RESUMEN
The emergence of drug-resistant tuberculosis has created an urgent need for new anti-tubercular agents. Here, we report the discovery of a series of macrolides called sequanamycins with outstanding in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb). Sequanamycins are bacterial ribosome inhibitors that interact with the ribosome in a similar manner to classic macrolides like erythromycin and clarithromycin, but with binding characteristics that allow them to overcome the inherent macrolide resistance of Mtb. Structures of the ribosome with bound inhibitors were used to optimize sequanamycin to produce the advanced lead compound SEQ-9. SEQ-9 was efficacious in mouse models of acute and chronic TB as a single agent, and it demonstrated bactericidal activity in a murine TB infection model in combination with other TB drugs. These results support further investigation of this series as TB clinical candidates, with the potential for use in new regimens against drug-susceptible and drug-resistant TB.
Asunto(s)
Antituberculosos , Mycobacterium tuberculosis , Animales , Ratones , Antituberculosos/farmacología , Macrólidos , Farmacorresistencia Bacteriana , ClaritromicinaRESUMEN
Widespread resistance to first-line TB drugs is a major problem that will likely only be resolved through the development of new drugs with novel mechanisms of action. We have used structure-guided methods to develop a lead molecule that targets the thioesterase activity of polyketide synthase Pks13, an essential enzyme that forms mycolic acids, required for the cell wall of Mycobacterium tuberculosis. Our lead, TAM16, is a benzofuran class inhibitor of Pks13 with highly potent in vitro bactericidal activity against drug-susceptible and drug-resistant clinical isolates of M. tuberculosis. In multiple mouse models of TB infection, TAM16 showed in vivo efficacy equal to the first-line TB drug isoniazid, both as a monotherapy and in combination therapy with rifampicin. TAM16 has excellent pharmacological and safety profiles, and the frequency of resistance for TAM16 is â¼100-fold lower than INH, suggesting that it can be developed as a new antitubercular aimed at the acute infection. PAPERCLIP.
Asunto(s)
Antituberculosos/farmacología , Benzofuranos/farmacología , Diseño de Fármacos , Farmacorresistencia Bacteriana , Mycobacterium tuberculosis/efectos de los fármacos , Piperidinas/farmacología , Tuberculosis/microbiología , Animales , Antituberculosos/química , Benzofuranos/química , Benzofuranos/farmacocinética , Línea Celular , Femenino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Piperidinas/química , Piperidinas/farmacocinética , Organismos Libres de Patógenos EspecíficosRESUMEN
Bacteria that cause disease rely on their ability to counteract and overcome host defenses. Here, we present a genome-scale study of Mycobacterium tuberculosis (Mtb) that uncovers the bacterial determinants of surviving host immunity, sets of genes we term "counteractomes." Through this analysis, we found that CD4 T cells attempt to contain Mtb growth by starving it of tryptophan--a mechanism that successfully limits infections by Chlamydia and Leishmania, natural tryptophan auxotrophs. Mtb, however, can synthesize tryptophan under stress conditions, and thus, starvation fails as an Mtb-killing mechanism. We then identify a small-molecule inhibitor of Mtb tryptophan synthesis, which converts Mtb into a tryptophan auxotroph and restores the efficacy of a failed host defense. Together, our findings demonstrate that the Mtb immune counteractomes serve as probes of host immunity, uncovering immune-mediated stresses that can be leveraged for therapeutic discovery.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Mycobacterium tuberculosis/metabolismo , Triptófano/biosíntesis , Tuberculosis/inmunología , Tuberculosis/microbiología , Animales , Vías Biosintéticas/efectos de los fármacos , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón gamma/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/tratamiento farmacológico , Factores de Virulencia/metabolismo , ortoaminobenzoatos/farmacologíaRESUMEN
YisK is an uncharacterized protein in Bacillus subtilis previously shown to interact genetically with the elongasome protein Mbl. YisK overexpression leads to cell widening and lysis, phenotypes that are dependent on mbl and suppressed by mbl mutations. In the present work, we characterize YisK's localization, structure, and enzymatic activity. We show that YisK localizes as puncta that depend on Mbl. YisK belongs to the fumarylacetoacetate hydrolase (FAH) superfamily, and crystal structures revealed close structural similarity to two oxaloacetate (OAA) decarboxylases: human mitochondrial FAHD1 and Corynebacterium glutamicum Cg1458. We demonstrate that YisK can also catalyze the decarboxylation of OAA (K m = 134 µM, K cat = 31 min-1). A catalytic dead variant (YisK E148A, E150A) retains wild-type localization and still widens cells following overexpression, indicating these activities are not dependent on YisK catalysis. Conversely, a non-localizing variant (YisK E30A) retains wild-type enzymatic activity in vitro but localizes diffusely and no longer widens cells following overexpression. Together, these results suggest that YisK may be subject to spatial regulation that depends on the cell envelope synthesis machinery. IMPORTANCE The elongasome is a multiprotein complex that guides lengthwise growth in some bacteria. We previously showed that, in B. subtilis, overexpression of an uncharacterized putative enzyme (YisK) perturbed function of the actin-like elongasome protein Mbl. Here, we show that YisK exhibits Mbl-dependent localization. Through biochemical and structural characterization, we demonstrate that, like its mitochondrial homolog FAHD1, YisK can catalyze the decarboxylation of the oxaloacetate to pyruvate and CO2. YisK is the first example of an enzyme implicated in central carbon metabolism with subcellular localization that depends on Mbl.
Asunto(s)
Bacillus subtilis , Carboxiliasas , Humanos , Bacillus subtilis/metabolismo , Carboxiliasas/genética , Ácido Pirúvico , Oxaloacetatos , Hidrolasas/genéticaRESUMEN
Urinary tract infections (UTIs) are a major global health problem and are caused predominantly by uropathogenic Escherichia coli (UPEC). UTIs are a leading cause of prescription antimicrobial use. Incessant increase in antimicrobial resistance in UPEC and other uropathogens poses a serious threat to the current treatment practices. Copper is an effector of nutritional immunity that impedes the growth of pathogens during infection. We hypothesized that copper would augment the toxicity of select small molecules against bacterial pathogens. We conducted a small molecule screening campaign with a library of 51,098 molecules to detect hits that inhibit a UPEC ΔtolC mutant in a copper-dependent manner. A molecule, denoted as E. coli inhibitor or ECIN, was identified as a copper-responsive inhibitor of wild-type UPEC strains. Our gene expression and metal content analysis results demonstrate that ECIN works in concert with copper to exacerbate Cu toxicity in UPEC. ECIN has a broad spectrum of activity against pathogens of medical and veterinary significance including Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. Subinhibitory levels of ECIN eliminate UPEC biofilm formation. Transcriptome analysis of UPEC treated with ECIN reveals induction of multiple stress response systems. Furthermore, we demonstrate that L-cysteine rescues the growth of UPEC exposed to ECIN. In summary, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC.IMPORTANCEUrinary tract infection (UTI) is a ubiquitous infectious condition affecting millions of people annually. Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of UTI. However, UTIs are becoming increasingly difficult to resolve with antimicrobials due to increased antimicrobial resistance in UPEC and other uropathogens. Here, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC. In addition to E. coli, this small molecule also inhibits pathogens of medical and veterinary significance including Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus.
Asunto(s)
Antibacterianos , Cobre , Pruebas de Sensibilidad Microbiana , Infecciones Urinarias , Escherichia coli Uropatógena , Escherichia coli Uropatógena/efectos de los fármacos , Escherichia coli Uropatógena/genética , Cobre/farmacología , Antibacterianos/farmacología , Infecciones Urinarias/microbiología , Infecciones Urinarias/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/tratamiento farmacológicoRESUMEN
Catalytic promiscuity is the coincidental ability to catalyze nonbiological reactions in the same active site as the native biological reaction. Several lines of evidence show that catalytic promiscuity plays a role in the evolution of new enzyme functions. Thus, studying catalytic promiscuity can help identify structural features that predispose an enzyme to evolve new functions. This study identifies a potentially preadaptive residue in a promiscuous N-succinylamino acid racemase/o-succinylbenzoate synthase (NSAR/OSBS) enzyme from Amycolatopsis sp. T-1-60. This enzyme belongs to a branch of the OSBS family which includes many catalytically promiscuous NSAR/OSBS enzymes. R266 is conserved in all members of the NSAR/OSBS subfamily. However, the homologous position is usually hydrophobic in other OSBS subfamilies, whose enzymes lack NSAR activity. The second-shell amino acid R266 is close to the catalytic acid/base K263, but it does not contact the substrate, suggesting that R266 could affect the catalytic mechanism. Mutating R266 to glutamine in Amycolatopsis NSAR/OSBS profoundly reduces NSAR activity but moderately reduces OSBS activity. This is due to a 1000-fold decrease in the rate of proton exchange between the substrate and the general acid/base catalyst K263. This mutation is less deleterious for the OSBS reaction because K263 forms a cation-π interaction with the OSBS substrate and/or the intermediate, rather than acting as a general acid/base catalyst. Together, the data explain how R266 contributes to NSAR reaction specificity and was likely an essential preadaptation for the evolution of NSAR activity.
Asunto(s)
Isomerasas de Aminoácido/química , Isomerasas de Aminoácido/metabolismo , Liasas de Carbono-Carbono/química , Liasas de Carbono-Carbono/metabolismo , Isomerasas de Aminoácido/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Amycolatopsis/enzimología , Amycolatopsis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Liasas de Carbono-Carbono/genética , Dominio Catalítico/genética , Secuencia Conservada , Cristalografía por Rayos X , Estabilidad de Enzimas/genética , Evolución Molecular , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The isocitrate lyase paralogs of Mycobacterium tuberculosis (ICL1 and 2) are essential for mycobacterial persistence and constitute targets for the development of antituberculosis agents. We report that (2R,3S)-2-hydroxy-3-(nitromethyl)succinic acid (5-NIC) undergoes apparent retro-aldol cleavage as catalyzed by ICL1 to produce glyoxylate and 3-nitropropionic acid (3-NP), the latter of which is a covalent-inactivating agent of ICL1. Kinetic analysis of this reaction identified that 5-NIC serves as a robust and efficient mechanism-based inactivator of ICL1 (kinact/KI = (1.3 ± 0.1) × 103 M-1 s-1) with a partition ratio <1. Using enzyme kinetics, mass spectrometry, and X-ray crystallography, we identified that the reaction of the 5-NIC-derived 3-NP with the Cys191 thiolate of ICL1 results in formation of an ICL1-thiohydroxamate adduct as predicted. One aspect of the design of 5-NIC was to lower its overall charge compared to isocitrate to assist with cell permeability. Accordingly, the absence of the third carboxylate group will simplify the synthesis of pro-drug forms of 5-NIC for characterization in cell-infection models of M. tuberculosis.
Asunto(s)
Inhibidores Enzimáticos/química , Isocitratoliasa/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , Succinatos/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Glioxilatos/química , Glioxilatos/metabolismo , Isocitratoliasa/química , Isocitratoliasa/metabolismo , Cinética , Modelos Químicos , Nitrocompuestos/química , Nitrocompuestos/metabolismo , Propionatos/química , Propionatos/metabolismo , Unión Proteica , Succinatos/síntesis química , Succinatos/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1006939.].
RESUMEN
The pathogenicity of Mycobacterium tuberculosis depends upon its ability to catabolize host cholesterol. Upregulation of the methylcitrate cycle (MCC) is required to assimilate and detoxify propionyl-CoA, a cholesterol degradation product. The transcription of key genes prpC and prpD in MCC is activated by MtPrpR, a member of a family of prokaryotic transcription factors whose structures and modes of action have not been clearly defined. We show that MtPrpR has a novel overall structure and directly binds to CoA or short-chain acyl-CoA derivatives to form a homotetramer that covers the binding cavity and locks CoA tightly inside the protein. The regulation of this process involves a [4Fe4S] cluster located close to the CoA-binding cavity on a neighboring chain. Mutations in the [4Fe4S] cluster binding residues rendered MtPrpR incapable of regulating MCC gene transcription. The structure of MtPrpR without the [4Fe4S] cluster-binding region shows a conformational change that prohibits CoA binding. The stability of this cluster means it is unlikely a redox sensor but may function by sensing ambient iron levels. These results provide mechanistic insights into this family of critical transcription factors who share similar structures and regulate gene transcription using a combination of acyl-CoAs and [4Fe4S] cluster.
Asunto(s)
Acilcoenzima A/química , Proteínas Bacterianas/química , Mycobacterium tuberculosis/genética , Factores de Transcripción/química , Acilcoenzima A/genética , Proteínas Bacterianas/genética , Colesterol/genética , Regulación Bacteriana de la Expresión Génica/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/patogenicidad , Proteínas PrPC/química , Proteínas PrPC/genética , Factores de Transcripción/genética , Tuberculosis/genética , Tuberculosis/microbiologíaRESUMEN
The nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10-5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.
Asunto(s)
Mycobacterium tuberculosis , Nitroimidazoles , Animales , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Ratones , Mutación , Mycobacterium tuberculosis/genética , Nitroimidazoles/farmacología , OxazolesRESUMEN
Once considered a phenotypically monomorphic bacterium, there is a growing body of work demonstrating heterogeneity among Mycobacterium tuberculosis (Mtb) strains in clinically relevant characteristics, including virulence and response to antibiotics. However, the genetic and molecular basis for most phenotypic differences among Mtb strains remains unknown. To investigate the basis of strain variation in Mtb, we performed genome-wide transposon mutagenesis coupled with next-generation sequencing (TnSeq) for a panel of Mtb clinical isolates and the reference strain H37Rv to compare genetic requirements for in vitro growth across these strains. We developed an analytic approach to identify quantitative differences in genetic requirements between these genetically diverse strains, which vary in genomic structure and gene content. Using this methodology, we found differences between strains in their requirements for genes involved in fundamental cellular processes, including redox homeostasis and central carbon metabolism. Among the genes with differential requirements were katG, which encodes the activator of the first-line antitubercular agent isoniazid, and glcB, which encodes malate synthase, the target of a novel small-molecule inhibitor. Differences among strains in their requirement for katG and glcB predicted differences in their response to these antimicrobial agents. Importantly, these strain-specific differences in antibiotic response could not be predicted by genetic variants identified through whole genome sequencing or by gene expression analysis. Our results provide novel insight into the basis of variation among Mtb strains and demonstrate that TnSeq is a scalable method to predict clinically important phenotypic differences among Mtb strains.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Tuberculosis/genética , Elementos Transponibles de ADN , Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mycobacterium tuberculosis/clasificación , Fenotipo , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Secuenciación Completa del GenomaRESUMEN
Isocitrate lyase (ICL, types 1 and 2) is the first enzyme of the glyoxylate shunt, an essential pathway for Mycobacterium tuberculosis (Mtb) during the persistent phase of human TB infection. Here, we report 2-vinyl-d-isocitrate (2-VIC) as a mechanism-based inactivator of Mtb ICL1 and ICL2. The enzyme-catalyzed retro-aldol cleavage of 2-VIC unmasks a Michael substrate, 2-vinylglyoxylate, which then forms a slowly reversible, covalent adduct with the thiolate form of active-site Cys191 2-VIC displayed kinetic properties consistent with covalent, mechanism-based inactivation of ICL1 and ICL2 with high efficiency (partition ratio, <1). Analysis of a complex of ICL1:2-VIC by electrospray ionization mass spectrometry and X-ray crystallography confirmed the formation of the predicted covalent S-homopyruvoyl adduct of the active-site Cys191.
Asunto(s)
Proteínas Bacterianas/genética , Isocitratoliasa/genética , Isocitratos/química , Mycobacterium tuberculosis/enzimología , Tuberculosis/tratamiento farmacológico , Proteínas Bacterianas/antagonistas & inhibidores , Dominio Catalítico , Cristalografía por Rayos X , Cisteína/química , Glioxilatos/química , Humanos , Isocitratoliasa/antagonistas & inhibidores , Ligandos , Malatos/química , Microscopía Fluorescente , Simulación del Acoplamiento Molecular , Espectrometría de Masa por Ionización de Electrospray , Ácido Succínico/química , Compuestos de Sulfhidrilo/química , Tuberculosis/microbiología , Tuberculosis/prevención & controlRESUMEN
The glyoxylate shunt is a metabolic pathway of bacteria, fungi, and plants used to assimilate even-chain fatty acids (FAs) and has been implicated in persistence of Mycobacterium tuberculosis (Mtb). Recent work, however, showed that the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), may mediate survival of Mtb during the acute and chronic phases of infection in mice through physiologic functions apart from fatty acid metabolism. Here, we report that malate synthase (MS), the second enzyme of the glyoxylate shunt, is essential for in vitro growth and survival of Mtb on even-chain fatty acids, in part, for a previously unrecognized activity: mitigating the toxicity of glyoxylate excess arising from metabolism of even-chain fatty acids. Metabolomic profiling revealed that MS-deficient Mtb cultured on fatty acids accumulated high levels of the ICL aldehyde endproduct, glyoxylate, and increased levels of acetyl phosphate, acetoacetyl coenzyme A (acetoacetyl-CoA), butyryl CoA, acetoacetate, and ß-hydroxybutyrate. These changes were indicative of a glyoxylate-induced state of oxaloacetate deficiency, acetate overload, and ketoacidosis. Reduction of intrabacterial glyoxylate levels using a chemical inhibitor of ICL restored growth of MS-deficient Mtb, despite inhibiting entry of carbon into the glyoxylate shunt. In vivo depletion of MS resulted in sterilization of Mtb in both the acute and chronic phases of mouse infection. This work thus identifies glyoxylate detoxification as an essential physiologic function of Mtb malate synthase and advances its validation as a target for drug development.
Asunto(s)
Carbono/metabolismo , Glioxilatos/metabolismo , Inactivación Metabólica , Malato Sintasa/metabolismo , Mycobacterium tuberculosis/metabolismo , Animales , Modelos Animales de Enfermedad , Ácidos Grasos/metabolismo , Femenino , Técnicas de Inactivación de Genes , Macrófagos/inmunología , Macrófagos/metabolismo , Malato Sintasa/genética , Redes y Vías Metabólicas , Ratones , Mutación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Tuberculosis/patología , Virulencia/genéticaRESUMEN
Bacillus subtilis is a bacterium capable of differentiating into a spore form more resistant to environmental stress. Early in sporulation, each cell possesses two copies of a circular chromosome. A polar FtsZ ring (Z ring) directs septation over one of the chromosomes, generating two cell compartments. The smaller "forespore" compartment initially contains only 25 to 30% of one chromosome, and this transient genetic asymmetry is required for differentiation. Timely assembly of polar Z rings and precise capture of the chromosome in the forespore both require the DNA-binding protein RefZ. To mediate its role in chromosome capture, RefZ must bind to specific DNA motifs (RBMs) that localize near the poles at the time of septation. Cells artificially induced to express RefZ during vegetative growth cannot assemble Z rings, an effect that also requires DNA binding. We hypothesized that RefZ-RBM complexes mediate precise chromosome capture by modulating FtsZ function. To investigate, we isolated 10 RefZ loss-of-function (rLOF) variants unable to inhibit cell division yet still capable of binding RBMs. Sporulating cells expressing the rLOF variants in place of wild-type RefZ phenocopied a ΔrefZ mutant, suggesting that RefZ acts through an FtsZ-dependent mechanism. The crystal structure of RefZ was solved, and wild-type RefZ and the rLOF variants were further characterized. Our data suggest that RefZ's oligomerization state and specificity for the RBMs are critical determinants influencing RefZ's ability to affect FtsZ dynamics. We propose that RBM-bound RefZ complexes function as a developmentally regulated nucleoid occlusion system for fine-tuning the position of the septum relative to the chromosome during sporulation.IMPORTANCE The bacterial nucleoid forms a large, highly organized structure. Thus, in addition to storing the genetic code, the nucleoid harbors positional information that can be leveraged by DNA-binding proteins to spatially constrain cellular activities. During B. subtilis sporulation, the nucleoid undergoes reorganization, and the cell division protein FtsZ assembles polarly to direct septation over one chromosome. The TetR family protein RefZ binds DNA motifs (RBMs) localized near the poles at the time of division and is required for both timely FtsZ assembly and precise capture of DNA in the future spore compartment. Our data suggest that RefZ exploits nucleoid organization by associating with polarly localized RBMs to modulate the positioning of FtsZ relative to the chromosome during sporulation.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Esporas Bacterianas/crecimiento & desarrollo , Bacillus subtilis/química , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , División Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Esporas Bacterianas/química , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismoRESUMEN
Pseudomonas aeruginosa has been detected in the lungs of â¼50% of patients with cystic fibrosis (CF), including 20% of adult CF patients. The majority of these adult patients harbor multi-drug resistant (MDR) strains, limiting the available treatment options. Silver has long been used as a broad-spectrum antimicrobial agent with a low incidence of resistance. Despite low toxicity, poor availability of silver cations mandates a high dosage to effectively eradicate infections. To address this shortcoming of silver, nanoparticles have been used as delivery devices to improve treatment outcomes. Furthermore, studies have demonstrated that synergistic combinations with careful dose calibrations and efficient delivery systems result in superior antimicrobial activity while avoiding potential side effects of both therapeutics. Here 4-epi-minocycline, a metabolite of minocycline, was identified as an active antimicrobial against P. aeruginosa using a high-throughput screen. The antimicrobial activities of 4-epi-minocycline, minocycline, and silver acetate against clinical isolates of P. aeruginosa obtained from CF patients were evaluated in vitro. Next, the synergistic activity of the silver/minocycline combination against P. aeruginosa isolates was investigated using checkerboard assays and identified with end-point colony forming unit determination assays. Finally, nanoparticles coloaded with minocycline and silver were evaluated in vitro for antimicrobial activity. The results demonstrated that both silver and minocycline are potent antimicrobials alone and that the combination allows a reduced dosage of both therapeutics to achieve the same antimicrobial effect. Furthermore, the proposed synergistic silver/minocycline combination can be coloaded into nanoparticles as a next-generation antibiotic to combat the threats presented by MDR pathogens.
Asunto(s)
Farmacorresistencia Bacteriana/efectos de los fármacos , Nanopartículas del Metal/química , Minociclina/administración & dosificación , Polifosfatos/química , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Plata/química , Antibacterianos/administración & dosificación , Humanos , Infecciones por Pseudomonas/microbiologíaRESUMEN
Ribosomes from Mycobacterium tuberculosis (Mtb) possess species-specific ribosomal RNA (rRNA) expansion segments and ribosomal proteins (rProtein). Here, we present the near-atomic structures of the Mtb 50S ribosomal subunit and the complete Mtb 70S ribosome, solved by cryo-electron microscopy. Upon joining of the large and small ribosomal subunits, a 100-nt long expansion segment of the Mtb 23S rRNA, named H54a or the 'handle', switches interactions from with rRNA helix H68 and rProtein uL2 to with rProtein bS6, forming a new intersubunit bridge 'B9'. In Mtb 70S, bridge B9 is mostly maintained, leading to correlated motions among the handle, the L1 stalk and the small subunit in the rotated and non-rotated states. Two new protein densities were discovered near the decoding center and the peptidyl transferase center, respectively. These results provide a structural basis for studying translation in Mtb as well as developing new tuberculosis drugs.
Asunto(s)
Mycobacterium tuberculosis/química , Ribosomas/química , Microscopía por Crioelectrón , Modelos Moleculares , Movimiento (Física) , Mycobacterium smegmatis/química , Inhibidores de la Síntesis de la Proteína , Proteínas Ribosómicas/química , Subunidades Ribosómicas Grandes Bacterianas/química , Especificidad de la EspecieRESUMEN
The Anopheles mosquito that harbors the Plasmodium parasite contains a microbiota that can influence both the vector and the parasite. In recent years, insect-associated microbes have highlighted the untapped potential of exploiting interspecies interactions to discover bioactive compounds. In this study, we report the discovery of nonribosomal lipodepsipeptides that are produced by a Serratia sp. within the midgut and salivary glands of Anopheles stephensi mosquitoes. The lipodepsipeptides, stephensiolidesâ A-K, have antibiotic activity and facilitate bacterial surface motility. Bioinformatic analyses indicate that the stephensiolides are ubiquitous in nature and are likely important for Serratia spp. colonization within mosquitoes, humans, and other ecological niches. Our results demonstrate the usefulness of probing insect-microbiome interactions, enhance our understanding of the chemical ecology within Anopheles mosquitoes, and provide a secondary-metabolite scaffold for further investigate of this complex relationship.
Asunto(s)
Anopheles/microbiología , Antiinfecciosos/metabolismo , Depsipéptidos/metabolismo , Lipopéptidos/metabolismo , Mosquitos Vectores/microbiología , Serratia/metabolismo , Animales , Antiinfecciosos/química , Antiinfecciosos/aislamiento & purificación , Antiinfecciosos/farmacología , Bacterias/efectos de los fármacos , Depsipéptidos/química , Depsipéptidos/aislamiento & purificación , Depsipéptidos/farmacología , Células Hep G2 , Humanos , Lipopéptidos/química , Lipopéptidos/aislamiento & purificación , Lipopéptidos/farmacología , Malaria/parasitología , Malaria/transmisión , Malaria Falciparum/parasitología , Malaria Falciparum/transmisión , Plasmodium falciparum/efectos de los fármacosRESUMEN
Mycobacterium tuberculosis (Mtb) mutants lacking rv1411c, which encodes the lipoprotein LprG, and rv1410c, which encodes a putative efflux pump, are dramatically attenuated for growth in mice. Here we show that loss of LprG-Rv1410 in Mtb leads to intracellular triacylglyceride (TAG) accumulation, and overexpression of the locus increases the levels of TAG in the culture medium, demonstrating a role of this locus in TAG transport. LprG binds TAG within a large hydrophobic cleft and is sufficient to transfer TAG from donor to acceptor membranes. Further, LprG-Rv1410 is critical for broadly regulating bacterial growth and metabolism in vitro during carbon restriction and in vivo during infection of mice. The growth defect in mice is due to disrupted bacterial metabolism and occurs independently of key immune regulators. The in vivo essentiality of this locus suggests that this export system and other regulators of metabolism should be considered as targets for novel therapeutics.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Lipoproteínas/metabolismo , Espectrometría de Masas , Ratones , Ratones Mutantes , VirulenciaRESUMEN
Human infection by Mycobacterium tuberculosis (Mtb) continues to be a global epidemic. Computer-aided drug design (CADD) methods are used to accelerate traditional drug discovery efforts. One noncovalent interaction that is being increasingly identified in biological systems but is neglected in CADD is the anion-π interaction. The study reported herein supports the conclusion that anion-π interactions play a central role in directing the binding of phenyl-diketo acid (PDKA) inhibitors to malate synthase (GlcB), an enzyme required for Mycobacterium tuberculosis virulence. Using density functional theory methods (M06-2X/6-31+G(d)), a GlcB active site template was developed for a predictive model through a comparative analysis of PDKA-bound GlcB crystal structures. The active site model includes the PDKA molecule and the protein determinants of the electrostatic, hydrogen-bonding, and anion-π interactions involved in binding. The predictive model accurately determines the Asp 633-PDKA structural position upon binding and precisely predicts the relative binding enthalpies of a series of 2-ortho halide-PDKAs to GlcB. A screening model was also developed to efficiently assess the propensity of each PDKA analog to participate in an anion-π interaction; this method is in good agreement with both the predictive model and the experimental binding enthalpies for the 2-ortho halide-PDKAs. With the screening and predictive models in hand, we have developed an efficient method for computationally screening and evaluating the binding enthalpy of variously substituted PDKA molecules. This study serves to illustrate the contribution of this overlooked interaction to binding affinity and demonstrates the importance of integrating anion-π interactions into structure-based CADD.
Asunto(s)
Antituberculosos/farmacología , Malato Sintasa/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , Antituberculosos/química , Sitios de Unión , Simulación por Computador , Malato Sintasa/metabolismo , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
Peptidoglycan (PG), a complex polymer composed of saccharide chains cross-linked by short peptides, is a critical component of the bacterial cell wall. PG synthesis has been extensively studied in model organisms but remains poorly understood in mycobacteria, a genus that includes the important human pathogen Mycobacterium tuberculosis (Mtb). The principle PG synthetic enzymes have similar and, at times, overlapping functions. To determine how these are functionally organized, we carried out whole-genome transposon mutagenesis screens in Mtb strains deleted for ponA1, ponA2, and ldtB, major PG synthetic enzymes. We identified distinct factors required to sustain bacterial growth in the absence of each of these enzymes. We find that even the homologs PonA1 and PonA2 have unique sets of genetic interactions, suggesting there are distinct PG synthesis pathways in Mtb. Either PonA1 or PonA2 is required for growth of Mtb, but both genetically interact with LdtB, which has its own distinct genetic network. We further provide evidence that each interaction network is differentially susceptible to antibiotics. Thus, Mtb uses alternative pathways to produce PG, each with its own biochemical characteristics and vulnerabilities.