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1.
Front Immunol ; 12: 656419, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34745081

RESUMO

Tuberculosis (TB) is the global health problem with the second highest number of deaths from a communicable disease after COVID-19. Although TB is curable, poor health infrastructure, long and grueling TB treatments have led to the spread of TB pandemic with alarmingly increasing multidrug-resistant (MDR)-TB prevalence. Alternative host modulating therapies can be employed to improve TB drug efficacies or dampen the exaggerated inflammatory responses to improve lung function. Here, we investigated the adjunct therapy of natural immune-modulatory compound berberine in C57BL/6 mouse model of pulmonary TB. Berberine treatment did not affect Mtb growth in axenic cultures; however, it showed increased bacterial killing in primary murine bone marrow-derived macrophages and human monocyte-derived macrophages. Ad libitum berberine administration was beneficial to the host in combination with rifampicin and isoniazid. Berberine adjunctive treatment resulted in decreased lung pathology with no additive or synergistic effects on bacterial burdens in mice. Lung immune cell flow cytometry analysis showed that adjunctive berberine treatment decreased neutrophil, CD11b+ dendritic cell and recruited interstitial macrophage numbers. Late onset of adjunctive berberine treatment resulted in a similar phenotype with consistently reduced numbers of neutrophils both in lungs and the spleen. Together, our results suggest that berberine can be supplemented as an immunomodulatory agent depending on the disease stage and inflammatory status of the host.


Assuntos
Antituberculosos/uso terapêutico , Berberina/uso terapêutico , Fatores Imunológicos/uso terapêutico , Isoniazida/uso terapêutico , Rifampina/uso terapêutico , Tuberculose Pulmonar/tratamento farmacológico , Animais , Antituberculosos/farmacologia , Berberina/farmacologia , Citocinas/imunologia , Células Dendríticas/efeitos dos fármacos , Quimioterapia Combinada , Feminino , Humanos , Fatores Imunológicos/farmacologia , Isoniazida/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Rifampina/farmacologia , Baço/efeitos dos fármacos , Baço/imunologia , Baço/microbiologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia
2.
Nat Commun ; 12(1): 5548, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545082

RESUMO

Isoniazid (INH) is a first-line anti-tuberculosis drug used for nearly 70 years. However, the mechanism underlying the side effects of INH has remained elusive. Here, we report that INH and its metabolites induce a post-translational modification (PTM) of histones, lysine isonicotinylation (Kinic), also called 4-picolinylation, in cells and mice. INH promotes the biosynthesis of isonicotinyl-CoA (Inic-CoA), a co-factor of intracellular isonicotinylation. Mass spectrometry reveals 26 Kinic sites in histones in HepG2 cells. Acetyltransferases CREB-binding protein (CBP) and P300 catalyse histone Kinic, while histone deacetylase HDAC3 functions as a deisonicotinylase. Notably, MNase sensitivity assay and RNA-seq analysis show that histone Kinic relaxes chromatin structure and promotes gene transcription. INH-mediated histone Kinic upregulates PIK3R1 gene expression and activates the PI3K/Akt/mTOR signalling pathway in liver cancer cells, linking INH to tumourigenicity in the liver. We demonstrate that Kinic is a histone acylation mark with a pyridine ring, which may have broad biological effects. Therefore, INH-induced isonicotinylation potentially accounts for the side effects in patients taking INH long-term for anti-tuberculosis therapy, and this modification may increase the risk of cancer in humans.


Assuntos
Antituberculosos/farmacologia , Código das Histonas , Isoniazida/farmacologia , Ácidos Isonicotínicos/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Cromatina/metabolismo , Coenzima A/metabolismo , Células HeLa , Células Hep G2 , Histona Desacetilases/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Ácidos Isonicotínicos/química , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética , Regulação para Cima/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/metabolismo
3.
Biomed Res Int ; 2021: 9916328, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34541001

RESUMO

Ferroptosis and inflammation induced by cerebral hemorrhage result in an excessive inflammatory response and irreversible neuronal injury. Alleviating ferroptosis might be an effective way to prevent neuroinflammatory injury and promote neural functional recovery. Pyridoxal isonicotinoyl hydrazine (PIH), a lipophilic iron-chelating agent, has been reported to reduce excess iron-induced cytotoxicity. However, whether PIH could ameliorate the effects of hemorrhagic stroke is not completely understood. In the present study, the preventive effects of PIH in an intracerebral hemorrhage (ICH) mouse model were investigated. Neurological score, rotarod test, and immunofluorescence around the hematoma were assessed to evaluate the effects of PIH on hemorrhagic injury. The involvement of ferroptosis and inflammation was also examined in vitro to explore the underlying mechanism. Results showed that administration of PIH prevented neuronal cell death and reduced lipid peroxidation in Erastin-treated PC-12 cells. In vivo, mice treated with PIH after ICH attenuated neurological deficit scores. Additionally, we found PIH reduced ROS production, iron accumulation, and lipid peroxidation around the hematoma peripheral tissue. Meanwhile, ICH mice treated with PIH showed an upregulation of the key ferroptosis enzyme, glutathione peroxidase 4, and downregulation of cyclooxygenase-2. Moreover, PIH administration inhibited proinflammatory polarization and reduced interleukin-1 beta and tumor necrosis factor alpha in ICH mice. Collectively, these results demonstrated that PIH protects mice against hemorrhage stroke, which was associated with mitigation of inflammation and ferroptosis.


Assuntos
Hemorragia Cerebral/tratamento farmacológico , Ferroptose/efeitos dos fármacos , Isoniazida/análogos & derivados , Piridoxal/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Hemorragia Cerebral/metabolismo , Compostos Férricos/farmacologia , Ferroptose/fisiologia , Inflamação/tratamento farmacológico , Inflamação/prevenção & controle , Ferro/metabolismo , Quelantes de Ferro/farmacologia , Isoniazida/metabolismo , Isoniazida/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Piridoxal/metabolismo , Piridoxal/farmacologia
4.
BMC Infect Dis ; 21(1): 891, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34465301

RESUMO

BACKGROUND: Determining factors affecting the transmission of rifampicin (RR) and multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains under standardized tuberculosis (TB) treatment is key to control TB and prevent the evolution of drug resistance. METHODS: We combined bacterial whole genome sequencing (WGS) and epidemiological investigations for 37% (n = 195) of all RR/MDR-TB patients in Cameroon (2012-2015) to identify factors associated with recent transmission. RESULTS: Patients infected with a strain resistant to high-dose isoniazid, and ethambutol had 7.4 (95% CI 2.6-21.4), and 2.4 (95% CI 1.2-4.8) times increased odds of being in a WGS-cluster, a surrogate for recent transmission. Furthermore, age between 30 and 50 was positively correlated with recent transmission (adjusted OR 3.8, 95% CI 1.3-11.4). We found high drug-resistance proportions against three drugs used in the short standardized MDR-TB regimen in Cameroon, i.e. high-dose isoniazid (77.4%), ethambutol (56.9%), and pyrazinamide (43.1%). Virtually all strains were susceptible to fluoroquinolones, kanamycin, and clofazimine, and treatment outcomes were mostly favourable (87.5%). CONCLUSION: Pre-existing resistance to high-dose isoniazid, and ethambutol is associated with recent transmission of RR/MDR strains in our study. A possible contributing factor for this observation is the absence of universal drug susceptibility testing in Cameroon, likely resulting in prolonged exposure of new RR/MDR-TB patients to sub-optimal or failing first-line drug regimens.


Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Adulto , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Camarões/epidemiologia , Estudos Epidemiológicos , Genômica , Humanos , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Rifampina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia
5.
Chem Commun (Camb) ; 57(69): 8644-8647, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34369955

RESUMO

Effective monitoring of liver hypoxia status is crucial for the detection and treatment of drug-induced liver injury. Here, a novel photoacoustic and fluorescent dual-modal probe (NO2-CS) was rationally developed and applied to image isoniazid-induced liver hypoxia through detecting the over-expressed nitroreductase.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Corantes Fluorescentes/química , Hipóxia/metabolismo , Fígado/metabolismo , Xantenos/química , Animais , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Feminino , Hipóxia/enzimologia , Isoniazida/farmacologia , Fígado/enzimologia , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Nitrorredutases/metabolismo , Técnicas Fotoacústicas
6.
J Glob Antimicrob Resist ; 26: 207-218, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34214698

RESUMO

OBJECTIVES: Globally, the incidence and mortality of tuberculosis (TB) are declining; however, low detection of drug-resistant disease threatens to reverse current progress toward global TB control. Multiple rapid molecular diagnostic tests have recently been developed to detect genetic mutations in Mycobacterium tuberculosis (Mtb) known to confer drug resistance. However, their utility depends on the frequency and distribution of resistance-associated mutations in the pathogen population. This review aimed to assess the prevalence of gene mutations associated with rifampicin (RIF)- and isoniazid (INH)-resistant Mtb in Ethiopia. METHODS: We searched the literature in PubMed/MEDLINE, Web of Science, Scopus and Cochrane Library. Data analysis was conducted in Stata 11. RESULTS: Totally, 909 (95.8%) of 949 INH-resistant Mtb isolates had detectable gene mutations: 95.8% in katG315 and 5.9% in the inhA promoter region. Meta-analysis resulted in an estimated pooled prevalence of katGMUT1(S315T1) of 89.2% (95% CI 81.94-96.43%) and a pooled prevalence of inhAMUT1(C15T) of 77.5% (95% CI 57.84-97.13%). Moreover, 769 (90.8%) of 847 RIF-resistant strains had detectable rpoB gene mutations. Meta-analysis resulted in a pooled prevalence of rpoBMUT3(S531L) of 74.2% (95% CI 66.39-82.00%). CONCLUSION: RIF-resistant Mtb were widespread, particularly those harbouring rpoB(S531L) mutation. Similarly, INH-resistant Mtb with katG(S315T1) and inhA(C15T) mutations were common. Tracking S531L, S315T1 and C15T mutations among RIF- and INH-resistant isolates, respectively, would be diagnostically and epidemiologically valuable. Rapid diagnosis of RIF- and INH-resistant Mtb would expedite modification of TB treatment regimens, and proper timely infection control interventions could reduce the risk of development and transmission of multidrug-resistant TB.


Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Resistência a Medicamentos , Etiópia/epidemiologia , Humanos , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/genética , Prevalência , Rifampina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia
7.
J Pharm Biomed Anal ; 203: 114223, 2021 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-34214766

RESUMO

Isoniazid (INH) is the first-line anti-tubercular drug that is used both for the prophylaxis as well as the treatment of tuberculosis (TB). The patients with TB are more vulnerable to secondary infections and other health complications, hence, they are usually administered a cocktail of drugs. This increases the likelihood of drug-drug interactions (DDIs). INH is clinically proven to interact with drugs like phenytoin, carbamazepine, diazepam, triazolam, acetaminophen, etc. Most of such clinical observations have been supported by in vitro inhibition studies involving INH and cytochrome P450 (CYP) enzymes. A few published in vitro studies have explored the CYP2E1 inhibition potential of INH to explain its interactions with acetaminophen and other CY2E1 substrates, such as chlorzoxazone, but none of them were able to demonstrate any significant inhibition of the enzyme by the drug. It was reported that metabolites of INH, such as acetylhydrazine and hydrazine, were bioactivated by CYP2E1, highlighting that perhaps the drug metabolites were responsible for the mechanism based inhibition (MBI) of the enzyme. Therefore, the purpose of this investigation was to explore CYP2E1 enzyme inhibition potential of INH and its four major metabolites, viz., acetylisoniazid, isonicotinic acid, acetylhydrazine and hydrazine, using human liver microsomes (HLM). Additionally, we determined the fraction unbound in microsomal incubation (fumic) for all the five compounds using equilibrium dialysis assay. We observed that INH and its metabolites had lower propensity for microsomal binding, and the metabolites also lacked the potential to inhibit CYP2E1 enzyme, either by direct inhibition or through MBI. This suggests involvement of some other mechanism to explain interactions of INH with CY2E1 substrates, signifying need of further exploration.


Assuntos
Citocromo P-450 CYP2E1 , Isoniazida , Antituberculosos/farmacologia , Cromatografia Líquida , Humanos , Isoniazida/farmacologia , Microssomos Hepáticos , Espectrometria de Massas em Tandem
8.
Trop Med Int Health ; 26(9): 1057-1067, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34107112

RESUMO

OBJECTIVES: Differences among Mycobacterium tuberculosis complex (MTC) species may predict drug resistance or treatment success. Thus, we optimised and deployed the genotype MTBC assay (gMTBC) to identify MTC to the species level, and then performed comparative genotypic drug-susceptibility testing to anti-tuberculosis drugs from direct sputum of patients with presumed multidrug-resistant tuberculosis (MDR-TB) by the MTBDRplus/sl reference method. METHODS: Patients with positive Xpert® MTB/RIF (Xpert) results were consented to provide early-morning-sputum for testing by the gMTBC and the reference MTBDRplus/sl. Chi-square or Fisher's exact test compared proportions. Modified Poisson regression modelled detection of MTC by gMTBC. RESULTS: Among 73 patients, 53 (73%) were male and had a mean age of 43 (95% CI; 40-45) years. In total, 34 (47%), 36 (49%) and 38 (55%) had positive gMTBC, culture and MTBDR respectively. Forty patients (55%) had low quantity MTC by Xpert, including 31 (78%) with a negative culture. gMTBC was more likely to be positive in patients with chest cavity 4.18 (1.31-13.32, P = 0.016), high-quantity MTC by Xpert 3.03 (1.35-6.82, P = 0.007) and sputum smear positivity 1.93 (1.19-3.14, P = 0.008). The accuracy of gMTBC in detecting MTC was 95% (95% CI; 86-98; κ = 0.89) compared to MTBDRplus/sl. All M. tuberculosis/canettii identified by gMTB were susceptible to fluoroquinolone and aminoglycosides/capreomycin. CONCLUSIONS: The concordance between the gMTBC assay and MTBDRplus/sl in detecting MTC was high but lagged behind the yield of Xpert MTB/RIF. All M. tuberculosis/canettii were susceptible to fluoroquinolones, a core drug in MDR-TB treatment regimens.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Genótipo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Escarro/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/genética , Adulto , Antituberculosos/farmacologia , Estudos Transversais , Feminino , Fluoroquinolonas/farmacologia , Humanos , Isoniazida/farmacologia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/farmacologia , Especificidade da Espécie , Tanzânia/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia
9.
mBio ; 12(3): e0108821, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34060333

RESUMO

ATP/ADP depicts the bioenergetic state of Mycobacterium tuberculosis (Mtb). However, the metabolic state of Mtb during infection remains poorly defined due to the absence of appropriate tools. Perceval HR (PHR) was recently developed to measure intracellular ATP/ADP levels, but it cannot be employed in mycobacterial cells due to mycobacterial autofluorescence. Here, we reengineered the ATP/ADP sensor Perceval HR into PHR-mCherry to analyze ATP/ADP in fast- and slow-growing mycobacteria. ATP/ADP reporter strains were generated through the expression of PHR-mCherry. Using the Mtb reporter strain, we analyzed the changes in ATP/ADP levels in response to antimycobacterial agents. As expected, bedaquiline induced a decrease in ATP/ADP. Interestingly, the transcriptional inhibitor rifampicin led to the depletion of ATP/ADP levels, while the cell wall synthesis inhibitor isoniazid did not affect the ATP/ADP levels in Mtb. The usage of this probe revealed that Mtb faces depletion of ATP/ADP levels upon phagocytosis. Furthermore, we observed that the activation of macrophages with interferon gamma and lipopolysaccharides leads to metabolic stress in intracellular Mtb. Examination of the bioenergetics of mycobacteria residing in subvacuolar compartments of macrophages revealed that the bacilli residing in phagolysosomes and autophagosomes have significantly less ATP/ADP than the bacilli residing in phagosomes. These observations indicate that phagosomes represent a niche for metabolically active Mtb, while autophagosomes and phagolysosomes harbor metabolically quiescent bacilli. Interestingly, even in activated macrophages, Mtb residing in phagosomes remains metabolically active. We further observed that macrophage activation affects the metabolic state of intracellular Mtb through the trafficking of Mtb from phagosomes to autophagosomes and phagolysosomes. IMPORTANCE ATP/ADP levels guide bacterial cells, whether to replicate or to enter nonreplicating persistence. However, tools for measuring ATP/ADP levels with spatiotemporal resolution are lacking. Here, we describe a method for tracking ATP/ADP levels at the single-cell and population levels. Using this tool, we have demonstrated that the transcription inhibitor rifampicin induces metabolic stress. In contrast, the cell wall synthesis inhibitor isoniazid does not alter the metabolic state of the bacilli, suggesting that transcription is tightly intertwined with metabolism, while cell wall synthesis is not. Furthermore, we analyzed the metabolic state of mycobacteria residing in different compartments of macrophages. We observed that Mtb cells residing inside phagosomes have healthy ATP/ADP levels. In contrast, the bacteria residing inside phagolysosomes and autophagosomes face depletion of ATP. Interestingly, the activation of macrophages facilitates the trafficking of mycobacterial cells from metabolism-conducive phagosomes to metabolism-averse phagolysosomes and autophagosomes. We believe that this tool holds the key to the identification of inhibitors of mycobacterial metabolism.


Assuntos
Metabolismo Energético , Macrófagos/microbiologia , Mycobacterium tuberculosis/metabolismo , Fagossomos/microbiologia , Difosfato de Adenosina/análise , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/metabolismo , Antibióticos Antituberculose/farmacologia , Autofagossomos/microbiologia , Humanos , Isoniazida/farmacologia , Mycobacterium tuberculosis/genética , Rifampina/farmacologia , Estresse Fisiológico/efeitos dos fármacos
10.
FEMS Microbiol Lett ; 368(9)2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-33974050

RESUMO

Rhodococcus spp. have broad potential applications related to the degradation of organic contaminants and the transformation or synthesis of useful compounds. However, some Gram-positive bacteria are difficult to manipulate genetically due to low transformation efficiency. In this study, we investigated the effects of chemicals including glycine, isonicotinic acid hydrazide (INH), Tween 80 and penicillin G, as well as cell growth status, competent cell concentration, electroporation field strength, electroporation time and heat shock time, on the electrotransformation efficiency of the tetrahydrofuran-degrading bacterium Rhodococcus ruber YYL with low transformation efficiency. The highest electrotransformation efficiency was 1.60 × 106 CFU/µg DNA after parameter optimization. GmhD (D-glycero-D-manno-heptose 1-phosphate guanosyltransferase) gene, which is important in the biosynthesis of lipopolysaccharide, was deleted via the optimized electrotransformation method. Compared with wild-type strain, YYL ΔgmhD showed extremely high electrotransformation efficiency because the surface of it had no mushroom-like extracellular polymeric substances (EPS). In addition, the results showed that cell wall-weakening reagents might cause some translucent substances like EPS, to detach from the cells, increasing the electrotransformation efficiency of strain YYL. We propose that these results could provide a new strategy for unique bacteria that are rich in EPS, for which genetic manipulation systems are difficult to establish.


Assuntos
Eletroporação/métodos , Rhodococcus/genética , Rhodococcus/metabolismo , Parede Celular , DNA Bacteriano/genética , Matriz Extracelular de Substâncias Poliméricas , Glicina/farmacologia , Isoniazida/farmacologia , Penicilina G/farmacologia , Polissorbatos/farmacologia , Rhodococcus/efeitos dos fármacos , Rhodococcus/crescimento & desenvolvimento , Transformação Bacteriana
11.
Antimicrob Agents Chemother ; 65(8): e0001121, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-33972252

RESUMO

Tuberculosis remains a leading cause of mortality among infectious diseases worldwide, prompting the need to discover new drugs to fight this disease. We report here the design, synthesis, and antimycobacterial activity of isatin-mono/bis-isoniazid hybrids. Most of the compounds exhibited very high activity against Mycobacterium tuberculosis, with MICs in the range of 0.195 to 0.39 µg/ml, and exerted a more potent bactericidal effect than the standard antitubercular drug isoniazid (INH). Importantly, these compounds were found to be well tolerated at high doses (>200 µg/ml) on Vero kidney cells, leading to high selectivity indices. Two of the most promising hybrids were evaluated for activity in THP-1 macrophages infected with M. tuberculosis, among which compound 11e was found to be slightly more effective than INH. Overexpression of InhA along with cross-resistance determination of the most potent compounds, selection of resistant mutants, and biochemical analysis, allowed us to decipher their mode of action. These compounds effectively inhibited mycolic acid biosynthesis and required KatG to exert their biological effects. Collectively, this suggests that the synthesized isatin-INH hybrids are promising antitubercular molecules for further evaluation in preclinical settings.


Assuntos
Isatina , Mycobacterium tuberculosis , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Proteínas de Bactérias/genética , Catalase/genética , Humanos , Isatina/farmacologia , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana , Mutação
12.
Sci Rep ; 11(1): 10953, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34040029

RESUMO

DNA is basically an intracellular molecule that stores genetic information and carries instructions for growth and reproduction in all cellular organisms. However, in some bacteria, DNA has additional roles outside the cells as extracellular DNA (eDNA), which is an essential component of biofilm formation and hence antibiotic tolerance. Mycobacteria include life-threating human pathogens, most of which are slow growers. However, little is known about the nature of pathogenic mycobacteria's eDNA. Here we found that eDNA is present in slow-growing mycobacterial pathogens, such as Mycobacterium tuberculosis, M. intracellulare, and M. avium at exponential growth phase. In contrast, eDNA is little in all tested rapid-growing mycobacteria. The physiological impact of disrupted eDNA on slow-growing mycobacteria include reduced pellicle formation, floating biofilm, and enhanced susceptibility to isoniazid and amikacin. Isolation and sequencing of eDNA revealed that it is identical to the genomic DNA in M. tuberculosis and M. intracellulare. In contrast, accumulation of phage DNA in eDNA of M. avium, suggests that the DNA released differs among mycobacterial species. Our data show important functions of eDNA necessary for biofilm formation and drug tolerance in slow-growing mycobacteria.


Assuntos
Biofilmes/crescimento & desenvolvimento , DNA Bacteriano/genética , Líquido Extracelular/química , Mycobacterium tuberculosis/genética , Micobactérias não Tuberculosas/genética , Amicacina/farmacologia , Antibacterianos/farmacologia , Antituberculosos/farmacologia , DNA Bacteriano/isolamento & purificação , DNA Viral/genética , DNA Viral/isolamento & purificação , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Micobactérias não Tuberculosas/efeitos dos fármacos , Micobactérias não Tuberculosas/crescimento & desenvolvimento , Prófagos/genética , Especificidade da Espécie , Sequenciamento Completo do Genoma
13.
Int J Med Sci ; 18(9): 1935-1945, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33850462

RESUMO

The prevalence of drug-resistant Mycobacterium tuberculosis (Mtb) strains makes disease control more complicated, which is the main cause of death in tuberculosis (TB) patients. Early detection and timely standard treatment are the key to current prevention and control of drug-resistant TB. In recent years, despite the continuous advancement in drug-resistant TB diagnostic technology, the needs for clinical rapid and accurate diagnosis are still not fully met. With the development of sequencing technology, the research of human microecology has been intensified. This study aims to use 16 rRNA sequencing technology to detect and analyze upper respiratory flora of TB patients with anti-TB drug sensitivity (DS, n = 55), monoresistance isoniazide (MR-INH, n = 33), monoresistance rifampin (MR-RFP, n = 12), multidrug resistance (MDR, n = 26) and polyresistance (PR, n = 39) in southern China. Potential microbial diagnostic markers for different types of TB drug resistance are searched by screening differential flora, which provides certain guiding significance for drug resistance diagnosis and clinical drug use of TB. The results showed that the pulmonary microenvironment of TB patients was more susceptible to infection by external pathogens, and the infection of different drug-resistant Mtb leads to changes in different flora. Importantly, seven novel microorganisms (Leptotrichia, Granulicatella, Campylobacter, Delfitia, Kingella, Chlamydophila, Bordetella) were identified by 16S rRNA sequencing as diagnostic markers for different drug resistance types of TB. Leptotrichia, Granulicatella, Campylobacter were potential diagnostic marker for TB patients with INH single-resistance. Delftia was a potential diagnostic marker for TB patients with RFP single drug-resistance. Kingella and Chlamydophila can be used as diagnostic markers for TB patients with PR. Bordetella can be used as a potential diagnostic marker for identification of TB patients with MDR.


Assuntos
Antituberculosos/farmacologia , Microbiota , Escarro/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Pulmonar/diagnóstico , Adolescente , Adulto , Idoso , Antituberculosos/uso terapêutico , DNA Bacteriano/isolamento & purificação , Feminino , Humanos , Isoniazida/farmacologia , Isoniazida/uso terapêutico , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , RNA Ribossômico 16S/genética , Rifampina/farmacologia , Rifampina/uso terapêutico , Sensibilidade e Especificidade , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Pulmonar/tratamento farmacológico , Adulto Jovem
14.
Molecules ; 26(7)2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33916423

RESUMO

A total of fourteen pyrazoline derivatives were synthesized through cyclo-condensation reactions by chalcone derivatives with different types of semicarbazide. These compounds were characterized by IR, 1D-NMR (1H, 13C and Distortionless Enhancement by Polarization Transfer - DEPT-135) and 2D-NMR (COSY, HSQC and HMBC) as well as mass spectroscopy analysis (HRMS). The synthesized compounds were tested for their antituberculosis activity against Mycobacterium tuberculosis H37Ra in vitro. Based on this activity, compound 4a showed the most potent inhibitory activity, with a minimum inhibitory concentration (MIC) value of 17 µM. In addition, six other synthesized compounds, 5a and 5c-5g, exhibited moderate activity, with MIC ranges between 60 µM to 140 µM. Compound 4a showed good bactericidal activity with a minimum bactericidal concentration (MBC) value of 34 µM against Mycobacterium tuberculosis H37Ra. Molecular docking studies for compound 4a on alpha-sterol demethylase was done to understand and explore ligand-receptor interactions, and to hypothesize potential refinements for the compound.


Assuntos
Inibidores de 14-alfa Desmetilase/síntese química , Antituberculosos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Pirazóis/síntese química , Semicarbazidas/síntese química , Esterol 14-Desmetilase/química , Inibidores de 14-alfa Desmetilase/farmacologia , Antituberculosos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Fluconazol/química , Fluconazol/farmacologia , Isoniazida/química , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Pirazóis/farmacologia , Semicarbazidas/farmacologia , Esterol 14-Desmetilase/metabolismo , Homologia Estrutural de Proteína , Termodinâmica
15.
Biochem Pharmacol ; 188: 114545, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33831395

RESUMO

Human NAT2 is a polymorphic pharmacogene encoding for N-acetyltransferase 2, a hepatic enzyme active towards arylamine and arylhydrazine drugs, including the anti-tubercular antibiotic isoniazid. The isoenzyme also modulates susceptibility to chemical carcinogenesis, particularly of the bladder. Human NAT2 represents an ideal model for anthropological investigations into the demographic adaptation of worldwide populations to their xenobiotic environment. Its sequence appears to be subject to positive selection pressures that are population-specific and may be attributed to gene-environment interactions directly associated with exogenous chemical challenges. However, recent evidence suggests that the same evolutionary pattern may not be observed in other primates. Here, we report NAT2 polymorphism in 25 rhesus macaques (Macaca mulatta) and compare the frequencies and functional characteristics of 12 variants. Seven non-synonymous single nucleotide variations (SNVs) were identified, including one nonsense mutation. The missense SNVs were demonstrated to affect enzymatic function in a substrate-dependent manner, albeit more moderately than certain NAT1 SNVs recently characterised in the same cohort. Haplotypic and functional variability of NAT2 was comparable to that previously observed for NAT1 in the same population sample, suggesting that the two paralogues may have evolved under similar selective pressures in the rhesus macaque. This is different to the population variability distribution pattern reported for humans and chimpanzees. Recorded SNVs were also different from those found in other primates. The study contributes to further understanding of NAT2 functional polymorphism in the rhesus macaque, a non-human primate model used in biomedicine and pharmacology, indicating variability in xenobiotic acetylation that could affect drug metabolism.


Assuntos
Arilamina N-Acetiltransferase/genética , Arilamina N-Acetiltransferase/metabolismo , Variação Genética/fisiologia , Polimorfismo Genético/fisiologia , Sequência de Aminoácidos , Animais , Antituberculosos/farmacologia , Arilamina N-Acetiltransferase/química , Variação Genética/efeitos dos fármacos , Humanos , Isoniazida/farmacologia , Macaca mulatta , Polimorfismo Genético/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
16.
J Pharm Pharmacol ; 73(4): 473-486, 2021 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-33793834

RESUMO

OBJECTIVES: Anti-TB drugs-isoniazid and rifampicin induced hepatotoxicity present a significant clinical problem. We aimed to evaluate the beneficial effect of gallic acid in anti-TB drug-induced liver injury in vivo and for the mechanism of action, we explored the influence of gallic acid on Nrf2 and NF-κB pathways. METHODS: We assessed serum liver function tests and histopathological analysis for the preventive effect of gallic acid on liver injury. For exploring the beneficial mechanism, we studied Nrf2 and NF-κB signalling pathways using molecular assays. Subsequently, we conducted in vitro cytotoxicity assays with Nrf2(ML385) and NF-κB(BAY 11-7085) antagonists. KEY FINDINGS: Gallic acid co-administration attenuated the elevation of liver function enzymes, hepatic necrosis and inflammation compared to the anti-TB drug treatment alone. Mechanistic investigations reveal that gallic acid increased Nrf2 activation and induction of its downstream targets, preventing cytotoxicity by isoniazid and rifampicin. The protective effect of gallic acid diminished in the presence of Nrf2 antagonists in vitro. Furthermore, we found that gallic acid treatment inhibited NF-κB/TLR-4 axis upregulated by the anti-TB drugs. CONCLUSIONS: Gallic acid is effective in preventing isoniazid and rifampicin induced hepatotoxicity in vivo by improving the redox homeostasis by activating Nrf2 and inhibiting NF-κB signalling pathways.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Isoniazida , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Rifampina , Receptor 4 Toll-Like/metabolismo , Animais , Antibióticos Antituberculose/farmacologia , Antibióticos Antituberculose/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Ácido Gálico/farmacologia , Isoniazida/farmacologia , Isoniazida/toxicidade , Testes de Função Hepática/métodos , Oxirredução/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Ratos , Ratos Wistar , Rifampina/farmacologia , Rifampina/toxicidade , Transdução de Sinais/efeitos dos fármacos
17.
Sci Rep ; 11(1): 8331, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859215

RESUMO

Studies related to loading ability and delivery of clinically used first-line anti-tuberculosis drugs (ATDs) such as isoniazid, rifampicin, pyrazinamide and streptomycin on the surface of starch-derived bulk and nanopolyurethanes (SBPUs and SNPUs) as drug delivery systems (DDS) have been focused to minimise or remove the drug-associated adverse effects. The efficiencies of nanopolyurethanes obtained from the differently substituted cyclic aliphatic and aromatic isocyanates have been studied for drug loading and release purposes. Different advanced instrumental techniques analysed the structural and morphological properties, thermal stability and crystallinity of the starch nanopolyurethans. Average particle sizes ranging from 27.35-42.38 nm to 126.89-218.60 nm for starch nanopolyurethans, SNPU3i and SNPU4i, respectively, were determined by high-resolution transmission electron microscopy. Similarly, the loading efficiency of ATDs to the surfaces of SNPUs and SBPUs was observed in the range of 60-97% while ATDs-loaded SNPUs showed a sustainable release profile for all ATDs except for streptomycin. However, most SBPUs provided burst-release for all the above-mentioned ATDs in pH-dependent studies. The anti-tuberculosis assay against the Mycobacterium tuberculosis H37Rv strain revealed that streptomycin-loaded SNPU4i and isoniazid-loaded SNPU7i are approximately 42 and 7 times more active than the native streptomycin and isoniazid, respectively.


Assuntos
Antituberculosos/administração & dosagem , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Poliuretanos/química , Amido/química , Antituberculosos/efeitos adversos , Antituberculosos/farmacologia , Liberação Controlada de Fármacos , Farmacorresistência Bacteriana , Estabilidade de Medicamentos , Concentração de Íons de Hidrogênio , Isoniazida/administração & dosagem , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Tamanho da Partícula , Estreptomicina/administração & dosagem , Estreptomicina/farmacologia
18.
PLoS One ; 16(3): e0248707, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33735224

RESUMO

OBJECTIVES: Tuberculosis (TB) is the leading infectious cause of death in the world. Multi-drug resistant TB (MDR-TB) is a major public health problem as treatment is long, costly, and associated to poor outcomes. Here, we report epidemiological data on the prevalence of drug-resistant TB in Haiti. METHODS: This cross-sectional prevalence study was conducted in five health centers across Haiti. Adult, microbiologically confirmed pulmonary TB patients were included. Molecular genotyping (rpoB gene sequencing and spoligotyping) and phenotypic drug susceptibility testing were used to characterize rifampin-resistant MTB isolates detected by Xpert MTB/RIF. RESULTS: Between April 2016 and February 2018, 2,777 patients were diagnosed with pulmonary TB by Xpert MTB/RIF screening and positive MTB cultures. A total of 74 (2.7%) patients were infected by a drug-resistant (DR-TB) M. tuberculosis strain. Overall HIV prevalence was 14.1%. Patients with HIV infection were at a significantly higher risk for infection with DR-TB strains compared to pan-susceptible strains (28.4% vs. 13.7%, adjusted odds ratio 2.6, 95% confidence interval 1.5-4.4, P = 0.001). Among the detected DR-TB strains, T1 (29.3%), LAM9 (13.3%), and H3 (10.7%) were the most frequent clades. In comparison with previous spoligotypes studies with data collected in 2000-2002 and in 2008-2009 on both sensitive and resistant strains of TB in Haiti, we observed a significant increase in the prevalence of the drug-resistant MTB Spoligo-International-Types (SIT) 137 (X2 clade: 8.1% vs. 0.3% in 2000-02 and 0.9% in 2008-09, p<0.001), 5 (T1 clade: 6.8% vs 1.9 in 2000-02 and 1.7% in 2008-09, P = 0.034) and 455 (T1 clade: 5.4% vs 1.6% and 1.1%, P = 0.029). Newly detected spoligotypes (SIT 6, 7, 373, 909 and 1624) were also recorded. CONCLUSION: This study describes the genotypic and phenotypic characteristics of DR-TB strains circulating in Haiti from April 2016 to February 2018. Newly detected MTB clades harboring multi-drug resistance patterns among the Haitian population as well as the higher risk of MDR-TB infection in HIV-positive people highlights the epidemiological relevance of these surveillance data. The importance of detecting RIF-resistant patients, as proxy for MDR-TB in peripheral sites via molecular techniques, is particularly important to provide adequate patient case management, prevent the transmission of resistant strains in the community and to contribute to the surveillance of resistant strains.


Assuntos
Antituberculosos/farmacologia , Coinfecção/epidemiologia , Infecções por HIV/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Adulto , Antituberculosos/uso terapêutico , Coinfecção/diagnóstico , Coinfecção/tratamento farmacológico , Coinfecção/microbiologia , Estudos Transversais , Farmacorresistência Bacteriana Múltipla/genética , Feminino , Haiti/epidemiologia , Humanos , Isoniazida/farmacologia , Isoniazida/uso terapêutico , Masculino , Programas de Rastreamento/estatística & dados numéricos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Prevalência , Estudos Retrospectivos , Rifampina/farmacologia , Rifampina/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Adulto Jovem
19.
Bull Exp Biol Med ; 170(4): 453-457, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33725244

RESUMO

Administration of the liposome-encapsulated dextrazide (LEDZ) to mice 6 months after infection with mycobacterium BCG vaccine for 3 months modulated metabolism of collagens and the intensity of fibrosis of internal organs. In the liver, we observed a redistribution of glycosaminoglycans towards sulfated forms, a decrease in the content of hyaluronan, a change in the ratio of hydroxyproline fractions indicating a decrease in fibrosis via two mechanisms: suppression of synthesis and increased degradation of collagens. In the lungs, administration of LEDZ did not affect the content of sulfated glycosaminoglycans, but significantly reduced the content of hyaluronan, stimulated degradation of collagen, and reduced its synthesis, but these processes were insufficient for significant reduction of fibrotic complications in the lungs. In animals treated with LEDZ, the decrease in collagen synthesis in the liver was 2-fold more pronounced than in the lungs.


Assuntos
Dextranos/farmacologia , Isoniazida/farmacologia , Lipossomos/química , Fígado/metabolismo , Pulmão/metabolismo , Animais , Vacina BCG/imunologia , Dextranos/química , Combinação de Medicamentos , Matriz Extracelular/química , Fibrose/tratamento farmacológico , Fibrose/metabolismo , Glicosaminoglicanos/química , Isoniazida/química , Fígado/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C
20.
Nat Commun ; 12(1): 1606, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33707445

RESUMO

Tuberculosis is a chronic disease that displays several features commonly associated with biofilm-associated infections: immune system evasion, antibiotic treatment failures, and recurrence of infection. However, although Mycobacterium tuberculosis (Mtb) can form cellulose-containing biofilms in vitro, it remains unclear whether biofilms are formed during infection in vivo. Here, we demonstrate the formation of Mtb biofilms in animal models of infection and in patients, and that biofilm formation can contribute to drug tolerance. First, we show that cellulose is also a structural component of the extracellular matrix of in vitro biofilms of fast and slow-growing nontuberculous mycobacteria. Then, we use cellulose as a biomarker to detect Mtb biofilms in the lungs of experimentally infected mice and non-human primates, as well as in lung tissue sections obtained from patients with tuberculosis. Mtb strains defective in biofilm formation are attenuated for survival in mice, suggesting that biofilms protect bacilli from the host immune system. Furthermore, the administration of nebulized cellulase enhances the antimycobacterial activity of isoniazid and rifampicin in infected mice, supporting a role for biofilms in phenotypic drug tolerance. Our findings thus indicate that Mtb biofilms are relevant to human tuberculosis.


Assuntos
Biofilmes/crescimento & desenvolvimento , Celulose/metabolismo , Farmacorresistência Bacteriana Múltipla/fisiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Animais , Celulase/farmacologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Isoniazida/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium abscessus/crescimento & desenvolvimento , Mycobacterium avium/crescimento & desenvolvimento , Mycobacterium fortuitum/crescimento & desenvolvimento , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/farmacologia , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/patologia
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