolismo , Ajo/química , Glutatión/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacosRESUMEN
Fluoroquinolones (FQ) are powerful broad-spectrum antibiotics whose side effects include renal damage and, strangely, tendinopathies. The pathological mechanisms underlying these toxicities are poorly understood. Here, we show that the FQ drugs norfloxacin, ciprofloxacin, and enrofloxacin are powerful iron chelators comparable with deferoxamine, a clinically useful iron-chelating agent. We show that iron chelation by FQ leads to epigenetic effects through inhibition of α-ketoglutarate-dependent dioxygenases that require iron as a co-factor. Three dioxygenases were examined in HEK293 cells treated with FQ. At sub-millimolar concentrations, these antibiotics inhibited jumonji domain histone demethylases, TET DNA demethylases, and collagen prolyl 4-hydroxylases, leading to accumulation of methylated histones and DNA and inhibition of proline hydroxylation in collagen, respectively. These effects may explain FQ-induced nephrotoxicity and tendinopathy. By the same reasoning, dioxygenase inhibition by FQ was predicted to stabilize transcription factor HIF-1α by inhibition of the oxygen-dependent hypoxia-inducible transcription factor prolyl hydroxylation. In dramatic contrast to this prediction, HIF-1α protein was eliminated by FQ treatment. We explored possible mechanisms for this unexpected effect and show that FQ inhibit HIF-1α mRNA translation. Thus, FQ antibiotics induce global epigenetic changes, inhibit collagen maturation, and block HIF-1α accumulation. We suggest that these mechanisms explain the classic renal toxicities and peculiar tendinopathies associated with FQ antibiotics.
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Dioxigenasas/antagonistas & inhibidores , Epigénesis Genética/efectos de los fármacos , Fluoroquinolonas/farmacología , Quelantes del Hierro/farmacología , Antibacterianos/química , Antibacterianos/farmacología , Ciprofloxacina/química , Ciprofloxacina/farmacología , Metilación de ADN/efectos de los fármacos , Deferoxamina/química , Deferoxamina/farmacología , Dioxigenasas/genética , Dioxigenasas/metabolismo , Enrofloxacina , Epigénesis Genética/genética , Fluoroquinolonas/química , Células HEK293 , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Histonas/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Immunoblotting , Hierro/metabolismo , Quelantes del Hierro/química , Metilación/efectos de los fármacos , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Estructura Molecular , Norfloxacino/química , Norfloxacino/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We characterized the inhibition of Neisseria gonorrhoeae type II topoisomerases gyrase and topoisomerase IV by AZD0914 (AZD0914 will be henceforth known as ETX0914 (Entasis Therapeutics)), a novel spiropyrimidinetrione antibacterial compound that is currently in clinical trials for treatment of drug-resistant gonorrhea. AZD0914 has potent bactericidal activity against N. gonorrhoeae, including multidrug-resistant strains and key Gram-positive, fastidious Gram-negative, atypical, and anaerobic bacterial species (Huband, M. D., Bradford, P. A., Otterson, L. G., Basrab, G. S., Giacobe, R. A., Patey, S. A., Kutschke, A. C., Johnstone, M. R., Potter, M. E., Miller, P. F., and Mueller, J. P. (2014) In Vitro Antibacterial Activity of AZD0914: A New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-positive, Fastidious Gram-negative, and Atypical Bacteria. Antimicrob. Agents Chemother. 59, 467-474). AZD0914 inhibited DNA biosynthesis preferentially to other macromolecules in Escherichia coli and induced the SOS response to DNA damage in E. coli. AZD0914 stabilized the enzyme-DNA cleaved complex for N. gonorrhoeae gyrase and topoisomerase IV. The potency of AZD0914 for inhibition of supercoiling and the stabilization of cleaved complex by N. gonorrhoeae gyrase increased in a fluoroquinolone-resistant mutant enzyme. When a mutation, conferring mild resistance to AZD0914, was present in the fluoroquinolone-resistant mutant, the potency of ciprofloxacin for inhibition of supercoiling and stabilization of cleaved complex was increased greater than 20-fold. In contrast to ciprofloxacin, religation of the cleaved DNA did not occur in the presence of AZD0914 upon removal of magnesium from the DNA-gyrase-inhibitor complex. AZD0914 had relatively low potency for inhibition of human type II topoisomerases α and ß.
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Antibacterianos/farmacología , Barbitúricos/farmacología , Girasa de ADN/metabolismo , Topoisomerasa de ADN IV/antagonistas & inhibidores , ADN Bacteriano/química , Compuestos de Espiro/farmacología , Inhibidores de Topoisomerasa II/farmacología , Ciprofloxacina/farmacología , Ensayos Clínicos como Asunto , ADN/química , ADN/metabolismo , Girasa de ADN/genética , Topoisomerasa de ADN IV/genética , Topoisomerasa de ADN IV/metabolismo , ADN Bacteriano/metabolismo , Farmacorresistencia Bacteriana/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Fluoroquinolonas/farmacología , Expresión Génica , Humanos , Isoxazoles , Morfolinas , Mutación , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/enzimología , Neisseria gonorrhoeae/genética , Oxazolidinonas , Especificidad de la EspecieRESUMEN
The antibiotic fusidic acid (FA) targets elongation factor G (EF-G) and inhibits ribosomal peptide elongation and ribosome recycling, but deeper mechanistic aspects of FA action have remained unknown. Using quench flow and stopped flow experiments in a biochemical system for protein synthesis and taking advantage of separate time scales for inhibited (10 s) and uninhibited (100 ms) elongation cycles, a detailed kinetic model of FA action was obtained. FA targets EF-G at an early stage in the translocation process (I), which proceeds unhindered by the presence of the drug to a later stage (II), where the ribosome stalls. Stalling may also occur at a third stage of translocation (III), just before release of EF-G from the post-translocation ribosome. We show that FA is a strong elongation inhibitor (K50% ≈ 1 µm), discuss the identity of the FA targeted states, and place existing cryo-EM and crystal structures in their functional context.
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Antibacterianos/farmacología , Ácido Fusídico/farmacología , Factor G de Elongación Peptídica/antagonistas & inhibidores , Inhibidores de la Síntesis de la Proteína/farmacología , Relación Dosis-Respuesta a Droga , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Factor G de Elongación Peptídica/metabolismo , Ribosomas/metabolismoRESUMEN
Aminoglycosides are a well known antibiotic family used to treat bacterial infections in humans and animals, but which can be toxic. By binding to the decoding site of helix44 of the small subunit RNA of the bacterial ribosome, the aminoglycoside antibiotics inhibit protein synthesis, cause misreading, or obstruct peptidyl-tRNA translocation. Although aminoglycosides bind helix69 of the bacterial large subunit RNA as well, little is known about their interaction with the homologous human helix69. To probe the role this binding event plays in toxicity, changes to thermal stability, base stacking, and conformation upon aminoglycoside binding to the human cytoplasmic helix69 were compared with those of the human mitochondrial and Escherichia coli helix69. Surprisingly, binding of gentamicin and kanamycin A to the chemically synthesized terminal hairpins of the human cytoplasmic, human mitochondrial, and E. coli helix69 revealed similar dissociation constants (1.3-1.7 and 4.0-5.4 µM, respectively). In addition, aminoglycoside binding enhanced conformational stability of the human mitochondrial helix69 by increasing base stacking. Proton one-dimensional and two-dimensional NMR suggested significant and specific conformational changes of human mitochondrial and E. coli helix69 upon aminoglycoside binding, as compared with human cytoplasmic helix69. The conformational changes and similar aminoglycoside binding affinities observed for human mitochondrial helix69 and E. coli helix69, as well as the increase in structural stability shown for the former, suggest that this binding event is important to understanding aminoglycoside toxicity.