RESUMO
BACKGROUND: Galatella is a genus in the family Asteraceae, represented by 35-45 species. Considering the high effectiveness of the ethyl acetate (EtOAc) fraction of G. grimmii against Mycobacterium tuberculosis (MIC = 0.5 µg/mL), a bioassay-directed fractionation of this extract was carried out. METHODS: The methanolic extract of the aerial parts of G. grimmii was obtained using maceration, then it was suspended in water and partitioned with petroleum ether, dichloromethane (CH2Cl2), EtOAc, and n-butanol (n-BuOH), successively. The most potent fraction (EtOAc), was selected for further isolation by Sephadex LH-20 and semi-preparative HPLC to obtain active compounds. RESULTS: Fractionation of the EtOAc solvent fraction resulted in the characterization of five compounds, among them, compounds 1 and 2 showed the highest anti-mycobacterial effects with MICs of 0.062 and 1.00 µg/mL against H37Rv M. tuberculosis, respectively, which were higher than those of rifampin (MIC of 1.25 µg/mL) and isoniazid (MIC of 0.31 µg/mL), as positive controls. Also, compound 1 ââinhibited all tested strains of drug-resistant Mycobacterium (MDR and XDR). Notably, the isolated compounds have been reported for the first time from G. grimmii. CONCLUSION: Due to the potent anti-mycobacterial effect of isolated compounds from G. grimmii, this study could pave the way for developing a novel class of natural anti-tuberculosis compounds.
Assuntos
Antituberculosos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Extratos Vegetais , Mycobacterium tuberculosis/efeitos dos fármacos , Antituberculosos/farmacologia , Antituberculosos/química , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Asteraceae/química , Bioensaio , Componentes Aéreos da Planta/químicaRESUMO
Tuberculosis, caused by Mycobacterium tuberculosis, remains an enduring global health challenge due to the limited efficacy of existing treatments. Although much research has focused on immune failure, the role of host macrophage biology in controlling the disease remains underappreciated. Here we show, through multi-modal single-cell RNA sequencing in a murine model, that different alveolar macrophage subsets play distinct roles in either advancing or controlling the disease. Initially, alveolar macrophages that are negative for the CD38 marker are the main infected population. As the infection progresses, CD38+ monocyte-derived and tissue-resident alveolar macrophages emerge as significant controllers of bacterial growth. These macrophages display a unique chromatin organization pre-infection, indicative of epigenetic priming for pro-inflammatory responses. Moreover, intranasal BCG immunization increases the numbers of CD38+ macrophages, enhancing their capability to restrict Mycobacterium tuberculosis growth. Our findings highlight the dynamic roles of alveolar macrophages in tuberculosis and open pathways for improved vaccines and therapies.
Assuntos
ADP-Ribosil Ciclase 1 , Pulmão , Macrófagos Alveolares , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis , Animais , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/microbiologia , Macrófagos Alveolares/metabolismo , Mycobacterium tuberculosis/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , ADP-Ribosil Ciclase 1/metabolismo , ADP-Ribosil Ciclase 1/genética , Modelos Animais de Doenças , Feminino , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/imunologia , Vacina BCG/imunologia , Análise de Célula Única , Tuberculose/imunologia , Tuberculose/microbiologiaRESUMO
BACKGROUND: Drug-resistant tuberculosis (DR-TB) remains a threat to public health. Shorter regimens have been proposed as potentially valuable treatments for multidrug or rifampicin resistant tuberculosis (MDR/RR-TB). We undertook a systematic review and network meta-analysis to evaluate the efficacy and safety of shorter MDR/RR-TB regimens. METHODS: We searched PubMed/MEDLINE, Cochrane Center for Clinical Trials (CENTRAL), Scopus, ClinicalTrials.gov, WHO International Clinical Trials Registry Platform, US Food and Drug Administration, and Chinese Clinical Trial Registry for primary articles published from 2013 to July 2023. Favorable (cured and treatment completed) and unfavorable (treatment failure, death, loss to follow-up, and culture conversion) outcomes were assessed as the main efficacy outcomes, while adverse events were assessed as the safety outcomes. The network meta-analysis was performed using R Studio version 4.3.1 and the Netmeta package. The study protocol adhered to the PRISMA-NMA guidelines and was registered in PROSPERO (CRD42023434050). RESULT: We included 11 eligible studies (4 randomized control trials and 7 cohorts) that enrolled 3,548 patients with MDR/RR-TB. Treatment with a 6-month combination of BdqLzdLfxZTrd/Eto/H had two times more favorable outcomes [RR 2.2 (95% CI 1.22, 4.13), P = 0.0094], followed by a 9-11 month combination of km/CmMfx/LfxPtoCfzZEHh [RR1.67 (95% CI 1.45, 1.92), P < 0.001] and a 6-month BdqPaLzdMfx [RR 1.64 (95% CI 1.24, 2.16), P < 0.0005] compared to the standard longer regimens. Treatment with 6 months of BdqPaLzdMfx [RR 0.33 (95% CI 0.2, 0.55), P < 0.0001] had a low risk of severe adverse events, followed by 6 months of BdqPaLzd [RR 0.36 (95% CI 0.22, 0.59), P ≤ 0.001] and BdqPaLzdCfz [RR 0.54 (95% CI 0.37, 0.80), P < 0.0001] than standard of care. CONCLUSION: Treatment of patients with RR/MDR-TB using shorter regimens of 6 months BdqLzdLfxZTrd/Eto/H, 9-11 months km/CmMfx/LfxPtoCfzZEHh, and 6 months BdqPaLzdMfx provides significantly higher cure and treatment completion rates compared to the standard longer MDR/RR-TB. However, 6BdqPaLzdMfx, 6BdqPaLzd, and 6BdqPaLzdCfz short regimens are significantly associated with decreased severity of adverse events. The findings are in support of the current WHO-recommended 6-month shorter regimens.
Assuntos
Antituberculosos , Metanálise em Rede , Rifampina , Tuberculose Resistente a Múltiplos Medicamentos , Humanos , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Rifampina/uso terapêutico , Antituberculosos/uso terapêutico , Antituberculosos/efeitos adversos , Resultado do Tratamento , Mycobacterium tuberculosis/efeitos dos fármacosRESUMO
INTRODUCTION: Tuberculosis (TB) is a serious global health problem in Indonesia, which is the country with the secondhighest TB burden after India. Accuracy in TB diagnosis is the key to effective treatment and decreased transmission rate. One of the latest diagnostic methods is interferon gamma release assay (IGRA), which measures the interferon-γ release associated with Mycobacterium tuberculosis (MTB) infection. This study aims to determine the diagnostic value of IGRA-TB using IchromaTM IGRA-TB diagnostic kit (sensitivity, specificity, positive predictive value [PPV], and negative predictive value [NPV]), compared to Ziehl-Neelsen (AFB) staining, nucleic acid amplificationbased test (Xpert-MTB) and chest-X Ray as the gold standard in TB diagnosis. MATERIALS AND METHODS: A cross-sectional observational study design was used. Patients were recruited through purposive sampling from pulmonology outpatient clinic and inpatient ward at Jemursari Islamic Hospital (RSI Jemursari), Surabaya from July 2023 to December 2023. All enrolled patients should have been previously tested positive or negative for pulmonary TB using AFB staining, Xpert MTB and chest x-ray. Blood samples of the patients were collected and processed using the IchromaTM IGRA-TB diagnostic kit. The results were then compared with gold standard methods for calculating the IGRA-TB diagnostic value. RESULTS: A total of 56 adult patients were enrolled in this study. The sensitivity, specificity, PPV, NPV and accuracy rate of IGRA-TB using IchromaTM IGRA-TB diagnostic kit were 80.56%, 85%, 90.62%, 70.83% and 82.14%, respectively. CONCLUSION: IchromaTM IGRA-TB showed reasonably high diagnostic sensitivity and specificity, indicating that this method can be further utilised as a diagnostic and screening tool for pulmonary TB.
Assuntos
Testes de Liberação de Interferon-gama , Sensibilidade e Especificidade , Tuberculose Pulmonar , Humanos , Testes de Liberação de Interferon-gama/métodos , Tuberculose Pulmonar/diagnóstico , Feminino , Adulto , Masculino , Estudos Transversais , Pessoa de Meia-Idade , Indonésia , Adulto Jovem , Mycobacterium tuberculosis/isolamento & purificação , Idoso , Fluorimunoensaio/métodosAssuntos
Antituberculosos , Isoniazida , Tuberculose Resistente a Múltiplos Medicamentos , Humanos , Isoniazida/uso terapêutico , Antituberculosos/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Mycobacterium tuberculosis/efeitos dos fármacos , EpidemiasRESUMO
Introduction. The discordance between phenotypic and molecular methods of rifampicin (RIF) drug susceptibility testing (DST) in Mycobacterium tuberculosis poses a significant challenge, potentially resulting in misdiagnosis and inappropriate treatment.Hypothesis/gap statement. A comparison of RIF phenotypic and molecular methods for DST, including whole genome sequencing (WGS), may provide a better understanding of resistance mechanisms.Aim. This study aims to compare RIF DST in M. tuberculosis using two phenotypic and molecular methods including the GeneXpert RIF Assay (GX) and WGS for better understanding.Methodology. The study evaluated two phenotypic liquid medium methods [Lowenstein-Jensen (LJ) and Mycobacterium Growth Indicator Tube (MGIT)], one targeted molecular method (GX), and one WGS method. Moreover, mutational frequency in ponA1 and ponA2 was also screened in the current and previous RIF resistance M. tuberculosis genomic isolates to find their compensatory role.Results. A total of 25 RIF-resistant isolates, including nine from treatment failures and relapse cases with both discordant and concordant DST results on LJ, MGIT and GX, were subjected to WGS. The phenotypic DST results indicated that 11 isolates (44%) were susceptible on LJ and MGIT but resistant on GX. These isolates exhibited multiple mutations in rpoB, including Thr444>Ala, Leu430>Pro, Leu430>Arg, Asp435>Gly, His445>Asn and Asn438>Lys. Conversely, four isolates that were susceptible on GX and MGIT but resistant on LJ were wild type for rpoB in WGS. However, these isolates possessed several novel mutations in the PonA1 gene, including a 10 nt insertion and two nonsynonymous mutations (Ala394>Ser, Pro631>Ser), as well as one nonsynonymous mutation (Pro780>Arg) in PonA2. The discordance rate of RIF DST is higher on MGIT than on LJ and GX when compared to WGS. These discordances in the Delhi/CAS lineages were primarily associated with failure and relapse cases.Conclusion. The WGS of RIF resistance is relatively expensive, but it may be considered for isolates with discordant DST results on MGIT, LJ and GX to ensure accurate diagnosis and appropriate treatment options.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Rifampina , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Rifampina/farmacologia , Humanos , Sequenciamento Completo do Genoma , Mutação , Farmacorresistência Bacteriana/genética , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Tuberculose/microbiologiaRESUMO
BACKGROUND: Control of rifampicin-resistant tuberculosis (RR-MTB) requires novel technologies for restoring the anti-TB efficacy of priority drugs. We sought to evaluate the ability of nanotechnology application in the recovery of the anti-tuberculosis efficacy of rifampicin. METHODS: Nanocomposite- standard dose of rifampicin and 20 nm silver nanoparticles (AgNPs) suspension solution of 6 different concentrations: 0.25%; 0.5%; 1%; 2.5%; 5%; and 10%, were supplemented to 70 rifampicin-resistant mycobacterium tuberculosis (RR-MTB) isolates. The control arm consisted of 35 RR-MTB isolates and AgNPs suspension with identical concentrations. The inhibitory effect of nanocomposites was evaluated by MTB growth rate using the BACTECTM MGIT 960TM. The safety assessment of single-use AgNPs was conducted in experimental animals. RESULTS: The suppression process of AgNPs on RR-MTB isolates started with 2,5% nanocomposite solution application and full suppression was achieved in 5% and 10% nanocomposite solutions. A standard dose of rifampicin and a 2.5% solution of AgNPs increased the minimal inhibitory effect on RR-MTB by 10% (total 80%) vs the isolated use of a 2.5% solution of AgNPs (70%). An experiment on animals revealed the complete safety of a single injection of ultra-high doses of AgNPs. CONCLUSION: The study showed the potentiating effect of AgNPs in overcoming the resistance of MTB to rifampicin providing a scientific basis for further research.
Assuntos
Nanopartículas Metálicas , Mycobacterium tuberculosis , Nanocompostos , Rifampina , Prata , Rifampina/farmacologia , Prata/química , Prata/farmacologia , Nanocompostos/química , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Animais , Humanos , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Antituberculosos/farmacologia , Antibióticos Antituberculose/farmacologiaRESUMO
Background: To effectively control tuberculosis (TB), it is crucial to distinguish between active TB disease and latent TB infection (LTBI) to provide appropriate treatment. However, no such tests are currently available. Immune responses associated with active TB and LTBI are dynamic and exhibit distinct patterns. Comparing these differences is crucial for developing new diagnostic methods and understanding the etiology of TB. This study aimed to investigate the relationship between pro- and anti-inflammatory CD4+ cytokine production following stimulation with two types of latency-associated Mycobacterium tuberculosis (M.tb) antigens to allow differentiation between active TB and LTBI. Methods: Cryopreserved PBMCs from patients with active TB disease or LTBI were stimulated overnight with replication-related antigen [ESAT-6/CFP-10 (E/C)] or two latency-associated antigens [heparin-binding hemagglutinin (HBHA) and alpha-crystallin-like protein (Acr)]. Responses were analyzed using multiparameter flow cytometry: active TB disease (n=15), LTBI (n=15) and ELISA: active TB disease (n=26) or LTBI (n=27). Results: CD4+ central memory T cells (Tcm) specific to E/C and CD4+ effector memory T cells specific to Acr and HBHA were higher in LTBI than in TB patients. IFN-γ+Tcm and IL-17+ Tem cells was higher in the LTBI group (p= 0.012 and p=0.029 respectively), but IL-10+ Tcm was higher in the active TB group (p= 0.029) following HBHA stimulation. Additionally, following stimulation with HBHA, IL-10 production from CD4+ T cells was significantly elevated in patients with active TB compared to those with LTBI (p= 0.0038), while CD4+ T cell production of IL-17 and IFN-γ was significantly elevated in LTBI compared to active TB (p= 0.0076, p< 0.0001, respectively). HBHA also induced more CCR6+IL-17+CD4Tcells and IL-17+FoxP3+CD25+CD4Tcells in LTBI than in TB patients (P=0.026 and P=0.04, respectively). HBHA also induced higher levels of IFN-γ+IL-10+CD4+ T cells in patients with active TB (Pp=0.03) and higher levels of IFN-γ+IL-17+ CD4+ T cells in those with LTBI (p=0.04). HBHA-specific cytokine production measured using ELISA showed higher levels of IFN-γ in participants with LTBI (P=0.004) and higher levels of IL-10 in those with active TB (P=0.04). Conclusion: Stimulation with HBHA and measurement of CD4+ T cell production of IFN-γ, IL-10, and IL-17 could potentially differentiate active TB from LTBI. The characteristics of cytokine-expressing cells induced by HBHA also differed between participants with active TB and LTBI.
Assuntos
Antígenos de Bactérias , Linfócitos T CD4-Positivos , Interferon gama , Interleucina-10 , Interleucina-17 , Tuberculose Latente , Mycobacterium tuberculosis , Tuberculose , Humanos , Masculino , Feminino , Linfócitos T CD4-Positivos/imunologia , Adulto , Interleucina-17/imunologia , Interleucina-17/metabolismo , Mycobacterium tuberculosis/imunologia , Interleucina-10/imunologia , Interferon gama/metabolismo , Interferon gama/imunologia , Pessoa de Meia-Idade , Tuberculose Latente/imunologia , Tuberculose/imunologia , Antígenos de Bactérias/imunologia , Idoso , Adulto Jovem , LectinasRESUMO
New Biginelli adducts were rationalised, via the introduction of selected anti-tubercular (TB) pharmacophores into the dihydropyrimidine (DHPM) ring of deoxythymidine monophosphate (dTMP), the natural substrate of Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt). Repurposing was one of the design rationale strategies for some selected mimics of the designed compounds. The anti-TB activity was screened against the Mtb H37Rv strain where 11a was superior to ethambutol (EMB), and was 9-fold more potent than pyrazinamide (PZA). Additionally, compounds 11b, 4a, 4b, 13a, 13b and 14a elicited higher anti-TB activity than PZA, showing better safety profiles than EMB against RAW 264.7 cells' growth. The in vitro TMPKmt inhibition assay released compounds 11a, 11b and 13b as the most potent inhibitors. Docking studies presumed the binding modes and molecular dynamics (MD) simulation revealed the dynamic stability of 11a-TMPKmt complex over 100 ns. In silico prediction of the chemo-informatics properties of the most active compounds was conducted.
Assuntos
Antituberculosos , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Núcleosídeo-Fosfato Quinase , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Antituberculosos/farmacologia , Antituberculosos/química , Antituberculosos/síntese química , Núcleosídeo-Fosfato Quinase/antagonistas & inibidores , Núcleosídeo-Fosfato Quinase/metabolismo , Relação Estrutura-Atividade , Estrutura Molecular , Camundongos , Modelos Moleculares , Animais , Células RAW 264.7 , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese químicaRESUMO
BACKGROUND: Respiratory microbiota is closely related to tuberculosis (TB) initiation and progression. However, the dynamic changes of respiratory microbiota during treatment and its association with TB progression remains unclear. METHODS: A total of 16 healthy individuals and 16 TB patients (10 drug-sensitive TB (DS-TB) and 6 drug-resistant TB (DR-TB)) were recruited. Sputum samples were collected at baseline for all anticipants and after anti-TB treatment at Month-6 for TB patients. High throughput 16 S RNA sequencing was used to characterize the respiratory microbiota composition. RESULTS: Compared to the healthy individuals, TB patients exhibited lower respiratory microbiota diversity (p < 0.05). This disruption was alleviated after anti-TB treatment, especially for DS-TB patients. Parvimonas spp. numbers significantly increased after six months of anti-TB treatment in both DS-TB and DR-TB patients (p < 0.05). Rothia spp. increase during treatment was associated with longer sputum-culture conversion time and worse pulmonary lesion absorption (p < 0.05). Besides, Moraxella spp. prevalence was associated with longer sputum-culture conversion time, while Gemella spp. increase was associated with worsening resolving of pulmonary lesions (p < 0.05). CONCLUSION: Dynamic changes of respiratory microbiota during anti-TB treatment is closely related to TB progression. The involvement of critical microorganisms, such as Parvimonas spp., Rothia spp., Moraxella, and Gemella spp., appears to be associated with pulmonary inflammatory conditions, particularly among DR-TB. These microorganisms could potentially serve as biomarkers or even as targets for therapeutic intervention to enhance the prognosis of tuberculosis patients.
Assuntos
Antituberculosos , Microbiota , Escarro , Tuberculose Resistente a Múltiplos Medicamentos , Tuberculose Pulmonar , Humanos , Escarro/microbiologia , Masculino , Feminino , Antituberculosos/uso terapêutico , Microbiota/efeitos dos fármacos , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologia , Adulto , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Pessoa de Meia-Idade , Resultado do Tratamento , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/classificação , Bactérias/isolamento & purificação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , RNA Ribossômico 16S/genéticaRESUMO
The control of bacterial growth is key to the prevention and treatment of tuberculosis (TB). Granulomas represent independent foci of the host immune response that present heterogeneous capacity for control of bacterial growth. At the whole tissue level, B cells and CD4 or CD8 T cells have an established role in immune protection against TB. Immune cells interact within each granuloma response, but the impact of granuloma immune composition on bacterial replication remains unknown. Here we investigate the associations between immune cell composition, including B cell, CD4, and CD8 T cells, and the state of replicating Mycobacterium tuberculosis (Mtb) within the granuloma. A measure of ribosomal RNA synthesis, the RS ratio®, represents a proxy measure of Mtb replication at the whole tissue level. We adapted the RS ratio through use of in situ hybridization, to identify replicating and non-replicating Mtb within each designated granuloma. We applied a regression model to characterize the associations between immune cell populations and the state of Mtb replication within each respective granuloma. In the evaluation of nearly 200 granulomas, we identified heterogeneity in both immune cell composition and proportion of replicating bacteria. We found clear evidence of directional associations between immune cell composition and replicating Mtb. Controlling for vaccination status and endpoint post-infection, granulomas with lower CD4 or higher CD8 cell counts are associated with a higher percent of replicating Mtb. Conversely, changes in B cell proportions were associated with little change in Mtb replication. This study establishes heterogeneity across granulomas, demonstrating that certain immune cell types are differentially associated with control of Mtb replication. These data suggest that evaluation at the granuloma level may be imperative to identifying correlates of immune protection.
Assuntos
Linfócitos T CD8-Positivos , Granuloma , Mycobacterium tuberculosis , Mycobacterium tuberculosis/imunologia , Humanos , Granuloma/imunologia , Granuloma/microbiologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Linfócitos T CD4-Positivos/imunologia , Linfócitos B/imunologia , Masculino , Tuberculose/imunologia , Tuberculose/microbiologiaRESUMO
We have been trying to find a miRNA that can specifically regulate the function of mycobacterial host cells to achieve the purpose of eliminating Mycobacterium tuberculosis. The purpose of this study is to investigate the regulation of mmu-let-7a-5p on macrophages apoptosis and its effect on intracellular BCG clearance. After a series of in vitro experiments, we found that mmu-let-7a-5p could negatively regulate the apoptosis of macrophages by targeting Caspase-3. The extrinsic apoptosis signal axis TNFR1/FADD/Caspase-8/Caspase-3 was inhibited after BCG infection. Up-regulated the expression level of mmu-let-7a-5p increase the cell proliferation viability and inhibit apoptosis rate of macrophages, but down-regulated its level could apparently reduce the bacterial load of intracellular Mycobacteria and accelerate the clearance of residual Mycobacteria effectively. Mmu-let-7a-5p has great potential to be utilized as an optimal candidate exosomal loaded miRNA for anti-tuberculosis immunotherapy in our subsequent research.
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Apoptose , Carga Bacteriana , Caspase 3 , Macrófagos , MicroRNAs , Animais , Camundongos , Caspase 3/metabolismo , Proliferação de Células , Macrófagos/microbiologia , Macrófagos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Mycobacterium bovis/fisiologia , Mycobacterium tuberculosis , Células RAW 264.7RESUMO
As the first identified multidrug efflux pump in Mycobacterium tuberculosis (Mtb), EfpA is an essential protein and promising drug target. However, the functional and inhibitory mechanisms of EfpA are poorly understood. Here we report cryo-EM structures of EfpA in outward-open conformation, either bound to three endogenous lipids or the inhibitor BRD-8000.3. Three lipids inside EfpA span from the inner leaflet to the outer leaflet of the membrane. BRD-8000.3 occupies one lipid site at the level of inner membrane leaflet, competitively inhibiting lipid binding. EfpA resembles the related lysophospholipid transporter MFSD2A in both overall structure and lipid binding sites and may function as a lipid flippase. Combining AlphaFold-predicted EfpA structure, which is inward-open, we propose a complete conformational transition cycle for EfpA. Together, our results provide a structural and mechanistic foundation to comprehend EfpA function and develop EfpA-targeting anti-TB drugs.
Assuntos
Proteínas de Bactérias , Mycobacterium tuberculosis , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Transporte Biológico , Microscopia Crioeletrônica , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Conformação ProteicaRESUMO
The clinical data of a child with disseminated tuberculosis with osteomyelitis of the right little finger as the first manifestation who was admitted to Tianjin Children's Hospital on April 8, 2023 were retrospectively analyzed. The child, a 14-year-old female, presented with osteomyelitis of the right little finger as the first manifestation. She still had recurrent fever after focal incision and drainage. She was referred to our hospital. The samples from multiple sites were positive for molecular biology detection of Mycobacterium tuberculosis. She was considered as disseminated tuberculosis and was given anti-tuberculosis treatment. The child has recovered well. Pediatric disseminated tuberculosis has variable clinical manifestations and lacks specificity. It is often misdiagnosed and has a high mortality rate. Clinicians should improve their understanding of the disease and ensure early diagnosis and treatment.
Assuntos
Dedos , Osteomielite , Humanos , Feminino , Adolescente , Osteomielite/diagnóstico , Osteomielite/microbiologia , Tuberculose Osteoarticular/diagnóstico , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/diagnóstico , Antituberculosos/uso terapêuticoRESUMO
Culturing and genomic sequencing of Mycobacterium tuberculosis (MTB) from tuberculosis (TB) cases is the basis for many research and clinical applications. The alternative, culture-free sequencing from diagnostic samples, is promising but poses challenges to obtain and analyse the MTB genome. Paradoxically, culture is assumed to impose a diversity bottleneck, which, if true, would entail unexplored consequences. To unravel this paradox we generate high-quality genomes of sputum-culture pairs from two different settings after developing a workflow for sequencing from sputum and a tailored bioinformatics analysis. Careful downstream comparisons reveal sources of sputum-culture incongruences due to false positive/negative variation associated with factors like low input MTB DNA or variable genomic depths. After accounting for these factors, contrary to the bottleneck dogma, we identify a 97% variant agreement within sputum-culture pairs, with a high correlation also in the variants' frequency (0.98). The combined analysis from five different settings and more than 100 available samples shows that our results can be extrapolated to different TB epidemic scenarios, demonstrating that for the cases tested culture accurately mirrors clinical samples.
Assuntos
Variação Genética , Mycobacterium tuberculosis , Escarro , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Escarro/microbiologia , Humanos , Tuberculose/microbiologia , Tuberculose/diagnóstico , Genoma Bacteriano , DNA Bacteriano/genética , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/diagnósticoRESUMO
The recA gene, encoding Recombinase A (RecA) is one of three Mycobacterium tuberculosis (Mtb) genes encoding an in-frame intervening protein sequence (intein) that must splice out of precursor host protein to produce functional protein. Ongoing debate about whether inteins function solely as selfish genetic elements or benefit their host cells requires understanding of interplay between inteins and their hosts. We measured environmental effects on native RecA intein splicing within Mtb using a combination of western blots and promoter reporter assays. RecA splicing was stimulated in bacteria exposed to DNA damaging agents or by treatment with copper in hypoxic, but not normoxic, conditions. Spliced RecA was processed by the Mtb proteasome, while free intein was degraded efficiently by other unknown mechanisms. Unspliced precursor protein was not observed within Mtb despite its accumulation during ectopic expression of Mtb recA within E. coli. Surprisingly, Mtb produced free N-extein in some conditions, and ectopic expression of Mtb N-extein activated LexA in E. coli. These results demonstrate that the bacterial environment greatly impacts RecA splicing in Mtb, underscoring the importance of studying intein splicing in native host environments and raising the exciting possibility of intein splicing as a novel regulatory mechanism in Mtb.
Assuntos
Proteínas de Bactérias , Escherichia coli , Inteínas , Mycobacterium tuberculosis , Processamento de Proteína , Recombinases Rec A , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Recombinases Rec A/metabolismo , Recombinases Rec A/genética , Inteínas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exteínas/genética , Dano ao DNA , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Serina EndopeptidasesRESUMO
Tuberculosis(TB) of the Central nervous system (CNS) is a rare and highly destructive disease. The emergence of drug resistance has increased treatment difficulty, leaving the Bacillus Calmette-Guérin (BCG) vaccine as the only licensed preventative immunization available. This study focused on identifying the epitopes of PknD (Rv0931c) and Rv0986 from Mycobacterium tuberculosis(Mtb) strain H37Rv using an in silico method. The goal was to develop a therapeutic mRNA vaccine for preventing CNS TB. The vaccine was designed to be non-allergenic, non-toxic, and highly antigenic. Codon optimization was performed to ensure effective translation in the human host. Additionally, the secondary and tertiary structures of the vaccine were predicted, and molecular docking with TLR-4 was carried out. A molecular dynamics simulation confirmed the stability of the complex. The results indicate that the vaccine structure shows effectiveness. Overall, the constructed vaccine exhibits ideal physicochemical properties, immune response, and stability, laying a theoretical foundation for future laboratory experiments.
Assuntos
Simulação por Computador , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis , Tuberculose do Sistema Nervoso Central , Humanos , Mycobacterium tuberculosis/imunologia , Tuberculose do Sistema Nervoso Central/prevenção & controle , Tuberculose do Sistema Nervoso Central/imunologia , Vacinas contra a Tuberculose/imunologia , Epitopos/imunologia , Epitopos/química , Vacinas de mRNA , Vacinas Sintéticas/imunologiaRESUMO
BACKGROUND: Rifampicin resistant tuberculosis remains a global health problem with almost half a million new cases annually. In high-income countries patients empirically start a standardized treatment regimen, followed by an individualized regimen guided by drug susceptibility test (DST) results. In most settings, DST information is not available or is limited to isoniazid and fluoroquinolones. Whole genome sequencing could more accurately guide individualized treatment as the full drug resistance profile is obtained with a single test. Whole genome sequencing has not reached its full potential for patient care, in part due to the complexity of translating a resistance profile into the most effective individualized regimen. METHODS: We developed a treatment recommender clinical decision support system (CDSS) and an accompanying web application for user-friendly recommendation of the optimal individualized treatment regimen to a clinician. RESULTS: Following expert stakeholder meetings and literature review, nine drug features and 14 treatment regimen features were identified and quantified. Using machine learning, a model was developed to predict the optimal treatment regimen based on a training set of 3895 treatment regimen-expert feedback pairs. The acceptability of the treatment recommender CDSS was assessed as part of a clinical trial and in a routine care setting. Within the clinical trial setting, all patients received the CDSS recommended treatment. In 8 of 20 cases, the initial recommendation was recomputed because of stock out, clinical contra-indication or toxicity. In routine care setting, physicians rejected the treatment recommendation in 7 out of 15 cases because it deviated from the national TB treatment guidelines. A survey indicated that the treatment recommender CDSS is easy to use and useful in clinical practice but requires digital infrastructure support and training. CONCLUSIONS: Our findings suggest that global implementation of the novel treatment recommender CDSS holds the potential to improve treatment outcomes of patients with RR-TB, especially those with 'difficult-to-treat' forms of RR-TB.
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Antituberculosos , Sistemas de Apoio a Decisões Clínicas , Aprendizado de Máquina , Rifampina , Tuberculose Resistente a Múltiplos Medicamentos , Humanos , Rifampina/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Antituberculosos/uso terapêutico , Antituberculosos/administração & dosagem , Mycobacterium tuberculosis/efeitos dos fármacos , Medicina de Precisão/métodos , Testes de Sensibilidade Microbiana , Masculino , Feminino , AdultoRESUMO
Introduction: Ehrlich-Ziehl-Neelsen (EZN) staining and culture methods are often used to diagnose tuberculosis. This study aimed to determine the acidfast bacteria (AFS) positivity rates in various clinical samples sent to our laboratory over five years and the growth and resistance rates in two different (solid and liquid) cultures and compare them with the data from Türkiye and the world. Materials and Methods: A total of 62.456 clinic samples were accepted in the microbiology laboratory between 2019 and 2024. The mycobacterial culture was performed by searching for acid-resistant bacilli microscopically and parallel inoculation media [solid Löwenstein-Jensen (L-J) and MGIT 960 liquid]. Those growing in the MGIT 960 system were identified using BD MGIT TBC Identification test kits that detect the MPT64 antigen. AFS and MPT64 antigenpositive samples were identified as Mycobacterium tuberculosis complex (MTBC) while AFS-positive samples and MPT64 antigen-negative results were classified as non-tuberculous mycobacteria (NTM). Drug susceptibility testing was performed with the BACTEC MGIT 960 SIRE kit. Susceptibility to NTM samples was not performed. Result: Out of a total of 120.829 samples, 95.101 were lung samples and 25.728 were extrapulmonary samples. AFS positivity was detected in 2961 (2.4%) samples. MTBC grew in 6854 (5.6%) samples, and NTM grew in 1506 (1.24%) samples. Contamination was detected in 7171 (5.9%) media. Two thousand one hundred and sixty-nine susceptibility tests were performed. Considering antibiotic resistance rates, isoniazid resistance was detected in 154 (7%), rifampicin resistance in 140 (6.4%), ethambutol resistance in 18 (0.8%), and streptomycin resistance in 120 (0.5%) samples. All four-drug resistance was observed in 91 (4.1%) samples. AFP positivity and resistance rates for rifampicin have decreased significantly, while there have been no significant changes in NTM rates over the years. Conclusions: When our data was determined, the sensitivity of microscopy was low. It is understood that mycobacterial culture and microscopy must be evaluated together to exclude tuberculosis infection. The high mycobacterial culture positivity rate, which is 5.6%, is due to the high number of follow-up patients and new referrals. It is seen that the change in sensitivity rates is due to the period of the COVID-19 epidemic, and it is similar to World Health Organization (WHO) data.