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1.
Microbiol Spectr ; 12(5): e0383723, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38619262

RESUMO

Mycobacterium abscessus, an emerging pathogen responsible for severe pulmonary infections in cystic fibrosis patients, displays either a smooth (S) or a rough (R) morphotype. Infections with M. abscessus R are associated with increased pathogenicity in animal models and humans. While the S-to-R transition correlating with reduced glycopeptidolipid (GPL) production is well-documented, the recent screening of a transposon library revealed additional gene candidates located outside of the GPL locus involved in this transition. These genes include MAB_1470c, encoding the putative lipoprotein peptidase LpqM. However, experimental confirmation of the implication of this gene in the morphotype switch is lacking. Herein, we re-examined the role of MAB_1470c, and its homolog MAB_1466c, in colonial morphotype changes by generating unmarked deletion mutants in M. abscessus S. Our results indicate that the morphotype of these mutants stayed smooth in different media. Unexpectedly, the intracellular growth of ΔMAB_1470c and ΔMAB_1466c in THP-1 macrophages was significantly reduced as compared to the parental S strain, and these defects were rescued upon complementation with their corresponding genes. Strikingly, the intracellular survival defect was further exacerbated in a mutant lacking both MAB_1470c and MAB_1466c genes. This implies that, despite their primary sequence relatedness, the two proteins are not functionally redundant. Collectively, this suggests that these two LpqM-related lipoproteins are unlikely to be involved in the S-to-R transition but are key players for intramacrophage survival of M. abscessus. IMPORTANCE: Mycobacterium abscessus causes persistent infections in patients with underlying pulmonary diseases, resulting in progressive lung function deterioration. The rough (R) morphotype is well-established as associated with chronic and more aggressive infections in patients. In this study, we individually and simultaneously deleted the MAB_1470c and MAB_1466c genes in M. abscessus S, without observing changes in colony morphotypes. However, these mutants exhibited a severe impairment in their ability to survive within human macrophages, highlighting the critical role of these two lipoproteins in M. abscessus virulence.


Assuntos
Proteínas de Bactérias , Macrófagos , Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Mycobacterium abscessus/genética , Mycobacterium abscessus/metabolismo , Mycobacterium abscessus/crescimento & desenvolvimento , Humanos , Macrófagos/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Mycobacterium não Tuberculosas/microbiologia , Células THP-1 , Virulência/genética
2.
Antimicrob Agents Chemother ; 67(4): e0160722, 2023 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-36920188

RESUMO

Mycobacterium fortuitum represents one of the most clinically relevant rapid-growing mycobacterial species. Treatments are complex due to antibiotic resistance and to severe side effects of effective drugs, prolonged time of treatment, and co-infection with other pathogens. Herein, we explored the activity of NITD-916, a direct inhibitor of the enoyl-ACP reductase InhA of the type II fatty acid synthase in Mycobacterium tuberculosis. We found that this compound displayed very low MIC values against a panel of M. fortuitum clinical strains and exerted potent antimicrobial activity against M. fortuitum in macrophages. Remarkably, the compound was also highly efficacious in a zebrafish model of infection. Short duration treatments were sufficient to significantly protect the infected larvae from M. fortuitum-induced killing, which correlated with reduced bacterial burdens and abscesses. Biochemical analyses demonstrated an inhibition of de novo synthesis of mycolic acids. Resolving the crystal structure of the InhAMFO in complex with NAD and NITD-916 confirmed that NITD-916 is a direct inhibitor of InhAMFO. Importantly, single nucleotide polymorphism leading to a G96S substitution in InhAMFO conferred high resistance levels to NITD-916, thus resolving its target in M. fortuitum. Overall, these findings indicate that NITD-916 is highly active against M. fortuitum both in vitro and in vivo and should be considered in future preclinical evaluations for the treatment of M. fortuitum pulmonary diseases.


Assuntos
Mycobacterium fortuitum , Mycobacterium tuberculosis , Animais , Peixe-Zebra , Ácidos Micólicos/farmacologia , Oxirredutases
3.
ACS Infect Dis ; 8(10): 2171-2186, 2022 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-36107992

RESUMO

There is an unmet medical need for effective treatments against Mycobacterium abscessus pulmonary infections, to which cystic fibrosis (CF) patients are particularly vulnerable. Recent studies showed that the antitubercular drug isoniazid is inactive against M. abscessus due to the incapacity of the catalase-peroxidase to convert the pro-drug into a reactive metabolite that inhibits the enoyl-ACP reductase InhA. To validate InhAMAB as a druggable target in M. abscessus, we assayed the activity of NITD-916, a 4-hydroxy-2-pyridone lead candidate initially described as a direct inhibitor of InhA that bypasses KatG bioactivation in Mycobacterium tuberculosis. The compound displayed low MIC values against rough and smooth clinical isolates in vitro and significantly reduced the bacterial burden inside human macrophages. Moreover, treatment with NITD-916 reduced the number and size of intracellular mycobacterial cords, regarded as markers of the severity of the infection. Importantly, NITD-916 significantly lowered the M. abscessus burden in CF-derived lung airway organoids. From a mechanistic perspective, NITD-916 abrogated de novo synthesis of mycolic acids and NITD-916-resistant spontaneous mutants harbored point mutations in InhAMAB at residue 96. That NITD-916 targets InhAMAB directly without activation requirements was confirmed genetically and by resolving the crystal structure of the protein in complex with NADH and NITD-916. These findings collectively indicate that InhAMAB is an attractive target to be exploited for future chemotherapeutic developments against this difficult-to-treat mycobacterium and highlight the potential of NITD-916 derivatives for further evaluation in preclinical settings.


Assuntos
Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Pró-Fármacos , Antituberculosos/química , Proteínas de Bactérias/metabolismo , Catalase/metabolismo , Catalase/farmacologia , Catalase/uso terapêutico , Humanos , Isoniazida/química , Isoniazida/farmacologia , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium abscessus/genética , Ácidos Micólicos/metabolismo , NAD/metabolismo , Pró-Fármacos/farmacologia
4.
Eur J Med Chem ; 239: 114531, 2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-35759907

RESUMO

Isoniazid is a cornerstone of modern tuberculosis (TB) therapy and targets the enoyl ACP reductase InhA, a key enzyme in mycolic acid biosynthesis. InhA is still a promising target for the development of new anti-TB drugs. Herein, we report the design, synthesis, and anti-tubercular activity of new isoniazid hybrids. Among these, 1H-1,2,3 triazole-tethered quinoline-isoniazid conjugates 16a to 16g exhibited high activity against Mycobacterium tuberculosis with minimal inhibitory concentrations in the 0.25-0.50 µg/mL range and were bactericidal in vitro. Importantly, these compounds were well tolerated at high doses on mammalian cells, leading to high selectivity indices. The hybrids were dependent on functional KatG production to inhibit mycolic acid biosynthesis. Moreover, overexpression of InhA in M. tuberculosis resulted in high resistance levels to 16a-16g and reduced mycolic acid biosynthesis inhibition, similar to isoniazid. Overall, these findings suggest that the synthesized quinoline-isoniazid hybrids are promising anti-tubercular molecules, which require further pre-clinical evaluation.


Assuntos
Mycobacterium tuberculosis , Quinolinas , Tuberculose , Animais , Antituberculosos/farmacologia , Proteínas de Bactérias , Isoniazida/farmacologia , Mamíferos , Ácidos Micólicos , Quinolinas/farmacologia
5.
Med Sci (Paris) ; 37(11): 993-1001, 2021 Nov.
Artigo em Francês | MEDLINE | ID: mdl-34851275

RESUMO

Mycobacterium abscessus is an environmental fast-growing, non-tuberculous mycobacterium responsible for severe lung infections, especially in patients with underlying lung disorders such as cystic fibrosis. The standard chemotherapy combines a b-lactam (imipenem or cefoxitin), an aminoglycoside (amikacin) and a macrolide (clarithromycin or azithromycin). However, resistance of this bacterium to most antibiotic classes, including nearly all anti-tubercular drugs, leads frequently to treatment failure and considerably reduces the therapeutic arsenal available to the clinician. A comprehensive understanding of the innate and acquired resistance mechanisms is thus necessary to counteract M. abscessus lung infections.


TITLE: Mycobacterium abscessus, un modèle de résistance aux différentes classes d'antibiotiques. ABSTRACT: Mycobacterium abscessus est une bactérie non tuberculeuse, environnementale, à croissance rapide, qui est responsable d'infections pulmonaires sévères, notamment chez les patients atteints de mucoviscidose. Le traitement actuel combine l'utilisation d'une b-lactamine et d'un aminoglycoside associés à un macrolide. Cette bactérie est polyrésistante à la plupart des antibiotiques utilisés en clinique. Les mécanismes de résistance, innés ou acquis, qu'elle a développés, conduisent fréquemment à des échecs thérapeutiques, ce qui limite considérablement les moyens de lutte disponibles pour le clinicien. Une compréhension globale des mécanismes de résistance de cette bactérie s'avère ainsi nécessaire pour contrer les infections pulmonaires qu'elle provoque.


Assuntos
Antibacterianos , Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Amicacina , Antibacterianos/farmacologia , Claritromicina , Farmacorresistência Bacteriana , Humanos , Testes de Sensibilidade Microbiana , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico
6.
Dis Model Mech ; 14(9)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34530447

RESUMO

Infection by multidrug-resistant Mycobacterium abscessus is increasingly prevalent in cystic fibrosis (CF) patients, leaving clinicians with few therapeutic options. A compassionate study showed the clinical improvement of a CF patient with a disseminated M. abscessus (GD01) infection, following injection of a phage cocktail, including phage Muddy. Broadening the use of phage therapy in patients as a potential antibacterial alternative necessitates the development of biological models to improve the reliability and successful prediction of phage therapy in the clinic. Herein, we demonstrate that Muddy very efficiently lyses GD01 in vitro, an effect substantially increased with standard drugs. Remarkably, this cooperative activity was retained in an M. abscessus model of infection in CFTR-depleted zebrafish, associated with a striking increase in larval survival and reduction in pathological signs. The activity of Muddy was lost in macrophage-ablated larvae, suggesting that successful phage therapy relies on functional innate immunity. CFTR-depleted zebrafish represent a practical model to rapidly assess phage treatment efficacy against M. abscessus isolates, allowing the identification of drug combinations accompanying phage therapy and treatment prediction in patients. This article has an associated First Person interview with the first author of the paper.


Assuntos
Micobacteriófagos , Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Humanos , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Reprodutibilidade dos Testes , Peixe-Zebra
7.
Bioorg Med Chem Lett ; 30(22): 127576, 2020 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-32980514

RESUMO

A series of 4-aminoquinoline-isoindoline-dione-isoniazid triads were synthesized and assessed for their anti-mycobacterial activities and cytotoxicity. Most of the synthesized compounds exhibited promising activities against the mc26230 strain of M. tuberculosis with MIC in the range of 5.1-11.9 µM and were non-cytotoxic against Vero cells. The conjugates lacking either isoniazid or quinoline core in their structural framework failed to inhibit the growth of M. tuberculosis; thus, further strengthening the proposed design of triads in the present study.


Assuntos
Aminoquinolinas/farmacologia , Antituberculosos/farmacologia , Desenho de Fármacos , Indóis/farmacologia , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Aminoquinolinas/química , Antituberculosos/síntese química , Antituberculosos/química , Relação Dose-Resposta a Droga , Indóis/química , Isoniazida/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade
8.
Antimicrob Agents Chemother ; 64(11)2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-32816730

RESUMO

Mycobacterium abscessus is increasingly recognized as an emerging opportunistic pathogen causing severe lung diseases. As it is intrinsically resistant to most conventional antibiotics, there is an unmet medical need for effective treatments. Repurposing of clinically validated pharmaceuticals represents an attractive option for the development of chemotherapeutic alternatives against M. abscessus infections. In this context, rifabutin (RFB) has been shown to be active against M. abscessus and has raised renewed interest in using rifamycins for the treatment of M. abscessus pulmonary diseases. Here, we compared the in vitro and in vivo activity of RFB against the smooth and rough variants of M. abscessus, differing in their susceptibility profiles to several drugs and physiopathologial characteristics. While the activity of RFB is greater against rough strains than in smooth strains in vitro, suggesting a role of the glycopeptidolipid layer in susceptibility to RFB, both variants were equally susceptible to RFB inside human macrophages. RFB treatment also led to a reduction in the number and size of intracellular and extracellular mycobacterial cords. Furthermore, RFB was highly effective in a zebrafish model of infection and protected the infected larvae from M. abscessus-induced killing. This was corroborated by a significant reduction in the overall bacterial burden, as well as decreased numbers of abscesses and cords, two major pathophysiological traits in infected zebrafish. This study indicates that RFB is active against M. abscessus both in vitro and in vivo, further supporting its potential usefulness as part of combination regimens targeting this difficult-to-treat mycobacterium.


Assuntos
Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Animais , Antibacterianos/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Rifabutina/farmacologia , Peixe-Zebra
9.
Artigo em Inglês | MEDLINE | ID: mdl-32041716

RESUMO

New drugs or therapeutic combinations are urgently needed against Mycobacterium abscessus Previously, we demonstrated the potent activity of indole-2-carboxamides 6 and 12 against M. abscessus We show here that these compounds act synergistically with imipenem and cefoxitin in vitro and increase the bactericidal activity of the ß-lactams against M. abscessus In addition, compound 12 also displays synergism with imipenem and cefoxitin within infected macrophages. The clinical potential of these new drug combinations requires further evaluation.


Assuntos
Antibacterianos/farmacologia , Indóis/farmacologia , Mycobacterium abscessus/efeitos dos fármacos , beta-Lactamas/farmacologia , Cefoxitina/farmacologia , Contagem de Colônia Microbiana , Sinergismo Farmacológico , Humanos , Imipenem/farmacologia , Macrófagos/microbiologia , Testes de Sensibilidade Microbiana , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia
10.
ACS Infect Dis ; 6(2): 324-337, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31860799

RESUMO

The prevalence of pulmonary infections due to nontuberculous mycobacteria such as Mycobacterium abscessus has been increasing and surpassing tuberculosis (TB) in some industrialized countries. Because of intrinsic resistance to most antibiotics that drastically limits conventional chemotherapeutic treatment options, new anti-M. abscessus therapeutics are urgently needed against this emerging pathogen. Extensive screening of a library of benzimidazole derivatives that were previously shown to be active against Mycobacterium tuberculosis led to the identification of a lead compound exhibiting very potent in vitro activity against a wide panel of M. abscessus clinical strains. Designated EJMCh-6, this compound, a 2-(2-cyclohexylethyl)-5,6-dimethyl-1H-benzo[d]imidazole), also exerted very strong activity against intramacrophage-residing M. abscessus. Moreover, the treatment of infected zebrafish embryos with EJMCh-6 was correlated with significantly increased embryo survival and a decrease in the bacterial burden as compared to those for untreated fish. Insights into the mechanism of action were inferred from the generation of spontaneous benzimidazole-resistant strains and the identification of a large set of missense mutations in MmpL3, the mycolic acid transporter in mycobacteria. Overexpression of the mutated mmpL3 alleles in a susceptible M. abscessus strain was associated with high resistance levels to EJMCh-6 and to other known MmpL3 inhibitors. Mapping the mutations conferring resistance on an MmpL3 three-dimensional homology model defined a potential EJMCh-6-binding cavity. These data emphasize a yet unexploited chemical structure class against M. abscessus with promising translational development for the treatment of M. abscessus lung diseases.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Benzimidazóis/química , Benzimidazóis/farmacologia , Mycobacterium abscessus/efeitos dos fármacos , Animais , Antituberculosos/química , Proteínas de Bactérias/genética , Transporte Biológico , Farmacorresistência Bacteriana/genética , Humanos , Pneumopatias/tratamento farmacológico , Pneumopatias/microbiologia , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Mutação , Infecções por Mycobacterium não Tuberculosas/microbiologia , Relação Estrutura-Atividade , Células THP-1 , Tuberculose/microbiologia , Peixe-Zebra/microbiologia , Peixe-Zebra/fisiologia
11.
J Biol Chem ; 294(46): 17512-17523, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31562241

RESUMO

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.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Ácidos Micólicos/metabolismo , Piperidinas/farmacologia , Antituberculosos/química , Proteínas de Bactérias/metabolismo , Transporte Biológico/efeitos dos fármacos , Fatores Corda/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Piperidinas/química , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia
12.
Artigo em Inglês | MEDLINE | ID: mdl-31332077

RESUMO

Mycobacterium abscessus is a human pathogen responsible for severe respiratory infections, particularly in patients with underlying lung disorders. Notorious for being highly resistant to most antimicrobials, new therapeutic approaches are needed to successfully treat M. abscessus-infected patients. Clofazimine (CFZ) and bedaquiline (BDQ) are two antibiotics used for the treatment of multidrug-resistant tuberculosis and are considered alternatives for the treatment of M. abscessus pulmonary disease. To get insights into their mechanisms of resistance in M. abscessus, we previously characterized the TetR transcriptional regulator MAB_2299c, which controls expression of the MAB_2300-MAB_2301 genes, encoding an MmpS-MmpL efflux pump. Here, in silico studies identified a second mmpS-mmpL (MAB_1135c-MAB_1134c) target of MAB_2299c. A palindromic DNA sequence upstream of MAB_1135c, sharing strong homology with the one located upstream of MAB_2300, was found to form a complex with the MAB_2299c regulator in electrophoretic mobility shift assays. Deletion of MAB_1135c-1134c in a wild-type strain led to increased susceptibility to both CFZ and BDQ. In addition, deletion of these genes in a CFZ/BDQ-susceptible mutant lacking MAB_2299c as well as MAB_2300-MAB_2301 further exacerbated the sensitivity of this strain to both drugs in vitro and inside macrophages. Overall, these results indicate that MAB_1135c-1134c encodes a new MmpS-MmpL efflux pump system involved in the intrinsic resistance to CFZ and BDQ. They also support the view that MAB_2299c controls the expression of two separate MmpS-MmpL efflux pumps, substantiating the importance of MAB_2299c as a marker of resistance to be considered when assessing drug susceptibility in clinical isolates.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Clofazimina/farmacologia , Diarilquinolinas/farmacologia , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium abscessus/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase em Tempo Real , Células THP-1 , Fatores de Transcrição/genética
13.
Artigo em Inglês | MEDLINE | ID: mdl-31209005

RESUMO

Due to intrinsic multidrug resistance, pulmonary infections with Mycobacterium abscessus are extremely difficult to treat. Previously, we demonstrated that bedaquiline is highly effective against Mycobacterium abscessus both in vitro and in vivo Here, we report that verapamil improves the efficacy of bedaquiline activity against M. abscessus clinical isolates and low-level resistant strains, both in vitro and in macrophages. Verapamil may have clinical potential as adjunctive therapy provided that sufficiently high doses can be safely achieved.


Assuntos
Antibacterianos/farmacologia , Diarilquinolinas/farmacologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium abscessus/efeitos dos fármacos , Verapamil/farmacologia , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Linhagem Celular , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Sinergismo Farmacológico , Humanos , Macrófagos/microbiologia , Testes de Sensibilidade Microbiana , Mutação , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Mycobacterium abscessus/genética , Mycobacterium abscessus/isolamento & purificação
14.
Artigo em Inglês | MEDLINE | ID: mdl-30323043

RESUMO

New therapeutic approaches are needed against Mycobacterium abscessus, a respiratory mycobacterial pathogen that evades efforts to successfully treat infected patients. Clofazimine and bedaquiline, two drugs used for the treatment of multidrug-resistant tuberculosis, are being considered as alternatives for the treatment of lung diseases caused by M. abscessus With the aim to understand the mechanism of action of these agents in M. abscessus, we sought herein to determine the means by which M. abscessus can develop resistance. Spontaneous resistant strains selected on clofazimine, followed by whole-genome sequencing, identified mutations in MAB_2299c, encoding a putative TetR transcriptional regulator. Unexpectedly, mutants with these mutations were also cross-resistant to bedaquiline. MAB_2299c was found to bind to its target DNA, located upstream of the divergently oriented MAB_2300-MAB_2301 gene cluster, encoding MmpS/MmpL membrane proteins. Point mutations or deletion of MAB_2299c was associated with the concomitant upregulation of the mmpS and mmpL transcripts and accounted for this cross-resistance. Strikingly, deletion of MAB_2300 and MAB_2301 in the MAB_2299c mutant strain restored susceptibility to bedaquiline and clofazimine. Overall, these results expand our knowledge with respect to the regulatory mechanisms of the MmpL family of proteins and a novel mechanism of drug resistance in this difficult-to-treat respiratory mycobacterial pathogen. Therefore, MAB_2299c may represent an important marker of resistance to be considered in the treatment of M. abscessus diseases with clofazimine and bedaquiline in clinical settings.


Assuntos
Antituberculosos/farmacologia , Clofazimina/farmacologia , Diarilquinolinas/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium abscessus/genética , Resistência a Tetraciclina/genética , Genoma Bacteriano/genética , Humanos , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Transativadores/genética , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Sequenciamento Completo do Genoma
15.
Artigo em Inglês | MEDLINE | ID: mdl-28807917

RESUMO

Pulmonary infections caused by Mycobacterium abscessus are emerging as a global threat, especially in cystic fibrosis patients. Further intensifying the concern of M. abscessus infection is the recent evidence of human-to-human transmission of the infection. M. abscessus is a naturally multidrug-resistant fast-growing pathogen for which pharmacological options are limited. Repurposing antitubercular drugs represents an attractive option for the development of chemotherapeutic alternatives against M. abscessus infections. Bedaquiline (BDQ), an ATP synthase inhibitor, has recently been approved for the treatment of multidrug-resistant tuberculosis. Herein, we show that BDQ has a very low MIC against a vast panel of clinical isolates. Despite being bacteriostatic in vitro, BDQ was highly efficacious in a zebrafish model of M. abscessus infection. Remarkably, a very short period of treatment was sufficient to protect the infected larvae from M. abscessus-induced killing. This was corroborated with reduced numbers of abscesses and cords, considered to be major pathophysiological signs in infected zebrafish. Mode-of-action studies revealed that BDQ triggered a rapid depletion of ATP in M. abscessusin vitro, consistent with the drug targeting the FoF1 ATP synthase. Importantly, despite a failure to select in vitro for spontaneous mutants that are highly resistant to BDQ, the transfer of single nucleotide polymorphisms leading to D29V or A64P substitutions in atpE conferred high resistance, thus resolving the target of BDQ in M. abscessus Overall, this study indicates that BDQ is active against M. abscessusin vitro and in vivo and should be considered for clinical use against the difficult-to-manage M. abscessus pulmonary infections.


Assuntos
Antituberculosos/farmacologia , ATPases Bacterianas Próton-Translocadoras/antagonistas & inibidores , Diarilquinolinas/farmacologia , Mycobacterium abscessus/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , ATPases Bacterianas Próton-Translocadoras/genética , ATPases Bacterianas Próton-Translocadoras/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium abscessus/metabolismo , Polimorfismo de Nucleotídeo Único , Peixe-Zebra/microbiologia
16.
Nucleic Acids Res ; 42(16): 10399-408, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25122744

RESUMO

RbpA, a transcriptional activator that is essential for Mycobacterium tuberculosis replication and survival during antibiotic treatment, binds to RNA polymerase (RNAP) in the absence of promoter DNA. It has been hypothesized that RbpA stimulates housekeeping gene expression by promoting assembly of the σ(A) subunit with core RNAP. Here, using a purified in vitro transcription system of M. tuberculosis, we show that RbpA functions in a promoter-dependent manner as a companion of RNAP essential for promoter DNA unwinding and formation of the catalytically active open promoter complex (RPo). Screening for RbpA activity using a full panel of the M. tuberculosis σ subunits demonstrated that RbpA targets σ(A) and stress-response σ(B), but not the alternative σ subunits from the groups 3 and 4. In contrast to σ(A), the σ(B) subunit activity displayed stringent dependency upon RbpA. These results suggest that RbpA-dependent control of RPo formation provides a mechanism for tuning gene expression during the switch between different physiological states, and in the stress response.


Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/genética , Regiões Promotoras Genéticas , Fator sigma/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Holoenzimas/metabolismo , Mycobacterium tuberculosis/enzimologia
17.
J Neurosci ; 31(42): 14882-92, 2011 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-22016521

RESUMO

Accumulation of PrP(Sc), an abnormal form of cellular prion protein (PrP), in the brain of animals and humans leads to fatal neurodegenerative disorders known as prion diseases. Limited protease digestion of PrP(Sc) produces a truncated form called PrP(27-30) that retains prion infectivity and is the main marker of disease targeted in most diagnostic tests. In the search for new anti-prion molecules, drug-screening assays on prion-infected murine cells have been oriented toward decreasing levels of PrP(27-30). In contrast, we screened for drugs promoting multimers of PrP(27-30), illustrating a possible stabilization of mouse PrP(Sc) species, because recent studies aiming to characterize the conformational stability of various prion strains showed that stable recombinant amyloids produced more stable prion strain, leading to longest incubation time. We identified a family of thienyl pyrimidine derivatives that induce SDS-resistant dimers and trimers of PrP(27-30). Bioassays performed on mice brain homogenates treated with these compounds showed that these thienyl pyrimidine derivatives diminished prion infectivity in vivo. Oligomeric-induced activity by thienyl pyrimidine compounds is a promising approach not only to understanding the pathogenesis of prions but also for prion diagnostics. This approach could be extended to other neurodegenerative "prionopathies," such as Alzheimer's, Huntington, or Parkinson's diseases.


Assuntos
Encéfalo/efeitos dos fármacos , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Doenças Priônicas/patologia , Pirimidinas/farmacologia , Anilidas/farmacologia , Anilidas/uso terapêutico , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Endopeptidase K/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Camundongos , Modelos Moleculares , Neuroblastoma/patologia , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas PrPC/análise , Doenças Priônicas/tratamento farmacológico , Conformação Proteica/efeitos dos fármacos , Pirimidinas/uso terapêutico , Silício , Estatísticas não Paramétricas , Fatores de Tempo , Transfecção/métodos
18.
Int J Antimicrob Agents ; 35(6): 519-23, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20185278

RESUMO

The first antibiotic of the ansamycin family, rifampicin (RIF), was isolated in 1959 and was introduced into therapy in 1962; it is still a first-line agent in the treatment of diseases such as tuberculosis, leprosy and various biofilm-related infections. The antimicrobial activity of RIF is due to its inhibition of bacterial RNA polymerase (RNAP). Most frequently, bacteria become resistant to RIF through mutation of the target; however, this mechanism is not unique. Other mechanisms of resistance have been reported, such as duplication of the target, action of RNAP-binding proteins, modification of RIF and modification of cell permeability. We suggest that several of these alternative resistance strategies could reflect the ecological function of RIF, such as autoregulation and/or signalling to surrounding microorganisms. Very often, resistance mechanisms found in the clinic have an environmental origin. One may ask whether the introduction of the RIF analogues rifaximin, rifalazil, rifapentine and rifabutin in the therapeutic arsenal, together with the diversification of the pathologies treated by these molecules, will diversify the resistance mechanisms of human pathogens against ansamycins.


Assuntos
Antibacterianos/uso terapêutico , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Farmacorresistência Bacteriana , Rifampina/uso terapêutico , Antibacterianos/farmacologia , Bactérias/isolamento & purificação , Ecologia , Genes Bacterianos , Humanos , Rifampina/farmacologia , Seleção Genética
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