Mycobacterium tuberculosis inhibits METTL14-mediated m6A methylation of Nox2 mRNA and suppresses anti-TB immunity.

Internal N6-methyladenosine (m6A) modifications are among the most abundant modifications of messenger RNA, playing a critical role in diverse biological and pathological processes. However, the functional role and regulatory mechanism of m6A modifications in the immune response to Mycobacterium tuberculosis infection remains unknown. Here, we report that methyltransferase-like 14 (METTL14)-dependent m6A methylation of NAPDH oxidase 2 (Nox2) mRNA was crucial for the host immune defense against M. tuberculosis infection and that M. tuberculosis-secreted antigen EsxB (Rv3874) inhibited METTL14-dependent m6A methylation of Nox2 mRNA. Mechanistically, EsxB interacted with p38 MAP kinase and disrupted the association of TAB1 with p38, thus inhibiting the TAB1-mediated autophosphorylation of p38. Interaction of EsxB with p38 also impeded the binding of p38 with METTL14, thereby inhibiting the p38-mediated phosphorylation of METTL14 at Thr72. Inhibition of p38 by EsxB restrained liquid-liquid phase separation (LLPS) of METTL14 and its subsequent interaction with METTL3, preventing the m6A modification of Nox2 mRNA and its association with the m6A-binding protein IGF2BP1 to destabilize Nox2 mRNA, reduce ROS levels, and increase intracellular survival of M. tuberculosis. Moreover, deletion or mutation of the phosphorylation site on METTL14 impaired the inhibition of ROS level by EsxB and increased bacterial burden or histological damage in the lungs during infection in mice. These findings identify a previously unknown mechanism that M. tuberculosis employs to suppress host immunity, providing insights that may empower the development of effective immunomodulators that target M. tuberculosis.

Correlation between Water Environment and Prevalence of Nontuberculous Mycobacteria Pulmonary Disease: A Case-Control Study.

Objective: Nontuberculous mycobacteria (NTM) prevalence in water systems has raised concerns about Nontuberculous Mycobacteria Pulmonary Disease (NTM-PD). Understanding the relationship between NTM-PD, drinking water distribution systems (DWDS), and other epidemiological factors is crucial for public health. Methods: A case-control study was conducted at the Inpatient Department of Tuberculosis Department of Shanghai Pulmonary Hospital. Subjects were divided into the NTM-PD group (n = 314) and pulmonary tuberculosis (PTB) group (n = 308) at a 1:1 ratio. Data was collected through questionnaires covering general information, depression (Self-Rating Depression Scale, SDS), and anxiety (Self-Rating Anxiety Scale, SAS). Multivariate unconditional logistic regression analysis was employed for the study. Results: The average age of NTM-PD patients was 55.26±14.44, with clinical symptoms including chest tightness, shortness of breath, hemoptysis, fever, and expectoration. Risk factors for NTM-PD included age (>60 years old, OR=1.042), gender (female, OR = 3.089), secondary water supply system (OR = 7.813), occupation (farmer/flower farmer, OR=2.676), depression (OR = 2.956), recurrent bronchiectasis (OR = 6.314), chronic obstructive pulmonary disease (COPD, OR = 2.704), and autoimmune disease (OR = 13.588) (P < .05). Use of household water purifiers was identified as a protective factor (OR = 0.128, P < .001). Conclusion: DWDS, drinking water mode, soil-related occupation, bronchiectasis, COPD, age, sex, and depression were closely related to the risk of NTM-PD. It is suggested to pay attention to water hygiene and illness progress and regulate mood to prevent NTM-PD in daily life.

Metabolic Profiles of Clinical Isolates of Drug-Susceptible and Multidrug-Resistant Mycobacterium tuberculosis: A Metabolomics-Based Study.

Background: Mycobacterium tuberculosis (MTB) is a global and highly deleterious pathogen that creates an enormous pressure on global public health. Although several effective drugs have been used to treat tuberculosis, the emergence of multidrug-resistant Mycobacterium tuberculosis (MDR-MTB) has further increased the public health burden. The aim of this study was to describe in depth the metabolic changes in clinical isolates of drug-susceptible Mycobacterium tuberculosis (DS-MTB) and MDR-MTB and to provide clues to the mechanisms of drug resistance based on metabolic pathways. Methods: Based on the minimum inhibition concentration (MIC) of multiple anti-tuberculosis drugs, two clinical isolates were selected, one DS-MTB isolate (isoniazid MIC=0.06 mg/L, rifampin MIC=0.25 mg/L) and one MDR-MTB isolate (isoniazid MIC=4 mg/L, rifampin MIC=8 mg/L). Through high-throughput metabolomics, the metabolic profiles of the DS-MTB isolate and the MDR-MTB isolate and their cultured supernatants were revealed. Results: Compared with the DS-MTB isolate, 128 metabolites were significantly altered in the MDR-MTB isolate and 66 metabolites were significantly altered in the cultured supernatant. The differential metabolites were significantly enriched in pyrimidine metabolism, purine metabolism, nicotinate and nicotinamide metabolism, arginine acid metabolism, and phenylalanine metabolism. Furthermore, metabolomics analysis of the bacterial cultured supernatants showed a significant increase in 10 amino acids (L-citrulline, L-glutamic acid, L-aspartic acid, L-norleucine, L-phenylalanine, L-methionine, L-tyrosine, D-tryptophan, valylproline, and D-methionine) and a significant decrease in 2 amino acids (L-lysine and L-arginine) in MDR-MTB isolate. Conclusion: The present study provided a metabolite alteration profile as well as a cultured supernatant metabolite alteration profile of MDR-MTB clinical isolate, providing clues to the potential metabolic pathways and mechanisms of multidrug resistance.

Efficacy and safety of an innovative short-course regimen containing clofazimine for treatment of drug-susceptible tuberculosis: a clinical trial.

In preclinical studies, a new antituberculosis drug regimen markedly reduced the time required to achieve relapse-free cure. This study aimed to preliminarily evaluate the efficacy and safety of this four-month regimen, consisting of clofazimine, prothionamide, pyrazinamide and ethambutol, with a standard six-month regimen in patients with drug-susceptible tuberculosis. An open-label pilot randomized clinical trial was conducted among the patients with newly diagnosed bacteriologically-confirmed pulmonary tuberculosis. The primary efficacy end-point was sputum culture negative conversion. Totally, 93 patients were included in the modified intention-to-treat population. The rates of sputum culture conversion were 65.2% (30/46) and 87.2% (41/47) for short-course and standard regimen group, respectively. There was no difference on two-month culture conversion rates, time to culture conversion, nor early bactericidal activity (P > 0.05). However, patients on short-course regimen were observed with lower rates of radiological improvement or recovery and sustained treatment success, which was mainly attributed to higher percent of patients permanently changed assigned regimen (32.1% vs. 12.3%, P = 0.012). The main cause for it was drug-induced hepatitis (16/17). Although lowering the dose of prothionamide was approved, the alternative option of changing assigned regimen was chosen in this study. While in per-protocol population, sputum culture conversion rates were 87.0% (20/23) and 94.4% (34/36) for the respective groups. Overall, the short-course regimen appeared to have inferior efficacy and higher incidence of hepatitis but desired efficacy in per-protocol population. It provides the first proof-of-concept in humans of the capacity of the short-course approach to identify drug regimens that can shorten the treatment time for tuberculosis.

Antimicrobial Susceptibility Testing Using the MYCO Test System and MIC Distribution of 8 Drugs against Clinical Isolates of Nontuberculous Mycobacteria from Shanghai.

Given the increased incidence and prevalence of nontuberculous mycobacterial (NTM) diseases and the natural resistance of NTM to multiple antibiotics, in vitro susceptibility testing of different NTM species against drugs from the MYCO test system and new applied drugs is required. A total of 241 NTM clinical isolates were analyzed, including 181 slowly growing mycobacteria (SGM) and 60 rapidly growing mycobacteria (RGM). The Sensititre SLOMYCO and RAPMYCO panels were used for testing susceptibility to commonly used anti-NTM antibiotics. Furthermore, MIC distributions were determined against 8 potential anti-NTM drugs, including vancomycin (VAN), bedaquiline (BDQ), delamanid (DLM), faropenem (FAR), meropenem (MEM), clofazimine (CLO), cefoperazone-avibactam (CFP-AVI), and cefoxitin (FOX), and epidemiological cutoff values (ECOFFs) were analyzed using ECOFFinder. The results showed that most of the SGM strains were susceptible to amikacin (AMK), clarithromycin (CLA), and rifabutin (RFB) from the SLOMYCO panels and BDQ and CLO from the 8 applied drugs, while RGM strains were susceptible to tigecycline (TGC) from the RAPMYCO panels and also BDQ and CLO. The ECOFFs of CLO were 0.25, 0.25, 0.5, and 1 µg/mL for the mycobacteria M. kansasii, M. avium, M. intracellulare, and M. abscessus, respectively, and the ECOFF of BDQ was 0.5 µg/mL for the same four prevalent NTM species. Due to the weak activity of the other 6 drugs, no ECOFF was determined. This study on the susceptibility of NTM includes 8 potential anti-NTM drugs and a large sample size of Shanghai clinical isolates and demonstrates that BDQ and CLO had efficient activities against different NTM species in vitro, which can be applied to the treatment of NTM diseases. IMPORTANCE We designed customized panel that contains 8 repurposed drugs, including vancomycin (VAN), bedaquiline (BDQ), delamanid (DLM), faropenem (FAR), meropenem (MEM), clofazimine (CLO), cefoperazone-avibactam (CFP-AVI), and cefoxitin (FOX) from the MYCO test system. To better understand the efficacy of these 8 drugs against different NTM species, we determined the MICs of 241 NTM isolates collected in Shanghai, China. We attempted to define the tentative epidemiological cutoff values (ECOFFs) for the most prevalent NTM species, which is an important factor in setting up the breakpoint for a drug susceptibility testing. We used the MYCO test system as an automatic quantitative drug sensitivity test of NTM and extended the method to BDQ and CLO in this study. The MYCO test system complements commercial microdilution systems that currently lack BDQ and CLO detection.

In vitro Synergism of Six Antituberculosis Agents Against Drug-Resistant Mycobacterium tuberculosis Isolated from Retreatment Tuberculosis Patients.

BACKGROUND: Retreatment tuberculosis (TB) has become a major source of drug-resistant TB. In contrast to the combination of isoniazid (INH) and rifampicin (RIF), that of pasiniazid (Pa) and rifabutin (RFB) or rifapentine (RFP) appears to have better activity in vitro against drug-resistant Mycobacterium tuberculosis (MTB), especially when combined with moxifloxacin (MXF). However, there has been limited study of potential synergism among Pa, RFB, RFP, and MXF, or simultaneous comparison with the standard INH and RIF combination. METHODS: In vitro synergism of four two-drug combinations (INH and RIF, Pa and RFB, Pa and RFP, MXF and Pa) and two three-drug combinations (MXF and Pa combined with RFB or RFP) was evaluated against 90 drug-resistant MTB strains isolated from retreatment TB patients by the checkerboard method. The fractional inhibitory concentration index (FICI) was calculated for each combination. RESULTS: The synergistic activity of the combination of Pa with RFB or RFP was higher than that of INH and RIF or MXF and Pa, and the synergistic activity of Pa in combination with RFP was even higher than that of RFB, although RFP yielded an MIC90 of 64 mg/liter, higher than that of RFB of 8 mg/liter against 90 drug-resistant MTB strains. Meanwhile, for three-drug combinations, the synergistic effects of MXF and Pa combined with RFB or RFP were similar. Further stratification analysis showed that, for XDR-MTB strains, the synergistic effect of the Pa and RFP combination was also better than those of other two-drug combinations. CONCLUSION: The combination of Pa with RFP shows better in vitro synergism than Pa with RFB and standard INH with RIF combinations, which can provide a reference for new regimens for retreatment TB patients.

TB-QUICK: CRISPR-Cas12b-assisted rapid and sensitive detection of Mycobacterium tuberculosis.

OBJECTIVES: Tuberculosis (TB) remains one of the public health problems worldwide. Rapid, sensitive and cost-effective diagnosis of Mycobacterium tuberculosis (M.tb) is critical for TB control. METHODS: We developed a novel M.tb DNA detection platform (nominated as TB-QUICK) which combined loop-mediated isothermal amplification (LAMP) and CRISPR-Cas12b detection. TB-QUICK was performed on pulmonary or plasma samples collected from 138 pulmonary TB (PTB) patients, 21 non-TB patients and 61 close contacts to TB patients. Acid-fast bacillus (AFB) smear, M.tb culture and GeneXpert MTB/RIF (Xpert) assays were routinely conducted in parallel. RESULTS: By targeting M.tb IS6110, TB-QUICK platform could detect as low as 1.3 copy/µL M.tb DNA within 2 h. In pulmonary TB samples, TB-QUICK exhibited improved overall sensitivity of 86.8% over M.tb culture (66.7%) and Xpert (70.4%), with the specificity of 95.2%. More significantly, TB-QUICK exhibited a superior sensitivity in AFB-negative samples (80.5%) compared to Xpert (57.1%) and M.tb culture (46.2%). In the detection of plasma M.tb DNA by TB-QUICK, 41.2% sensitivity for AFB-positive and 31.7% for AFB-negative patients were achieved. CONCLUSION: In conclusion, TB-QUICK exhibits rapidity and sensitivity for M.tb DNA detection with the superiority in smear-negative paucibacillary TB patients. The clinical application of TB-QUICK in TB diagnosis needs to be further validated in larger cohort.