RESUMO
Tuberculosis (TB) caused by bacteria of the Mycobacterium tuberculosis complex remains one of the most important infectious diseases of mankind. Rifampicin is a first line drug used in multi-drug treatment of TB, however, the necessary duration of treatment with these drugs is long and development of resistance is an increasing impediment to treatment programmes. As a result, there is a requirement for research and development of new TB drugs, which can form the basis of new drug combinations, either due to their own anti-mycobacterial activity or by augmenting the activity of existing drugs such as rifampicin. This study describes a TnSeq analysis to identify mutants with enhanced sensitivity to sub-minimum inhibitory concentrations (MIC) of rifampicin. The rifampicin-sensitive mutants were disrupted in genes of a variety of functions and the majority fitted into three thematic groups: firstly, genes that were involved in DNA/RNA metabolism, secondly, genes involved in sensing and regulating mycobacterial cellular systems, and thirdly, genes involved in the synthesis and maintenance of the cell wall. Selection at two concentrations of rifampicin (1/250 and 1/62 MIC) demonstrated a dose response for mutants with statistically significant sensitivity to rifampicin. The dataset reveals mechanisms of how mycobacteria are innately tolerant to and initiate an adaptive response to rifampicin; providing putative targets for the development of adjunctive therapies that potentiate the action of rifampicin.
Assuntos
Testes de Sensibilidade Microbiana , Mycobacterium bovis , Rifampina , Rifampina/farmacologia , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/genética , Antituberculosos/farmacologia , Mutação , Farmacorresistência Bacteriana/genéticaRESUMO
In vitro biofilm models have allowed researchers to investigate the role biofilms play in the pathogenesis, virulence, and antimicrobial drug susceptibility of a wide range of bacterial pathogens. Rotary cell culture systems create three-dimensional cellular structures, primarily applied to eukaryotic organoids, that better capture characteristics of the cells in vivo. Here, we describe how to apply a low-shear, detergent-free rotary cell culture system to generate biofilms of Mycobacterium bovis BCG. The three-dimensional biofilm model forms mycobacterial cell aggregates in suspension as surface-detached biomass, without severe nutrient starvation or environmental stress, that can be harvested for downstream experiments. Mycobacterium bovis BCG derived from cell clusters display antimicrobial drug tolerance, presence of an extracellular matrix, and evidence of cell wall remodeling, all features of biofilm-associated bacteria that may be relevant to the treatment of tuberculosis.
Assuntos
Biofilmes , Mycobacterium bovis , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/fisiologia , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células em Três Dimensões/métodosRESUMO
Delivering novel antimycobacterial agents through the pulmonary route using nanoparticle-based systems shows promise for treating diseases like tuberculosis. However, creating dry powder inhaler (DPI) with suitable aerodynamic characteristics while preserving nanostructure integrity and maintaining bioactivity until the active ingredient travels deeply into the lungs is a difficult challenge. We developed DPI formulations containing levofloxacin-loaded solid lipid nanoparticles (SLNs) via spray-drying technique with tailored aerosolization characteristics for effective inhalation therapy. A range of biophysical techniques, including transmission electron microscopy, confocal microscopy, and scanning electron microscopy were used to measure the morphologies and sizes of the spray-dried microparticles that explored both the geometric and aerodynamic properties. Spray drying substantially reduced the particle sizes of the SLNs while preserving their nanostructural integrity and enhancing aerosol dispersion with efficient mucus penetration. Despite a slower uptake rate compared to plain SLNs, the polyethylene glycol modified formulations exhibited enhanced cellular uptake in both A549 and NR8383 cell lines. The percent viability of Mycobacterium bovis had dropped to nearly 0 % by day 5 for both types of SLNs. Interestingly, the levofloxacin-loaded SLNs demonstrated a lower minimum bactericidal concentration (0.25 µg/mL) compared with pure levofloxacin (1 µg/mL), which indicated the formulations have potential as effective treatments for tuberculosis.
Assuntos
Antituberculosos , Inaladores de Pó Seco , Levofloxacino , Nanopartículas , Tamanho da Partícula , Tuberculose , Levofloxacino/administração & dosagem , Levofloxacino/química , Levofloxacino/farmacologia , Nanopartículas/química , Administração por Inalação , Humanos , Antituberculosos/administração & dosagem , Antituberculosos/química , Antituberculosos/farmacologia , Antituberculosos/farmacocinética , Tuberculose/tratamento farmacológico , Lipídeos/química , Mycobacterium bovis/efeitos dos fármacos , Linhagem Celular , Aerossóis , Células A549 , Animais , Secagem por Atomização , Testes de Sensibilidade Microbiana , Portadores de Fármacos/química , Polietilenoglicóis/química , Antibacterianos/administração & dosagem , Antibacterianos/química , Antibacterianos/farmacologia , LipossomosRESUMO
BACKGROUND: Tuberculosis currently stands as the second leading cause of deaths worldwide due to single infectious agent after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The current challenges of drug resistance in tuberculosis highlight an urgent need to develop newer anti-mycobacterial compounds. In the present study, we report the serendipitous discovery of a bacterial laboratory contaminant (LC-1) exhibiting a zone of growth inhibition on an agar plate seeded with Mycobacterium tuberculosis. RESULTS: We utilized microbiological, biochemical and biophysical approaches to characterize LC-1 and anti-mycobacterial compound(s) in its secretome. Based on 16S rRNA sequencing and BIOLOG analysis, LC-1 was identified as Staphylococcus hominis, a human bacterial commensal. Anti-mycobacterial activity was initially found in 30 kDa retentate that was obtained by ultrafiltration of culture filtrate (CF). SDS-PAGE analysis of peak fractions obtained by size exclusion chromatography of 30 kDa retentate confirmed the presence of high molecular weight (≥ 30 kDa) proteins. Peak fraction-1 (F-1) exhibited inhibitory activity against M. bovis BCG, but not against M. smegmatis, E. coli and S. aureus. The active fraction F-1 was inactivated by treatment with Proteinase K and α-chymotrypsin. However, it retained its anti-mycobacterial activity over a wide range of heat and pH treatment. The anti-mycobacterial activity of F-1 was found to be maintained even after a long storage (~12 months) at - 20 °C. Mass spectrometry analysis revealed that the identified peptide masses do not match with any previously known bacteriocins. CONCLUSIONS: The present study highlights the anti-mycobacterial activity of high molecular weight protein(s) present in culture filtrate of LC-1, which may be tested further to target M. tuberculosis. The heat and pH stability of these proteins add to their characteristics as therapeutic proteins and may contribute to their long shelf life. LC-1 being a human commensal can be tested in future for its potential as a probiotic to treat tuberculosis.
Assuntos
Antituberculosos/química , Proteínas de Bactérias/química , Antituberculosos/isolamento & purificação , Antituberculosos/farmacologia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Estabilidade de Medicamentos , Endopeptidase K/metabolismo , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Mycobacterium bovis/efeitos dos fármacos , Staphylococcus hominis/metabolismoRESUMO
During our screening for antibiotics against Mycobacterium avium complex (MAC) with a mass spectrometry network-based indexing approach, a new compound named kimidinomycin was isolated from the culture broth of Streptomyces sp. KKTA-0263 by solvent extraction, HP20 column chromatography, and preparative HPLC. From the structural elucidation, the compound possesses a 38-membered macrolide structure with an N-methylguanidyl group at the terminal side chain. The compound exhibited antimycobacterial activity against M. avium, M. intracellulare, M. smegmatis, and M. bovis BCG with respective MIC values of 12.5, 0.78, 12.5, and 25.0 µg ml-1.
Assuntos
Antibióticos Antituberculose , Complexo Mycobacterium avium , Streptomyces , Animais , Cricetinae , Humanos , Antibióticos Antituberculose/biossíntese , Antibióticos Antituberculose/farmacologia , Antibióticos Antituberculose/toxicidade , Células CHO , Cromatografia Líquida de Alta Pressão , Cricetulus , Fermentação , Células HeLa , Macrolídeos/química , Testes de Sensibilidade Microbiana , Complexo Mycobacterium avium/efeitos dos fármacos , Infecção por Mycobacterium avium-intracellulare , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium smegmatis/efeitos dos fármacos , Streptomyces/metabolismoRESUMO
Indolmycin (IND) is a microbial natural product that selectively inhibits bacterial tryptophanyl-tRNA synthetase (TrpRS). The tryptophan biosynthesis pathway was recently shown to be an important target for developing new antibacterial agents against Mycobacterium tuberculosis (Mtb). We investigated the antibacterial activity of IND against several mycobacterial model strains. A TrpRS biochemical assay was developed to analyze a library of synthetic IND analogues. The 4â³-methylated IND compound, Y-13, showed improved anti-Mtb activity with a minimum inhibitory concentration (MIC) of 1.88 µM (â¼0.5 µg/mL). The MIC increased significantly when overexpression of TrpRS was induced in the genetically engineered surrogate M. bovis BCG. The cocrystal structure of Mtb TrpRS complexed with IND and ATP has revealed that the amino acid pocket is in a state between the open form of apo protein and the closed complex with the reaction intermediate. In whole-cell-based experiments, we studied the combination effect of Y-13 paired with different antibacterial agents. We evaluated the killing kinetics, the frequency of resistance to INDs, and the mode of resistance of IND-resistant mycobacteria by genome sequencing. The synergistic interaction of Y-13 with the TrpE allosteric inhibitor, indole propionic acid, suggests that prospective IND analogues could shut down tryptophan biosynthesis and protein biosynthesis in pathogens, leading to a new class of antibiotics. Finally, we discuss a strategy to expand the genome mining of antibiotic-producing microbes specifically for antimycobacterial development.
Assuntos
Antibacterianos/farmacologia , Produtos Biológicos/farmacologia , Mycobacterium bovis/efeitos dos fármacos , Antibacterianos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Produtos Biológicos/química , Clonagem Molecular , Escherichia coli , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Indóis/química , Indóis/farmacologia , Estrutura Molecular , MutaçãoRESUMO
Drug-resistant tuberculosis (DR-TB) poses a new threat to global health; to improve the treatment outcome, therapeutic vaccines are considered the best chemotherapy adjuvants. Unfortunately, there is no therapeutic vaccine approved against DR-TB. Our study assessed the therapeutic efficacy of a recombinant drug-resistant BCG (RdrBCG) vaccine in DR-TB. We constructed the RdrBCG overexpressing Ag85B and Rv2628 by selecting drug-resistant BCG strains and transformed them with plasmid pEBCG or pIBCG to create RdrBCG-E and RdrBCG-I respectively. Following successful stability testing, we tested the vaccine's safety in severe combined immune deficient (SCID) mice that lack both T and B lymphocytes plus immunoglobulins. Finally, we evaluated the RdrBCG's therapeutic efficacy in BALB/c mice infected with rifampin-resistant M. tuberculosis and treated with a second-line anti-TB regimen. We obtained M. bovis strains which were resistant to several second-line drugs and M. tuberculosis resistant to rifampin. Notably, the exogenously inserted genes were lost in RdrBCG-E but remained stable in the RdrBCG-I both in vitro and in vivo. When administered adjunct to a second-line anti-TB regimen in a murine model of DR-TB, the RdrBCG-I lowered lung M. tuberculosis burden by 1 log10. Furthermore, vaccination with RdrBCG-I adjunct to chemotherapy minimized lung tissue pathology in mice. Most importantly, the RdrBCG-I showed almost the same virulence as its parent BCG Tice strain in SCID mice. Our findings suggested that the RdrBCG-I was stable, safe and effective as a therapeutic vaccine. Hence, the "recombinant" plus "drug-resistant" BCG strategy could be a useful concept for developing therapeutic vaccines against DR-TB.
Assuntos
Antituberculosos/farmacologia , Vacina BCG/imunologia , Farmacorresistência Bacteriana/genética , Mycobacterium bovis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Pulmonar/prevenção & controle , Vacinas Sintéticas/imunologia , Amicacina/farmacologia , Amicacina/uso terapêutico , Animais , Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Antituberculosos/uso terapêutico , Vacina BCG/biossíntese , Vacina BCG/genética , Vacina BCG/uso terapêutico , Modelos Animais de Doenças , Levofloxacino/farmacologia , Levofloxacino/uso terapêutico , Camundongos Endogâmicos BALB C , Camundongos SCID , Mycobacterium bovis/química , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Plasmídeos , Protionamida/farmacologia , Protionamida/uso terapêutico , Pirazinamida/farmacologia , Pirazinamida/uso terapêutico , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/patologia , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/genética , Vacinas Sintéticas/uso terapêutico , VirulênciaRESUMO
Mycobacterium bovis is the causative agent of bovine tuberculosis and also responsible for serious threat to public health. Koumiss is a fermented mare's milk product, used as traditional drink. Here, we explored the effect of koumiss on gut microbiota and the host immune response against M bovis infection. Therefore, mice were treated with koumiss and fresh mare milk for 14 days before M bovis infection and continue for 5 weeks after infection. The results showed a clear change in the intestinal flora of mice treated with koumiss, and the lungs of mice treated with koumiss showed severe edema, inflammatory infiltration, and pulmonary nodules in M bovis-infected mice. Notably, we found that the content of short-chain fatty acids was significantly lower in the koumiss-treated group compared with the control group. However, the expression of endoplasmic reticulum stress and apoptosis-related proteins in the lungs of koumiss-treated mice were significantly decreased. Collectively, these findings suggest that koumiss treatment disturb the intestinal flora of, which is associated with disease severity and the possible mechanism that induces lungs pathology. Our current findings can be exploited further to establish the "gut-lung" axis which might be a novel strategy for the control of tuberculosis.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Kumis/efeitos adversos , Mycobacterium bovis/efeitos dos fármacos , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Ácidos Graxos/análise , Fezes/química , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/imunologia , Cavalos , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/imunologia , Tuberculose Pulmonar/dietoterapia , Tuberculose Pulmonar/metabolismoRESUMO
Context: Disseminated infections due to Mycobacterium bovis Bacillus Calmette-Guérin (BCG) are unusual and occur mostly in patients with inborn error of immunity (IEI) or acquired immunodeficiency. However, cases of secondary BCGosis due to intravesical BCG instillation have been described. Herein, we present a case of severe BCGosis occurring in an unusual situation. Case Description: We report one case of severe disseminated BCG disease occurring after hematological malignancy in a 48-year-old man without BCG instillation and previously vaccinated in infancy with no complication. Laboratory investigations demonstrated that he was not affected by any known or candidate gene of IEI or intrinsic cellular defect involving IFNγ pathway. Whole genome sequencing of the BCG strain showed that it was most closely related to the M. bovis BCG Tice strain, suggesting an unexpected relationship between the secondary immunodeficiency of the patient and the acquired BCG infection. Conclusion: This case highlights the fact that, in addition to the IEI, physicians, as well as microbiologists and pharmacists should be aware of possible acquired disseminated BCG disease in secondary immunocompromised patients treated in centers that administrate BCG for bladder cancers.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Hematológicas/tratamento farmacológico , Reconstituição Imune , Hospedeiro Imunocomprometido , Mycobacterium bovis/patogenicidade , Infecções Oportunistas/microbiologia , Tuberculose Pulmonar/microbiologia , Administração Intravesical , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Antituberculosos/uso terapêutico , Vacina BCG/administração & dosagem , Vacina BCG/efeitos adversos , Interações Hospedeiro-Patógeno , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/imunologia , Infecções Oportunistas/tratamento farmacológico , Infecções Oportunistas/imunologia , Fatores de Risco , Resultado do Tratamento , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/imunologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/imunologiaRESUMO
We herein report new 5-substituted uridine derivatives as potent inhibitors of mycobacteria - causative agents of tuberculosis. A series of new 5-alkynyl-substituted uridine derivatives were synthesised via palladium-catalysed Sonogashira cross-coupling reaction of 5-iodo-6-methylpyrimidine base with terminal acetylenes with good yields in DMF at room temperature. It was found that methyl group in C-6 position of pyrimidine ring had no impact on yields of target compounds. All obtained compounds were evaluated for their antimycobacterial activity against Mycobacetrium bovis and Mycobacterium tuberculosis at concentrations of 1-100 µg/ml using MABA test. Synthesized nucleosides showed high antimycobacterial activity against M. bovis and M. Tuberculosis. The MIC50 values of 11 and 13 were similar or close to that of the reference drug rifampicin.
Assuntos
Antituberculosos/farmacologia , Desenho de Fármacos , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Nucleosídeos de Pirimidina/farmacologia , Antituberculosos/síntese química , Antituberculosos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Nucleosídeos de Pirimidina/síntese química , Nucleosídeos de Pirimidina/química , Relação Estrutura-AtividadeRESUMO
To search for potent antimycobacterial lead compounds, a new series of 3-substituted phenyl-2-(2-(substituted phenyl)thiazol-4-yl) thiazolidin-4-one (5a-t) derivatives have been synthesized by the condensation of 2-substituted phenyl thiazole-4-carbaldehyde with aromatic amine followed by cyclocondensation with thioglycolic acid. The structure of the newly synthesized 2-(thiazol-4-yl)thiazolidin-4-one derivatives were characterized by the spectroscopic analysis. The synthesized compounds were screened for antimycobacterial activity against Mycobacterium tuberculosis H37Ra (MTB) (ATCC 25177) and Mycobacterium bovis BCG (BCG, ATCC 35743). Most of the 2-(thiazol-4-yl)thiazolidin-4-one derivatives showed good to excellent antimycobacterial activity against both the Mtb strains. Nine derivatives 5c, 5g, 5j, 5m, 5n, 5o, 5p, 5s, and 5t showed excellent activity against M. bovis BCG with MIC 4.43 to 24.04 µM were further evaluated for the cytotoxicity activity against HeLa A549, and HCT-116 cell lines and showed no significant cytotoxic activity at the maximum concentration evaluated. The potential antimycobacterial activities enforced that the thiazolyl-thiazolidin-4-one derivatives could lead to compounds that could treat tuberculosis.
Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Tiazóis/química , Tiazóis/farmacologia , Animais , Antituberculosos/síntese química , Linhagem Celular , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Relação Estrutura-Atividade , Tiazóis/síntese química , Tuberculose/tratamento farmacológico , Tuberculose/veterináriaRESUMO
Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) still remains a devastating infectious disease in the world. There has been a daunting increase in the incidence of Type 2 Diabetes Mellitus (T2DM) worldwide. T2DM patients are three times more vulnerable to M. tb infection compared to healthy individuals. TB-T2DM coincidence is a challenge for global health control. Despite some progress in the research, M. tb still has unexplored characteristics in successfully evading host defenses. The lengthy duration of treatment, the emergence of multi-drug-resistant strains and extensive-drug-resistant strains of M. tb have made TB treatment very challenging. Previously, we have tested the antimycobacterial effects of everolimus within in vitro granulomas generated from immune cells derived from peripheral blood of healthy subjects. However, the effectiveness of everolimus treatment against mycobacterial infection in individuals with T2DM is unknown. Furthermore, the effectiveness of the combination of in vivo glutathione (GSH) supplementation in individuals with T2DM along with in vitro treatment of isolated immune cells with everolimus against mycobacterial infection has never been tested. Therefore, we postulated that liposomal glutathione (L-GSH) and everolimus would offer great hope for developing adjunctive therapy for mycobacterial infection. L-GSH or placebo was administered to T2DM individuals orally for three months. Study subjects' blood was drawn pre- and post-L-GSH/or placebo supplementation, where Peripheral Blood Mononuclear Cells (PBMCs) were isolated from whole blood to conduct in vitro studies with everolimus. We found that in vitro treatment with everolimus, an mTOR (membrane target of rapamycin) inhibitor, significantly reduced intracellular M. bovis BCG infection alone and in conjunction with L-GSH supplementation. Furthermore, we found L-GSH supplementation coupled with in vitro everolimus treatment produced a greater effect in inhibiting the growth of intracellular Mycobacterium bovis BCG, than with the everolimus treatment alone. We also demonstrated the functions of L-GSH along with in vitro everolimus treatment in modulating the levels of cytokines such as IFN-γ, TNF-α, and IL-2 and IL-6, in favor of improving control of the mycobacterial infection. In summary, in vitro everolimus-treatment alone and in combination with oral L-GSH supplementation for three months in individuals with T2DM, was able to increase the levels of T-helper type 1 (Th1) cytokines IFN-γ, TNF-α, and IL-2 as well as enhance the abilities of granulomas from individuals with T2DM to improve control of a mycobacterial infection.
Assuntos
Vacina BCG/administração & dosagem , Diabetes Mellitus Tipo 2/imunologia , Everolimo/farmacologia , Glutationa/administração & dosagem , Leucócitos Mononucleares/imunologia , Mycobacterium bovis/imunologia , Tuberculose/imunologia , Administração Oral , Adolescente , Adulto , Idoso , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/microbiologia , Suplementos Nutricionais , Método Duplo-Cego , Feminino , Granuloma/imunologia , Humanos , Imunidade , Imunossupressores/farmacologia , Técnicas In Vitro , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/metabolismo , Tuberculose/tratamento farmacológico , Tuberculose/metabolismo , Tuberculose/microbiologia , Adulto JovemRESUMO
For over 50 years, patients with drug-sensitive and drug-resistant tuberculosis have undergone long, arduous, and complex treatment processes with several antimicrobials. With the prevalence of drug-resistant strains on the rise and new therapies for tuberculosis urgently required, we assessed whether manipulating iron levels in macrophages infected with mycobacteria offered some insight into improving current antimicrobials that are used to treat drug-resistant tuberculosis. We investigated if the iron chelator, desferrioxamine, can support the function of human macrophages treated with an array of second-line antimicrobials, including moxifloxacin, bedaquiline, amikacin, clofazimine, linezolid and cycloserine. Primary human monocyte-derived macrophages were infected with Bacillus Calmette-Guérin (BCG), which is pyrazinamide-resistant, and concomitantly treated for 5 days with desferrioxamine in combination with each one of the second-line tuberculosis antimicrobials. Our data indicate that desferrioxamine used as an adjunctive treatment to bedaquiline significantly reduced the bacterial load in human macrophages infected with BCG. Our findings also reveal a link between enhanced bactericidal activity and increases in specific cytokines, as the addition of desferrioxamine increased levels of IFN-γ, IL-6, and IL-1ß in BCG-infected human monocyte-derived macrophages (hMDMs) treated with bedaquiline. These results provide insight, and an in vitro proof-of-concept, that iron chelators may prove an effective adjunctive therapy in combination with current tuberculosis antimicrobials.
Assuntos
Antituberculosos/farmacologia , Desferroxamina/farmacologia , Diarilquinolinas/farmacologia , Quelantes de Ferro/farmacologia , Ferro/metabolismo , Macrófagos/efeitos dos fármacos , Mycobacterium bovis/efeitos dos fármacos , Amicacina/farmacologia , Carga Bacteriana/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Clofazimina/farmacologia , Ciclosserina/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Sinergismo Farmacológico , Expressão Gênica , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Linezolida/farmacologia , Macrófagos/imunologia , Macrófagos/microbiologia , Testes de Sensibilidade Microbiana , Moxifloxacina/farmacologia , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium bovis/metabolismo , Cultura Primária de Células , Pirazinamida/farmacologiaRESUMO
Genomic analysis revealed that the vaccine seed lot of Mycobacterium bovis bacillus Calmette-Guérin (BCG) Tokyo 172 contains two subclones (types I and II), but their phenotypic differences have not been elucidated. In this study, we compared the susceptibility of bacilli types I and II to oxidative stress in vitro and within host cells. Notably, the subclones displayed similar superoxide dismutase activity; however, foam height in the catalase test and lysate catalase/peroxidase activity were higher for type I bacilli than for type II bacilli. Additionally, type I bacilli were less susceptible to hydrogen peroxide (H2O2) than type II bacilli. After exposure to H2O2, antioxidative stress response genes katG, ahpC, sodA, and trxA were more strongly induced in type I bacilli than in type II bacilli. Further, we investigated cell survival in macrophages. Fewer type II bacilli were recovered than type I bacilli. However, in the presence of apocynin, a specific inhibitor of NADPH oxidase, type II recovery was greater than that of type I. The production of interleukin 1ß (IL-1ß), IL-12 p40, and tumor necrosis factor alpha (TNF-α) was higher in type I bacillus-infected macrophages than in type II bacillus-infected macrophages. The proportions of type I and type II bacilli in vaccine lots over 3 years (100 lots) were 97.6% ± 1.5% and 2.4% ± 1.5%, respectively. The study results illustrated that type I bacilli are more resistant to oxidative stress than type II bacilli. Overall, these findings provide important information in terms of the quality control and safety of BCG Tokyo 172 vaccine.IMPORTANCE This study revealed the difference of in vivo and in vitro antioxidative stress properties of BCG Tokyo 172 types I and II as one of the bacteriological characteristics. In particular, the bacilli exhibited differences in catalase/peroxidase activity, which could explain their different protective effects against infection. The differences correlated with survival in the host cell and the production of proinflammatory cytokines to protect against infection by Mycobacterium tuberculosis The proportion of bacilli types I and II in all commercial lots of BCG Tokyo 172 over 3 years (100 lots) was constant. The findings also highlighted the importance of analyzing their content for quality control during vaccine production.
Assuntos
Peróxido de Hidrogênio/farmacologia , Macrófagos/microbiologia , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/genética , Estresse Oxidativo/efeitos dos fármacos , Animais , Vacina BCG/imunologia , Citocinas/imunologia , Genoma Bacteriano , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Mycobacterium bovis/classificação , Mycobacterium tuberculosis/imunologia , Células RAW 264.7 , Células THP-1 , Tóquio , Tuberculose/imunologia , Tuberculose/microbiologia , Tuberculose/prevenção & controleRESUMO
Tryptophan biosynthesis represents an important potential drug target for new anti-TB drugs. We identified a series of indole-4-carboxamides with potent antitubercular activity. In vitro, Mycobacterium tuberculosis (Mtb) acquired resistance to these compounds through three discrete mechanisms: (1) a decrease in drug metabolism via loss-of-function mutations in the amidase that hydrolyses these carboxamides, (2) an increased biosynthetic rate of tryptophan precursors via loss of allosteric feedback inhibition of anthranilate synthase (TrpE), and (3) mutation of tryptophan synthase (TrpAB) that decreased incorporation of 4-aminoindole into 4-aminotryptophan. Thus, these indole-4-carboxamides act as prodrugs of a tryptophan antimetabolite, 4-aminoindole.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Indóis/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Triptofano/biossíntese , Animais , Antituberculosos/química , Antituberculosos/metabolismo , Relação Dose-Resposta a Droga , Indóis/química , Indóis/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismoRESUMO
An in situ screening assay for UDP-galactopyranose mutase (UGM, an essential enzyme of M. tuberculosis cell wall biosynthesis) has been developed to discover novel UGM inhibitors. The approach is based on the amide-forming reaction of an amino acid core with various cinnamic acids, followed by a direct fluorescence polarization assay to identify the best UGM binders without isolation and purification of the screened ligands. This assay allows us to perform one-pot high-throughput synthesis and screening of enzyme inhibitors in a 384-well plate format. UGM ligands were successfully identified by this technology and their inhibition levels were established from pure synthetic compounds in vitro and in a whole cell antibacterial assay. This study provides a blueprint for designing enamide structures as new UGM inhibitors and anti-mycobacterial agents.
Assuntos
Aminoácidos/farmacologia , Antituberculosos/farmacologia , Cinamatos/farmacologia , Inibidores Enzimáticos/farmacologia , Transferases Intramoleculares/antagonistas & inibidores , Aminoácidos/síntese química , Aminoácidos/metabolismo , Antituberculosos/síntese química , Antituberculosos/metabolismo , Cinamatos/síntese química , Cinamatos/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Transferases Intramoleculares/química , Transferases Intramoleculares/metabolismo , Cinética , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/enzimologia , Mycobacterium tuberculosis/enzimologia , Ligação ProteicaRESUMO
Acetyl Coenzyme A Carboxylase (AccD6) is a homodimeric protein which is involved in the carboxylation of acetyl coenzyme A to produce malonyl coenzyme A, which plays an important role in the biosynthesis of fatty acid chain. However, studies suggest that AccD6 in combination with AccA3 produces malonyl co-A. Certain herbicides are known to inhibit plant ACC. Among these herbicides, haloxyfop was found to inhibit AccD6 at IC50 of 21.1 ± 1 µM. In this study, we have performed molecular docking of the Maybridge database consisting of ~55,000 compounds in the active site of the protein with haloxyfop as a reference molecule, followed by molecular dynamics study and biological activity determination of prioritized compounds. Out of the nine compounds selected for biological evaluation, three compounds - CD07230, HTS08529 and KM08871 - were found to exhibit anti-mycobacterial activity.
Assuntos
Acetil-CoA Carboxilase/antagonistas & inibidores , Antituberculosos/farmacologia , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mycobacterium bovis/genética , Organismos Geneticamente Modificados , Piridinas/farmacologia , Relação Quantitativa Estrutura-AtividadeRESUMO
Identifying the Mycobacterium tuberculosis resistance mutation patterns is of the utmost importance to assure proper patient's management and devising of control programs aimed to limit spread of disease. Zoonotic Mycobacterium bovis infection still represents a threat to human health, particularly in dairy production regions. Routinary, molecular characterization of M. bovis is performed primarily by spoligotyping and mycobacterial interspersed repetitive units (MIRU) while next generation sequencing (NGS) approaches are often performed by reference laboratories. However, spoligotyping and MIRU methodologies lack the resolution required for the fine characterization of tuberculosis isolates, particularly in outbreak settings. In conjunction with sophisticated bioinformatic algorithms, whole genome sequencing (WGS) analysis is becoming the method of choice for advanced genetic characterization of tuberculosis isolates. WGS provides valuable information on drug resistance and compensatory mutations that other technologies cannot assess. Here, we performed an analysis of the most frequently identified mutations associated with tuberculosis drug resistance and their genetic relationship among 2,074 Mycobacterium bovis WGS recovered primarily from non-human hosts. Full-length gene sequences harboring drug resistant associated mutations and their phylogenetic relationships were analyzed. The results showed that M. bovis isolates harbor mutations conferring resistance to both first- and second-line antibiotics. Mutations conferring resistance for isoniazid, fluoroquinolones, streptomycin, and aminoglycosides were identified among animal strains. Our findings highlight the importance of molecular surveillance to monitor the emergence of mutations associated with multi and extensive drug resistance in livestock and other non-human mammals.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/genética , Tuberculose/veterinária , América/epidemiologia , Animais , Antituberculosos/farmacologia , Mutação , Filogenia , Tuberculose/microbiologia , Sequenciamento Completo do GenomaRESUMO
Present study addresses the challenge of incorporating hydrophilic streptomycin sulphate (STRS; log P -6.4) with high dose (1 g/day) into a lipid matrix of SLNs. Cold high-pressure homogenization technique used for SLN preparation achieved 30% drug loading and 51.17 ± 0.95% entrapment efficiency. Polyethylene glycol 600 as a supporting-surfactant assigned small size (218.1 ± 15.46 nm) and mucus-penetrating property. It was conceived to administer STRS-SLNs orally rather than intramuscularly. STRS-SLNs remained stable on incubation for varying times in SGF or SIF. STRS-SLNs were extensively characterised for microscopic (TEM and AFM), thermal (DSC), diffraction (XRD) and spectroscopic (NMR and FTIR) properties and showed zero-order controlled release. Enhanced (60 times) intracellular uptake was observed in THP-1 and Pgp expressing LoVo and DLD-1 cell lines, using fluorescein-SLNs. Presence of SLNs in LoVo cells was also revealed by TEM studies. STRS-SLNs showed 3 times reduction in MIC against Mycobacterium tuberculosis H37RV (256182) in comparison to free STRS. It also showed better activity against both M. bovis BCG and Mycobacterium tuberculosis H37RV (272994) in comparison to free STRS. Cytotoxicity and acute toxicity studies (OECD 425 guidelines) confirmed in vitro and in vivo safety of STRS-SLNs. Single-dose oral pharmacokinetic studies in rat plasma using validated LCMS/MS technique or the microbioassay showed significant oral absorption and bioavailability (160% - 710% increase than free drug).
Assuntos
Antituberculosos/administração & dosagem , Portadores de Fármacos/química , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Estreptomicina/administração & dosagem , Administração Oral , Animais , Antituberculosos/química , Antituberculosos/farmacocinética , Antituberculosos/toxicidade , Disponibilidade Biológica , Relação Dose-Resposta a Droga , Composição de Medicamentos/métodos , Liberação Controlada de Fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipídeos/química , Macrófagos/metabolismo , Masculino , Testes de Sensibilidade Microbiana , Nanopartículas/química , Tamanho da Partícula , Ratos , Solubilidade , Estreptomicina/química , Estreptomicina/farmacocinética , Estreptomicina/toxicidade , Células THP-1 , Testes de Toxicidade AgudaRESUMO
Based on the structural elements of bioactive indole-based compounds, a series of novel 1-substituted indole-3-carboxaldehyde thiosemicarbazones were synthesized as potential antimycobacterial and anticancer agents. The derivatives were prepared via a two-step methodology including N-alkylation(benzylation) of indole-3-carboxaldehyde and conversion of the intermediate aldehydes to corresponding thiosemicarbazones. The derivatives were evaluated for their antimycobacterial activity and compounds 3d (R = propyl) and 3q (R = 4-nitrobenzyl) were among the most potent and selective derivatives with IC50 values of 0.9 and 1.9 µg/mL respectively. The anticancer activity of the derivatives was also assessed against a panel of tumor cell lines. Compounds 3t, 3u, 3v and 3w efficiently inhibited the majority of the cancer cell lines with considerable selectivity.