RESUMEN
Bacteria belonging to the Burkholderia genus are extremely versatile and diverse. They can be environmental isolates, opportunistic pathogens in cystic fibrosis, immunocompromised or chronic granulomatous disease patients, or cause disease in healthy people (e.g., Burkholderia pseudomallei) or animals (as in the case of Burkholderia mallei). Since the genus was separated from the Pseudomonas one in the 1990s, the methodological tools to study and characterize these bacteria are evolving fast. Here we reviewed the techniques used in the last few years to update the taxonomy of the genus, to study gene functions and regulations, to deepen the knowledge on the drug resistance which characterizes these bacteria, and to elucidate their mechanisms to establish infections. The availability of these tools significantly impacts the quality of research on Burkholderia and the choice of the most appropriated is fundamental for a precise characterization of the species of interest.Key points⢠Updated techniques to study the genus Burkholderia were reviewed.⢠Taxonomy, genomics, assays, and animal models were described.⢠A comprehensive overview on recent advances in Burkholderia studies was made.
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Infecciones por Burkholderia , Burkholderia mallei , Burkholderia pseudomallei , Burkholderia , Fibrosis Quística , Animales , Burkholderia/genética , HumanosRESUMEN
C109 is a potent but poorly soluble FtsZ inhibitor displaying promising activity against Burkholderia cenocepacia, a high-risk pathogen for cystic fibrosis (CF) sufferers. To harness C109 for inhalation, we developed nanocrystal-embedded dry powders for inhalation suspension consisting in C109 nanocrystals stabilized with D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) embedded in hydroxypropyl-ß-cyclodextrin (CD). The powders could be safely re-dispersed in water for in vitro aerosolization. Owing to the presence of a PEG shell, the rod shape and the peculiar aspect ratio, C109 nanocrystals were able to diffuse through artificial CF mucus. The promising technological features were completed by encouraging in vitro/in vivo effects. The formulations displayed no toxicity towards human bronchial epithelial cells and were active against planktonic and sessile B. cenocepacia strains. The efficacy of C109 nanosuspensions in combination with piperacillin was confirmed in a Galleria mellonella infection model, strengthening their potential for combined therapy of B. cenocepacia lung infections.
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Antibacterianos , Proteínas Bacterianas/antagonistas & inhibidores , Bronquios/microbiología , Infecciones por Burkholderia/tratamiento farmacológico , Burkholderia cenocepacia/crecimiento & desarrollo , Fibrosis Quística/tratamiento farmacológico , Proteínas del Citoesqueleto/antagonistas & inhibidores , Sistemas de Liberación de Medicamentos , Células Epiteliales/microbiología , Nanopartículas , Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Bronquios/metabolismo , Bronquios/patología , Infecciones por Burkholderia/metabolismo , Infecciones por Burkholderia/patología , Línea Celular Tumoral , Fibrosis Quística/metabolismo , Fibrosis Quística/microbiología , Fibrosis Quística/patología , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Nanopartículas/química , Nanopartículas/uso terapéuticoRESUMEN
Combining antibiotics with potentiators that increase their activity is a promising strategy to tackle infections caused by antibiotic-resistant bacteria. As potentiators do not interfere with essential processes, it has been hypothesized that they are less likely to induce resistance. However, evidence supporting this hypothesis is lacking. In the present study, we investigated whether Burkholderia cenocepacia J2315 biofilms develop reduced susceptibility toward one such adjuvant, baicalin hydrate (BH). Biofilms were repeatedly and intermittently treated with tobramycin (TOB) alone or in combination with BH for 24 h. After treatment, the remaining cells were quantified using plate counting. After 15 cycles, biofilm cells were less susceptible to TOB and TOB+BH compared to the start population, and the potentiating effect of BH toward TOB was lost. Whole-genome sequencing was performed to probe which changes were involved in the reduced effect of BH, and mutations in 14 protein-coding genes were identified (including mutations in genes involved in central metabolism and in BCAL0296, encoding an ABC transporter). No changes in the MIC or MBC of TOB or changes in the number of persister cells were observed. However, basal intracellular levels of reactive oxygen species (ROS) and ROS levels found after treatment with TOB were markedly decreased in the evolved populations. In addition, in evolved cultures with mutations in BCAL0296, a significantly reduced uptake of TOB was observed. Our results indicate that B. cenocepacia J2315 biofilms rapidly lose susceptibility toward the antibiotic-potentiating activity of BH and point to changes in central metabolism, reduced ROS production, and reduced TOB uptake as mechanisms.
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Antibacterianos/farmacología , Biopelículas/crecimiento & desarrollo , Burkholderia cenocepacia/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Percepción de Quorum/efectos de los fármacos , Tobramicina/farmacología , Biopelículas/efectos de los fármacos , Burkholderia cenocepacia/crecimiento & desarrollo , Farmacorresistencia Bacteriana/fisiología , Quimioterapia Combinada , Genoma Bacteriano/genética , Pruebas de Sensibilidad Microbiana , Especies Reactivas de Oxígeno/metabolismo , Secuenciación Completa del GenomaRESUMEN
During phenylalanine catabolism, phenylacetic acid (PAA) is converted to phenylacetyl coenzyme A (PAA-CoA) by a ligase, PaaK, and then PAA-CoA is epoxidized by a multicomponent monooxygenase, PaaABCDE, before further degradation through the tricarboxylic acid (TCA) cycle. In the opportunistic pathogen Burkholderia cenocepacia, loss of paaABCDE attenuates virulence factor expression, which is under the control of the LuxIR-like quorum sensing (QS) system, CepIR. To further investigate the link between CepIR-regulated virulence and PAA catabolism, we created knockout mutants of the first step of the pathway (PAA-CoA synthesis by PaaK) and characterized them in comparison to a paaABCDE mutant using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and virulence assays. We found that while loss of PaaABCDE decreased virulence, deletion of the paaK genes resulted in a more virulent phenotype than that of the wild-type strain. Deletion of either paaK or paaABCDE led to higher levels of released PAA but no differences in levels of internal accumulation compared to the wild-type level. While we found no evidence of direct cepIR downregulation by PAA-CoA or PAA, a low-virulence cepR mutant reverted to a virulent phenotype upon removal of the paaK genes. On the other hand, removal of paaABCDE in the cepR mutant did not impact its attenuated phenotype. Together, our results suggest an indirect role for PAA-CoA in suppressing B. cenocepacia CepIR-activated virulence.IMPORTANCE The opportunistic pathogen Burkholderia cenocepacia uses a chemical signal process called quorum sensing (QS) to produce virulence factors. In B. cenocepacia, QS relies on the presence of the transcriptional regulator CepR which, upon binding QS signal molecules, activates virulence. In this work, we found that even in the absence of CepR, B. cenocepacia can elicit a pathogenic response if phenylacetyl-CoA, an intermediate of the phenylacetic acid degradation pathway, is not produced. Instead, accumulation of phenylacetyl-CoA appears to attenuate pathogenicity. Therefore, we have discovered that it is possible to trigger virulence in the absence of CepR, challenging the classical view of activation of virulence by this QS mechanism. Our work provides new insight into the relationship between metabolism and virulence in opportunistic bacteria. We propose that in the event that QS signaling molecules cannot accumulate to trigger a pathogenic response, a metabolic signal can still activate virulence in B. cenocepacia.
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Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/metabolismo , Fenilacetatos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biodegradación Ambiental , Cromatografía Liquida , Regulación Bacteriana de la Expresión Génica , Redes y Vías Metabólicas/genética , Percepción de Quorum , Eliminación de Secuencia , Espectrometría de Masas en Tándem , Transcriptoma , Virulencia/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Cystic fibrosis (CF) is an autosomal recessive genetic disorder which leads to the secretion of a viscous mucus layer on the respiratory epithelium that facilitates colonization by various bacterial pathogens. The problem of drug resistance has been reported for all the species able to colonize the lung of CF patients, so alternative treatments are urgently needed. In this context, a valid approach is to investigate new natural and synthetic molecules for their ability to counteract alternative pathways, such as virulence regulating quorum sensing (QS). In this review we describe the pathogens most commonly associated with CF lung infections: Staphylococcus aureus, Pseudomonas aeruginosa, species of the Burkholderia cepacia complex and the emerging pathogens Stenotrophomonas maltophilia, Haemophilus influenzae and non-tuberculous Mycobacteria. For each bacterium, the QS system(s) and the molecules targeting the different components of this pathway are described. The amount of investigations published in the last five years clearly indicate the interest and the expectations on antivirulence therapy as an alternative to classical antibiotics.
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Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/microbiología , Fibrosis Quística/complicaciones , Percepción de Quorum/efectos de los fármacos , Virulencia/efectos de los fármacos , Animales , Antibacterianos/química , Bacterias/genética , Bacterias/patogenicidad , Descubrimiento de Drogas , Humanos , Percepción de Quorum/genética , Virulencia/genética , Factores de Virulencia/genéticaRESUMEN
To streamline the elucidation of antibacterial compounds' mechanism of action, comprehensive high-throughput assays interrogating multiple putative targets are necessary. However, current chemogenomic approaches for antibiotic target identification have not fully utilized the multiplexing potential of next-generation sequencing. Here, we used Illumina sequencing of transposon insertions to track the competitive fitness of a Burkholderia cenocepacia library containing essential gene knockdowns. Using this method, we characterized a novel benzothiadiazole derivative, 10126109 (C109), with antibacterial activity against B. cenocepacia, for which whole-genome sequencing of low-frequency spontaneous drug-resistant mutants had failed to identify the drug target. By combining the identification of hypersusceptible mutants and morphology screening, we show that C109 targets cell division. Furthermore, fluorescence microscopy of bacteria harboring green fluorescent protein (GFP) cell division protein fusions revealed that C109 prevents divisome formation by altering the localization of the essential cell division protein FtsZ. In agreement with this, C109 inhibited both the GTPase and polymerization activities of purified B. cenocepacia FtsZ. C109 displayed antibacterial activity against Gram-positive and Gram-negative cystic fibrosis pathogens, including Mycobacterium abscessus C109 effectively cleared B. cenocepacia infection in the Caenorhabditis elegans model and exhibited additive interactions with clinically relevant antibiotics. Hence, C109 is an enticing candidate for further drug development.
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Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Burkholderia cenocepacia/genética , Proteínas del Citoesqueleto/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos/métodos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Infecciones por Burkholderia/tratamiento farmacológico , Infecciones por Burkholderia/microbiología , Burkholderia cenocepacia/efectos de los fármacos , Burkholderia cenocepacia/aislamiento & purificación , Caenorhabditis elegans/microbiología , Fibrosis Quística/microbiología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Técnicas de Silenciamiento del Gen , Genes Esenciales , Proteínas Fluorescentes Verdes/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Pruebas de Sensibilidad Microbiana , MutaciónRESUMEN
BACKGROUND: Antibiotic resistance is a major problem for human health. Multidrug resistance efflux pumps, especially those of the Resistance-Nodulation-Cell Division (RND) family, are major contributors to high-level antibiotic resistance in Gram-negative bacteria. Most bacterial genomes contain several copies of the different classes of multidrug resistance efflux pumps. Gene duplication and gain of function by the duplicate copies of multidrug resistance efflux pump genes plays a key role in the expansion and diversification of drug-resistance mechanisms. RESULTS: We used two members of the Burkholderia RND superfamily as models to understand how duplication events affect the antibiotic resistance of these strains. First, we analyzed the conservation and distribution of these two RND systems and their regulators across the Burkholderia genus. Through genetic manipulations, we identified both the exact substrate range of these transporters and their eventual interchangeability. We also performed a directed evolution experiment, combined with next generation sequencing, to evaluate the role of antibiotics in the activation of the expression of these systems. Together, our results indicate that the first step to diversify the functions of these pumps arises from changes in their regulation (subfunctionalization) instead of functional mutations. Further, these pumps could rewire their regulation to respond to antibiotics, thus maintaining high genomic plasticity. CONCLUSIONS: Studying the regulatory network that controls the expression of the RND pumps will help understand and eventually control the development and expansion of drug resistance.
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Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/genética , Fenómenos Fisiológicos Bacterianos , Farmacorresistencia Bacteriana Múltiple , Burkholderia/genética , Orden Génico , Genoma Bacteriano , Genómica/métodos , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Operón , Filogenia , PlásmidosRESUMEN
Burkholderia cenocepacia is a major concern among respiratory tract infections in cystic fibrosis patients. This pathogen is particularly difficult to treat because of its high level of resistance to the clinically relevant antimicrobial agents. In B. cenocepacia, the quorum sensing cell-cell communication system is involved in different processes that are important for bacterial virulence, such as biofilm formation and protease and siderophore production. Targeting the enzymes involved in this process represents a promising therapeutic approach. With the aim of finding effective quorum sensing inhibitors, we have determined the three-dimensional structure of B. cenocepacia diffusible factor synthase A, DfsA. This bifunctional crotonase (dehydratase/thioesterase) produces the characteristic quorum sensing molecule of B. cenocepacia, cis-2-dodecenoic acid or BDSF, starting from 3-hydroxydodecanoyl-acyl carrier protein. Unexpectedly, the crystal structure revealed the presence of a lipid molecule in the catalytic site of the enzyme, which was identified as dodecanoic acid. Our biochemical characterization shows that DfsA is able to use dodecanoyl-acyl carrier protein as a substrate, demonstrating that dodecanoic acid, the product of this reaction, is released very slowly from the DfsA active site, therefore acting as a DfsA inhibitor. This molecule shows an unprecedented conformational arrangement inside the DfsA active site. In contrast with previous hypotheses, our data illustrate how DfsA and closely related homologous enzymes can recognize long hydrophobic substrates without large conformational changes or assistance by additional regulator molecules. The elucidation of the substrate binding mode in DfsA provides the starting point for structure-based drug discovery studies targeting B. cenocepacia quorum sensing-assisted virulence.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Burkholderia cenocepacia/metabolismo , Ácidos Grasos/metabolismo , Percepción de Quorum , Secuencia de Aminoácidos , Cristalización , Cristalografía por Rayos X , Cromatografía de Gases y Espectrometría de Masas , Conformación Proteica , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Ionización de Electrospray , Especificidad por SustratoRESUMEN
The discovery of new compounds that are able to inhibit the growth of Burkholderia cenocepacia is of primary importance for cystic fibrosis patients. Here, the mechanism of resistance to a new pyridine derivative already shown to be effective against Mycobacterium tuberculosis and to have good activity toward B. cenocepacia was investigated. Increased expression of a resistance-nodulation-cell division (RND) efflux system was detected in the resistant mutants, thus confirming their important roles in B. cenocepacia antibiotic resistance.
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Antibacterianos/farmacología , Antituberculosos/farmacología , Burkholderia cenocepacia/efectos de los fármacos , Piridinas/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/metabolismo , Farmacorresistencia Bacteriana/genéticaRESUMEN
Burkholderia cenocepacia is notorious for causing respiratory tract infections in people with cystic fibrosis. Infections with this organism are particularly difficult to treat due to its high level of intrinsic resistance to most antibiotics. Multidrug resistance in B. cenocepacia can be ascribed to different mechanisms, including the activity of efflux pumps and biofilm formation. In the present study, the effects of deletion of the 16 operons encoding resistance-nodulation-cell division (RND)-type efflux pumps in B. cenocepacia strain J2315 were investigated by determining the MICs of various antibiotics and by investigating the antibiofilm effect of these antibiotics. Finally, the expression levels of selected RND genes in treated and untreated cultures were investigated using reverse transcriptase quantitative PCR (RT-qPCR). Our data indicate that the RND-3 and RND-4 efflux pumps are important for resistance to various antimicrobial drugs (including tobramycin and ciprofloxacin) in planktonic B. cenocepacia J2315 populations, while the RND-3, RND-8, and RND-9 efflux systems protect biofilm-grown cells against tobramycin. The RND-8 and RND-9 efflux pumps are not involved in ciprofloxacin resistance. Results from the RT-qPCR experiments on the wild-type strain B. cenocepacia J2315 suggest that there is little regulation at the level of mRNA expression for these efflux pumps under the conditions tested.
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Secuencia de Bases , Biopelículas/efectos de los fármacos , Burkholderia cenocepacia/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica , Genes MDR , Plancton/efectos de los fármacos , Eliminación de Secuencia , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Biopelículas/crecimiento & desarrollo , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/crecimiento & desarrollo , Ciprofloxacina/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Operón , Plancton/genética , Plancton/crecimiento & desarrollo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tobramicina/farmacologíaRESUMEN
People with cystic fibrosis (CF) suffer from recurrent bacterial infections which induce inflammation, lung tissue damage and failure of the respiratory system. Prolonged exposure to combinatorial antibiotic therapies triggers the appearance of multi-drug resistant (MDR) bacteria. The development of alternative antimicrobial strategies may provide a way to mitigate antimicrobial resistance. Here we discuss different alternative approaches to the use of classic antibiotics: anti-virulence and anti-biofilm compounds which exert a low selective pressure; phage therapies that represent an alternative strategy with a high therapeutic potential; new methods helping antibiotics activity such as adjuvants; and antimicrobial peptides and nanoparticle formulations. Their mechanisms and in vitro and in vivo efficacy are described, in order to figure out a complete landscape of new alternative approaches to fight MDR Gram-negative CF pathogens.
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Pseudomonas aeruginosa is a major human pathogen, able to establish difficult-to-treat infections in immunocompromised and people with cystic fibrosis (CF). The high rate of antibiotic treatment failure is due to its notorious drug resistance, often mediated by the formation of persistent biofilms. Alternative strategies, capable of overcoming P. aeruginosa resistance, include antivirulence compounds which impair bacterial pathogenesis without exerting a strong selective pressure, and the use of antimicrobial adjuvants that can resensitize drug-resistant bacteria to specific antibiotics. In this work, the dispirotripiperazine derivative PDSTP, already studied as antiviral, was characterized for its activity against P. aeruginosa adhesion to epithelial cells, its antibiotic adjuvant ability and its biofilm inhibitory potential. PDSTP was effective in impairing the adhesion of P. aeruginosa to various immortalized cell lines. Moreover, the combination of clinically relevant antibiotics with the compound led to a remarkable enhancement of the antibiotic efficacy towards multidrug-resistant CF clinical strains. PDSTP-ceftazidime combination maintained its efficacy in vivo in a Galleria mellonella infection model. Finally, the compound showed a promising biofilm inhibitory activity at low concentrations when tested both in vitro and using an ex vivo pig lung model. Altogether, these results validate PDSTP as a promising compound, combining the ability to decrease P. aeruginosa virulence by impairing its adhesion and biofilm formation, with the capability to increase antibiotic efficacy against antibiotic resistant strains.
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AIMS: The incidence of lung infections is increasing worldwide in individuals suffering from cystic fibrosis and chronic obstructive pulmonary diseases. Mycobacterium abscessus is associated to chronic lung deterioration in these populations. The intrinsic resistance of M. abscessus to most conventional antibiotics jeopardizes treatment success rates. To date no single drug has been developed targeting specifically M. abscessus. Our objective was to characterize the pyrithione-core drug-like small molecule named VOMG as a new compound active against M. abscessus and other pathogens. METHODS: We used a multidisciplinary approach including microbiological, chemical, biochemical and transcriptomics procedures to validate VOMG as a promising anti-M. abscessus drug candidate. RESULTS: We report for the first time the in vitro and in vivo bactericidal activity of VOMG against M. abscessus and other pathogens. Besides being active against M. abscessus biofilm, the compound showed a favourable pharmacology (ADME-Tox) profile. Frequency of resistance studies were unable to isolate resistant mutants. VOMG inhibits cell division, particularly the FtsZ enzyme. CONCLUSIONS: VOMG is a new drug-like molecule discovered against M. abscessus inhibiting cell division with broad spectrum activity against other microbial pathogens.
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The Burkholderia cepacia complex comprises environmental and clinical Gram-negative bacteria that infect particularly debilitated people, such as those with cystic fibrosis. Their high level of antibiotic resistance makes empirical treatments often ineffective, increasing the risk of worst outcomes and the diffusion of multi-drug resistance. However, the discovery of new antibiotics is not trivial, so an alternative can be the use of vaccination. Here, the reverse vaccinology approach has been used to identify antigen candidates, obtaining a short-list of 24 proteins. The localization and different aspects of virulence were investigated for three of them-BCAL1524, BCAM0949, and BCAS0335. The three antigens were localized in the outer membrane vesicles confirming that they are surface exposed. We showed that BCAL1524, a collagen-like protein, promotes bacteria auto-aggregation and plays an important role in virulence, in the Galleria mellonella model. BCAM0949, an extracellular lipase, mediates piperacillin resistance, biofilm formation in Luria Bertani and artificial sputum medium, rhamnolipid production, and swimming motility; its predicted lipolytic activity was also experimentally confirmed. BCAS0335, a trimeric adhesin, promotes minocycline resistance, biofilm organization in LB, and virulence in G. mellonella. Their important role in virulence necessitates further investigations to shed light on the usefulness of these proteins as antigen candidates.
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Benzothiazinones (BTZs) are antituberculosis drug candidates with nanomolar bactericidal activity against tubercle bacilli. Here we demonstrate that BTZs are suicide substrates of the FAD-dependent decaprenylphosphoryl-ß-D-ribofuranose 2'-oxidase DprE1, an enzyme involved in cell-wall biogenesis. BTZs are reduced by DprE1 to an electrophile, which then reacts in a near-quantitative manner with an active-site cysteine of DprE1, thus providing a rationale for the extraordinary potency of BTZs. Mutant DprE1 enzymes from BTZ-resistant strains reduce BTZs to inert metabolites while avoiding covalent inactivation. Our results explain the basis for drug sensitivity and resistance to an exceptionally potent class of antituberculosis agents.
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Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Mycobacterium/enzimología , Oxidorreductasas/antagonistas & inhibidores , Antituberculosos/química , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The 1,5-diarylpyrrole derivative BM212 was previously shown to be active against multidrug-resistant clinical isolates and Mycobacterium tuberculosis residing within macrophages as well as against Mycobacterium avium and other atypical mycobacteria. To determine its mechanism of action, we identified the cellular target. Spontaneous Mycobacterium smegmatis, Mycobacterium bovis BCG, and M. tuberculosis H37Rv mutants that were resistant to BM212 were isolated. By the screening of genomic libraries and by whole-genome sequencing, we found that all the characterized mutants showed mutations in the mmpL3 gene, allowing us to conclude that resistance to BM212 maps to the MmpL3 protein, a member of the MmpL (mycobacterial membrane protein, large) family. Susceptibility was unaffected by the efflux pump inhibitors reserpine, carbonylcyanide m-chlorophenylhydrazone, and verapamil. Uptake/efflux experiments with [(14)C]BM212 demonstrated that resistance is not driven by the efflux of BM212. Together, these data strongly suggest that the MmpL3 protein is the cellular target of BM212.
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Antituberculosos/farmacología , Genoma Bacteriano , Proteínas de Transporte de Membrana/genética , Mycobacterium bovis/genética , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Piperazinas/farmacología , Pirroles/farmacología , Animales , Radioisótopos de Carbono , Carbonil Cianuro m-Clorofenil Hidrazona/análogos & derivados , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Bovinos , Análisis Mutacional de ADN , Farmacorresistencia Bacteriana Múltiple , Biblioteca Genómica , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium bovis/efectos de los fármacos , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Reserpina/farmacología , Verapamilo/farmacologíaRESUMEN
Multidrug resistance is a major barrier in the battle against tuberculosis and still a leading cause of death worldwide. In order to fight this pathogen, two routes are practicable: vaccination or drug treatment. Vaccination against Mycobacterium tuberculosis with the current vaccine Mycobacterium bovis Bacillus Calmette-Guerin is partially successful, being its efficacy variable. A few new tuberculosis vaccines are now in various phases of clinical trials. The emergence of multidrug-resistant strains of M. tuberculosis gave the impulse to discover new effective antitubercular drugs, a few of which are in clinical development. Here we focus on three different classes of very promising antitubercular drugs recently discovered (benzothiazinones, dinitrobenzamides, and benzoquinoxalines) that share the same cellular target: a subunit of the heteromeric decaprenylphosphoryl-ß-D: -ribose 2'-epimerase, encoded by the dprE1 (or Rv3790) gene. This enzyme is involved in the biosynthesis of D: -arabinose which is crucial for the synthesis of the mycobacterial cell wall and essential for the pathogen's survival.
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Antituberculosos/farmacología , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Oxidorreductasas/antagonistas & inhibidores , Racemasas y Epimerasas/antagonistas & inhibidores , Arabinosa/antagonistas & inhibidores , Arabinosa/biosíntesis , Mycobacterium bovis , Mycobacterium tuberculosis/metabolismo , Quinoxalinas/farmacologíaRESUMEN
The spread of antimicrobial resistance (AMR) is still a major threat to global health that is likely to worsen also as a consequence of the COVID-19 pandemic. For this reason, there is an urgent need to develop new compounds and novel alternative treatments. Furthermore, the new lines of action must consider the issue of antibiotics' sustainability. Within this persrective, we have highlighted the main points on which actions in this perspective are possible.
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Antibacterianos , COVID-19 , Humanos , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana , PandemiasRESUMEN
In the last few years, Acinetobacter baumannii has ranked as a number one priority due to its Multi Drug Resistant phenotype. The different metabolic states, such as the one adopted when growing as biofilm, help the bacterium to resist a wide variety of compounds, placing the discovery of new molecules able to counteract this pathogen as a topic of utmost importance. In this context, bacterial cell division machinery and the conserved protein FtsZ are considered very interesting cellular targets. The benzothiadiazole compound C109 is able to inhibit bacterial growth and to block FtsZ GTPase and polymerization activities in Burkholderia cenocepacia, Pseudomonas aeruginosa, and Staphylococcus aureus. In this work, the activity of C109 was tested against a panel of antibiotic sensitive and resistant A. baumannii strains. Its ability to inhibit biofilm formation was explored, together with its activity against the A. baumannii FtsZ purified protein. Our results indicated that C109 has good MIC values against A. baumannii clinical isolates. Moreover, its antibiofilm activity makes it an interesting alternative treatment, effective against diverse metabolic states. Finally, its activity was confirmed against A. baumannii FtsZ.
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The first effective synthetic approach to naphthofuroquinones via a reaction involving lawsone, various aldehydes, and three isocyanides under microwave irradiation afforded derivatives in moderate to good yields. In addition, for less-reactive aldehydes, two naphtho-enaminodione quinones were obtained for the first time, as result of condensation between lawsone and isocyanides. X-ray structure determination for 9 and 2D-NMR spectra of 28 confirmed the obtained structures. All compounds were evaluated for their anti-infectious activities against Plasmodium falciparum, Leishmania donovani, and Mycobacterium tuberculosis. Among the naphthofuroquinone series, 17 exhibited comparatively the best activity against P. falciparum (IC50 = 2.5 µM) and M. tuberculosis (MIC = 9 µM) with better (P. falciparum) or equivalent (M. tuberculosis) values to already-known naphthofuroquinone compounds. Among the two naphtho-enaminodione quinones, 28 exhibited a moderate activity against P. falciparum with a good selectivity index (SI > 36) while also a very high potency against L. donovani (IC50 = 3.5 µM and SI > 28), rendering it very competitive to the reference drug miltefosine. All compounds were studied through molecular modeling on their potential targets for P. falciparum, Pfbc1, and PfDHODH, where 17 showed the most favorable interactions.