Objective The prevalence of drug-resistant Mycobacterium tuberculosis (Mtb) strains is exacerbating the global burden of tuberculosis (TB), highlighting the urgent need for new treatment strategies for TB. Methods The recombinant adenovirus vaccine expressing cyclic di-adenosine monophosphate (c-di-AMP) phosphodiesterase B (CnpB) (rAd-CnpB), was administered to normal mice via mucosal immunization, either alone or in combination with drug therapy, to treat Mtb respiratory infections in mice.Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of antibodies in serum and bronchoalveolar lavage fluid (BALF). Real-time quantitative PCR was performed to assess the transcription levels of cytokines interferon γ(IFN-γ) and interleukin 10(IL-10) in mouse lungs. Flow cytometry was used to determine the proportions of CD4⁺ and CD8⁺ T cell subsets in the lungs and spleens. ELISA was employed to measure the levels of cytokines IFN-γ, IL-2, IL-10, inflammatory factors IL-6, and tumor necrosis factor α (TNF-α) secreted by spleen cells following antigen stimulation. The bacteria loads in the lungs and spleens of Mtb-infected mice were enumerated by plate counting methods. Resluts Intranasal immunization with rAd-CnpB induced high titers of IgG in mouse serum and the production of IgG and IgA in BALF, along with alterations in T lymphocyte subsets in the lungs and spleens. Administration of rAd-CnpB, either alone or in combination with drugs, to Mtb-infected mice significantly increased serum IgG levels as well as IgA and IgG levels in BALF. rAd-CnpB immunization promoted the secretion of CnpB-specific cytokines and inflammatory factors by splenocytes in Mtb-infected mice. However, rAd-CnpB immunotherapy, either alone or combined with drugs, did not significantly affect the bacterial loads in the lungs and spleens of mice with Mtb respiratory infections. Conclusion Mucosal immunization with rAd-CnpB induced significant mucosal, humoral and cellular immune responses in mice, and significantly enhanced CnpB-specific cellular immune responses in Mtb-infected mice.
Animals ; Adenoviridae/immunology* ; Mycobacterium tuberculosis/genetics* ; Mice ; Female ; Phosphoric Diester Hydrolases/genetics* ; Tuberculosis Vaccines/administration & dosage* ; Tuberculosis/prevention & control* ; Mice, Inbred BALB C ; Cytokines ; Lung/microbiology* ; Immunization ; Bronchoalveolar Lavage Fluid/immunology* ; Immunity, Mucosal

Objective To explore the effect of miR-582-5p on Mycobacterium tuberculosis (Mtb)-infected macrophages by regulating dual specificity phosphatase 1 (DUSP1). Methods THP-1 macrophages were divided into six groups: control group, Mtb group, inhibitor-NC group, miR-582-5p inhibitor group, miR-582-5p inhibitor+si-NC group, and miR-582-5p inhibitor+si-DUSP1 group. QRT-PCR was applied to detect the gene expression of miR-582-5p and DUSP1 in cells. ELISA kit was used to detect the levels of interferon γ (IFN-γ), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and interleukin 1β (IL-1β). CCK-8 method was applied to detect cell proliferation. Flow cytometry was applied to detect cell apoptosis rate. Western blot analysis was used to measure the protein expression levels of B-cell lymphoma 2 (Bcl2), Bcl2-associated X (BAX), and cleaved-caspase 3 (c-caspase-3) in cells. In addition, the target relationship between miR-582-5p and DUSP1 was verified. Results Compared with the control group, the expression of miR-582-5p, levels of IFN-γ, IL-6, TNF-α, IL-1β, bacterial load and OD450 values (24 h, 48 h), and the protein expression of Bcl2 in macrophages were higher in the Mtb group, while the mRNA expression of DUSP1, apoptosis rate, and the protein expression levels of c-caspase-3, BAX and DUSP1 were lower. Compared with the Mtb group and the inhibitor-NC group, the above-mentioned indicators in the miR-582-5p inhibitor group were partially reversed. Down-regulation of DUSP1 expression partially reversed the inhibitory effect of down-regulation of miR-582-5p expression on Mtb-infected macrophages. Conclusion Inhibiting the expression of miR-582-5p can up-regulate DUSP1, thereby inhibiting the proliferation and inflammatory response of Mtb-infected macrophages and promoting cell apoptosis.
Humans ; Macrophages/metabolism* ; Dual Specificity Phosphatase 1/metabolism* ; MicroRNAs/metabolism* ; Mycobacterium tuberculosis/physiology* ; Tuberculosis/microbiology* ; Apoptosis/genetics* ; THP-1 Cells ; Cell Proliferation/genetics* ; Interferon-gamma/genetics* ; Tumor Necrosis Factor-alpha/genetics* ; Interleukin-1beta/genetics*

OBJECTIVES: This study aimed to investigate the impact of foam macrophages (FMs) on the intracellular survival of Mycobacterium tuberculosis (MTB) and identify the molecular mechanisms influencing MTB survival. METHODS: An in vitro FM model was established using oleic acid induction. Transcriptomic and metabolomic analyses were conducted to identify the key molecular pathways involved in FM-mediated MTB survival. RESULTS: Induced FMs effectively restricted MTB survival. Transcriptomic and metabolomic profiling revealed distinct changes in gene and metabolite expression in FMs during MTB infection compared with normal macrophages. Integrated analyses identified significant alterations in the cyclic adenosine monophosphate (cAMP) signaling pathway, indicating that its activation contributes to the FM-mediated restriction of MTB survival. CONCLUSIONS FMs inhibit MTB survival. The cAMP signaling pathway is a key contributor. These findings enhance the understanding of the role of FMs in tuberculosis progression, suggest potential targets for host-directed therapies, and offer new directions for developing diagnostic and therapeutic strategies against tuberculosis.
Mycobacterium tuberculosis/physiology* ; Transcriptome ; Metabolomics ; Foam Cells/microbiology* ; Humans ; Metabolome ; Tuberculosis/microbiology* ; Gene Expression Profiling

OBJECTIVE: Assessing the accuracy of automated EasyNAT system for rapidly detecting paraffin-embedded tissue for the diagnosis of tuberculosis. METHODS: A retrospective analysis was conducted on 134 patients, comprising 101 with confirmed tuberculosis and 33 without tuberculosis, treated at Beijing Chest Hospital, Capital Medical University, between 2018 and 2022.The clinical diagnostic results served as the standard for assessing the diagnostic performance of the EasyNAT system in comparison to quantitative real-time polymerase chain reaction (qPCR) for tuberculosis detection in paraffin-embedded tissues.The evaluation criteria included sensitivity, specificity, positive predictive value, negative predictive value, and accuracy rate. RESULTS: Based on the clinical diagnostic results, the EasyNAT assay demonstrated a sensitivity of 87.1%(88/101, 95%CI: 79.2%-92.3%)and a specificity of 100.0%(33/33, 95%CI: 89.6%-100.0%).The positive predictive value, negative predictive value, and accuracy rate were 100% (88/88, 95%CI: 95.8%-100.0%), 71.7%(33/46, 95%CI: 57.5%-82.7%), and 90.3%(121/134, 95%CI: 84.1%-94.2%), respectively.In comparison, the qPCR assay exhibited a sensitivity of 96.0%(90.3%-98.5%)and a specificity of 100.0%(89.6%-100.0%).The positive predictive value, negative predictive value, and accuracy rate for qPCR were 100.0%(96.2%-100.0%), 89.2%(75.3%- 95.7%), and 97.0%(92.6%-98.8%).The Cohen's kappa value of 0.84 indicated substantial agreement between EasyNAT and qPCR.The detection rate of tuberculosis using this method was 86.4%(38/44, 95%CI: 73.3%-93.6%), while the detection rate for extrapulmonary tuberculosis was 87.7%(50/57, 95%CI: 76.8%-93.9%).In comparison, qPCR showed a detection rate of 97.7%(88.2%- 99.6%) for pulmonary tuberculosis and 94.7%(85.6%-98.6%)for extrapulmonary tuberculosis.There was no statistically significant difference in the detection results between the method and qPCR for both pulmonary and extrapulmonary tuberculosis(P>0.05).Importantly, the EasyNAT detection combined nucleic acid extraction, amplification, and analysis into one process.Compared with traditional qPCR methods, manual operation time was reduced by 2 hours, leading to an overall reduction in total testing time by 3 hours. CONCLUSION The EasyNAT nucleic acid rapid detection system can quickly, conveniently, and accurately detect Mycobacterium tuberculosis DNA in paraffin-embedded tissues, demonstrating significant clinical utility in the pathological diagnosis of tuberculosis.
Humans ; Retrospective Studies ; Paraffin Embedding ; Sensitivity and Specificity ; Tuberculosis/microbiology* ; Real-Time Polymerase Chain Reaction ; Mycobacterium tuberculosis/genetics* ; Predictive Value of Tests ; Nucleic Acid Amplification Techniques/methods* ; Female ; Male

Humans ; Isoniazid/pharmacology* ; Mycobacterium tuberculosis/genetics* ; Rifampin/pharmacology* ; Antitubercular Agents/pharmacology* ; Mutation ; Microbial Sensitivity Tests ; Tuberculosis, Multidrug-Resistant/microbiology* ; Drug Resistance, Multiple, Bacterial/genetics* ; Bacterial Proteins/genetics*

Humans ; Mycobacterium tuberculosis/genetics* ; Microspheres ; Antitubercular Agents/pharmacology* ; Mutation ; Microbial Sensitivity Tests ; Fluoroquinolones ; Drug Resistance, Bacterial/genetics* ; Tuberculosis, Multidrug-Resistant/microbiology*

Tuberculosis (TB) is an ancient infectious disease. Before the availability of effective drug therapy, it had high morbidity and mortality. In the past 100 years, the discovery of revolutionary anti-TB drugs such as streptomycin, isoniazid, pyrazinamide, ethambutol and rifampicin, along with drug combination treatment, has greatly improved TB control globally. As anti-TB drugs were widely used, multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis emerged due to acquired genetic mutations, and this now presents a major problem for effective treatment. Genes associated with drug resistance have been identified, including katG mutations in isoniazid resistance, rpoB mutations in rifampin resistance, pncA mutations in pyrazinamide resistance, and gyrA mutations in quinolone resistance. The major mechanisms of drug resistance include loss of enzyme activity in prodrug activation, drug target alteration, overexpression of drug target, and overexpression of the efflux pump. During the disease process, Mycobacterium tuberculosis may reside in different microenvironments where it is expose to acidic pH, low oxygen, reactive oxygen species and anti-TB drugs, which can facilitate the development of non-replicating persisters and promote bacterial survival. The mechanisms of persister formation may include toxin-antitoxin (TA) modules, DNA protection and repair, protein degradation such as trans-translation, efflux, and altered metabolism. In recent years, the use of new anti-TB drugs, repurposed drugs, and their drug combinations has greatly improved treatment outcomes in patients with both drug-susceptible TB and MDR/XDR-TB. The importance of developing more effective drugs targeting persisters of Mycobacterium tuberculosis is emphasized. In addition, host-directed therapeutics using both conventional drugs and herbal medicines for more effective TB treatment should also be explored. In this article, we review historical aspects of the research on anti-TB drugs and discuss the current understanding and treatments of drug resistant and persistent tuberculosis to inform future therapeutic development.
Humans ; Pyrazinamide/therapeutic use* ; Isoniazid/therapeutic use* ; Antitubercular Agents/therapeutic use* ; Tuberculosis, Multidrug-Resistant/microbiology* ; Mycobacterium tuberculosis/genetics* ; Tuberculosis/drug therapy* ; Rifampin/therapeutic use* ; Mutation ; Drug Resistance, Multiple, Bacterial/genetics*

Objective: To analyze the genomic mutation of Mycobacterium tuberculosis (M. tuberculosis) isolated in endogenous activation period and estimate the molecular clock based on the whole genome sequencing data. Methods: Literatures of the whole genome research of endogenous reactivated tuberculosis were retrieved, and the corresponding whole genome sequencing data were downloaded. We extracted the single nucleotide polymorphisms (SNPs) and strain isolation time of initial treatment and relapse of tuberculosis cases, explored the relationship between the different SNPs and interval between initial treatment and relapse by Poisson regression model, calculated the M. tuberculosis molecular clock, and estimated the mutation rate. Results: When the generation time of M. tuberculosis was 18 hours, the mutation rate in 0-2 years, i.e. short-term endogenous activation, was 6.47×10^-10 (95%CI: 5.59×10^-10-7.44×10^-10), which was significantly higher than that in 2-14 years in long term endogenous activation (3.27×10^-10, 95%CI: 2.88×10^-10-3.69×10^-10). The mutation rates of 0-, 1-, 2-, 3-, 5- and 7-14 years were 7.10×10^-10, 6.06×10^-10, 4.24×10^-10, 5.34×10^-10, 2.59×10^-10 and 1.26×10^-10 respectively. Conclusions: In the period of endogenous reactivation, the mutation rate of M. tuberculosis decreases with the interval time between initial treatment and relapse, which verifies the clinically observed phenomenon that the relapse often occurs within two years after the initial treatment of tuberculosis.
Genome, Bacterial ; Humans ; Mycobacterium tuberculosis/genetics* ; Recurrence ; Tuberculosis/microbiology* ; Whole Genome Sequencing

Diagnostic Tests, Routine ; methods ; Humans ; Mycobacterium tuberculosis ; isolation & purification ; Sensitivity and Specificity ; Tuberculosis, Pulmonary ; diagnosis ; microbiology

Two new isomeric modified tripeptides, aspergillamides C and D (compounds 1 and 2), together with fifteen known compounds (compounds 3-17), were obtained from the marine sponge-derived fungus Aspergillus terreus SCSIO 41008. The structures of the new compounds, including absolute configurations, were determined by extensive analyses of spectroscopic data (NMR, MS, UV, and IR) and comparisons between the calculated and experimental electronic circular dichroism (ECD) spectra. Butyrolactone I (compound 11) exhibited strong inhibitory effects against Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) with the IC being 5.11 ± 0.53 μmol·L, and acted as a noncompetitive inhibitor based on kinetic analysis.
4-Butyrolactone ; analogs & derivatives ; chemistry ; isolation & purification ; pharmacology ; Animals ; Aspergillus ; chemistry ; Chemistry Techniques, Analytical ; Dipeptides ; chemistry ; isolation & purification ; pharmacology ; Enzyme Inhibitors ; chemistry ; isolation & purification ; pharmacology ; Indoles ; chemistry ; isolation & purification ; pharmacology ; Molecular Structure ; Mycobacterium tuberculosis ; drug effects ; Peptides ; chemistry ; isolation & purification ; pharmacology ; Polyketides ; chemistry ; isolation & purification ; pharmacology ; Porifera ; microbiology ; Protein Tyrosine Phosphatases ; chemistry