Mycobacterium Tuberculosis Research Articles

A pH-dependent direct sulfhydrylation pathway is required for the pathogenesis of Mycobacterium tuberculosis

Methionine is essential for the growth and survival of Mycobacterium tuberculosis (M. tuberculosis), however, the canonical transsulfuration pathway involved in the biosynthesis of methionine is dispensable, suggesting redundancy. This study explores the presence of an ortholog of O-succinyl homoserine sulfhydrylase in M. tuberculosis, which catalyses direct sulfhydrylation for methionine biosynthesis. Bioinformatics analysis of putative O-succinyl homoserine sulfhydrylase encoded by metZ in M. tuberculosis showed similarities with its orthologues in other bacterial species. Here, we show that metZ deletion in M. tuberculosis resulted in impaired growth under acidic conditions, which was reversed by methionine supplementation. Molecular dynamics simulation studies revealed improved binding of substrate, O-succinyl homoserine, to the active site of MetZ at low pH mimicking the phagosomal microenvironment. Intriguingly, despite higher ATP levels, metZ deletion reduced the frequency of Bedaquiline-induced persister formation. Finally, we demonstrate that loss of metZ hinders M. tuberculosis growth inside the host.

Exploration of 3-substituted benzofuran-tethered sulfamoyl triazoles as carbonic anhydrase inhibitors: Design, synthesis and biological evaluation.

This study reports the design and synthesis of benzofuran-tethered sulfamoyl triazoles. These compounds were evaluated against two human carbonic anhydrases (hCA I and II) and three carbonic anhydrases from the bacterial pathogen Mycobacterium TB, (mtCA1-3). The findings indicated that molecules featuring triazolyl benzene-3-sulfonamide were significantly more selective for mtCA 1-3 than hCA I and II. In contrast, across the generated molecules, benzofuran with triazolyl benzene-4-sulfonamide demonstrated greater selectivity for hCA II than mtCAs. Among these compounds, 3F4 showed the greatest suppression of mtCA2, with a Ki value of 0.0052 µM. 4F1 demonstrated essential and focused inhibition of hCA II, with a Ki value of 0.0094 µM, Compound 3F4 is 439 times more selective to mtCA2 than hCA I, and 47 times more selective than hCAII. Additionally, when examined for antitubercular activity, these compounds showed MIC values for Mtb H37Rv strain inhibition in the range of moderate to good with 4-128 µg/mL.

Assessment of Healthcare Professionals' Knowledge Regarding Nontuberculosis Mycobacterial Infections at Debre Tabor Comprehensive Specialized Hospital, Northwest Ethiopia, 2024: A Cross-Sectional Study.

Nontuberculosis mycobacteria (NTM) are environmental pathogens that can cause pulmonary infections, especially among the immunocompromised population. There is limited research on healthcare professionals' knowledge of NTM infections in Ethiopia. This study aimed to evaluate healthcare professionals' knowledge about NTM infections at Debre Tabor Comprehensive Specialized Hospital (DTCSH). A cross-sectional study was conducted from March 5 to July 30, 2024 at DTCSH to evaluate the knowledge of 292 healthcare professionals on NTM infections. A semi-structured questionnaire was used for data collection, and the data were analyzed using SPSS version 25. The study's findings were presented through texts, tables, and figures. Of all the participants, 160 (54.8%) were male, and the majority 153 (52.4%) were 36-45 years old. Nurses made up the largest professional group, 127 (43.5%). Approximately 59% of healthcare professionals showed good knowledge of NTM infections. In this study, a higher percentage of female [92 (69.7%)] participants demonstrated good knowledge compared to males [83 out of 160 males (51.9%)]. Similarly, 35 doctors (89.7%) and 80 nurses (63%) demonstrated favorable knowledge. Only 52.4% could distinguish between NTM and Mycobacterium tuberculosis infections. Notably, 77.4% recognized that NTM infections are more common in immunocompromised individuals, yet only 20.5% knew that NTM species are not typically treated with anti-tuberculosis drugs. The knowledge gaps among healthcare professionals regarding NTM infections necessitate focused educational initiatives to improve their ability to manage these infections. The focused training is essential for enhancing the management and identification of NTM infections.

Verapamil and its metabolite norverapamil inhibit the Mycobacterium tuberculosis MmpS5L5 efflux pump to increase bedaquiline activity

Bedaquiline is the cornerstone of a new regimen for the treatment of drug-resistant tuberculosis. However, its clinical use is threatened by the emergence of bedaquiline-resistant strains of Mycobacterium tuberculosis. Bedaquiline targets mycobacterial ATP synthase but the predominant route to clinical bedaquiline resistance is via upregulation of the MmpS5L5 efflux pump due to mutations that inactivate the transcriptional repressor Rv0678. Here, we show that the MmpS5L5 efflux pump reduces susceptibility to bedaquiline as well as its new, more potent derivative TBAJ-876 and other antimicrobial substrates, including clofazimine and the DprE1 inhibitors PBTZ-169 and OPC-167832. Furthermore, the increased resistance of Rv0678 mutants stems entirely from increased MmpS5L5 expression. These results highlight the potential of a pharmacological MmpS5L5 inhibitor to increase drug efficacy. Verapamil, primarily used as a calcium channel inhibitor, is known to inhibit diverse efflux pumps and to potentiate bedaquiline and clofazimine activity in M. tuberculosis. Here, we show that verapamil potentiates the activity of multiple diverse MmpS5L5 substrates. Using biochemical approaches, we demonstrate that verapamil does not exert this effect by acting as a disruptor of the protonmotive force used to power MmpS5L5, as previously proposed, suggesting that verapamil inhibits the function of the MmpS5L5 pump. Finally, norverapamil, the major verapamil metabolite, which has greatly reduced calcium channel activity, has equal potency in reducing resistance to MmpS5L5 substrates. Our findings highlight verapamil's potential for enhancing bedaquiline TB treatment, for preventing acquired resistance to bedaquiline and other MmpS5L5 substrates, while also providing the impetus to identify additional MmpS5L5 inhibitors.

A new target for drug repositioning: CEBPα elicits LL-37 expression in a vitamin D-independent manner promoting Mtb clearance.

A new target for drug repositioning: CEBPα elicits LL-37 expression in a vitamin D-independent manner promoting Mtb clearance.

A prospective study on enhanced recovery of Mycobacterium tuberculosis from children’s stool using novel power ultrasound decontamination

Stool specimen is commonly used for mycobacterial culture to provide critical microbiological evidence for diagnosing tuberculosis (TB) in pediatric patients, especially when obtaining sputum specimens is challenging. However, the utilization of stool specimens for mycobacterial culture continues to face significant challenges in clinical practice, mainly owing to the relatively low diagnostic sensitivity and the frequent contamination by intestinal flora during specimen processing. This study evaluated the effectiveness of power ultrasound (PU) method in improving the recovery of Mycobacterium tuberculosis (M. TB) from stool specimens collected from children suspected of pulmonary tuberculosis (PTB). A total of 252 stool specimens were collected, and each specimen was subsequently divided into approximately three equal groups. One stool specimen was subjected to direct acid-fast bacilli (AFB) smear microscopy, while the other two stool specimens were processed using different decontamination methods(NaOH-NALC method vs. PU method). Out of the 252 specimens, positive cultures were observed in 14 (5.56%) stool specimens treated with the NaOH-NALC method, including M. TB (n = 11, 4.37%) and non-tuberculous mycobacteria (NTM; n = 3, 1.19%). Similarly, among the stool specimens subjected to the PU method, culture positivity was found in 36 (14.28%), comprising of M. TB (n = 24, 9.52%) and NTM (n = 12, 4.76%). The statistical analysis revealed a significant difference in the isolation rate of M. TB and NTM between the two methods (P<0.001). Furthermore, the contamination rate of stool treated with the PU method (14.28%) is significantly lower than that of the NALC-NaOH method (22.62%). In conclusion, our findings indicate that the PU method is a rapid and effective decontamination method for stool in children, improving the detection of active TB among pediatric populations when compared to the NALC-NaOH method.

Corrigendum: Characterizing the immune response to Mycobacterium tuberculosis: a comprehensive narrative review and implications in disease relapse

Corrigendum: Characterizing the immune response to Mycobacterium tuberculosis: a comprehensive narrative review and implications in disease relapse

Value of contrast-enhanced ultrasound assisted core needle biopsy in the diagnosis of cervical lymph node tuberculosis

AimTo investigate the value of contrast-enhanced ultrasound (CEUS) assisted core needle biopsy (CNB) in the diagnosis of cervical lymph node tuberculosis (LN TB) and improve the positive rate of cervical LN TB.MethodsWe retrospectively analyzed 730 samples obtained from July 2010 to January 2025 from patients treated with effective antituberculosis therapy and with microbiologically confirmed and surgical pathologically proven cervical lymph node enlargement who had undergone CEUS- CNB at our hospital. All patients were divided into two groups according to the historical control method. The CEUS group (2017–2025) underwent CEUS- CNB (485 cases), whereas the US group (2010–2018) underwent US-guided CNB (245 cases). The positive rates of pathological diagnosis and Xpert Mycobacterium tuberculosis complex (MTBC) and resistance to rifampin (RIF) (MTB/RIF) assay diagnoses were compared between the groups.ResultsThe specimens’ integrity was significantly higher after CNB in the CEUS group than in the US group (CEUS group: 72.30%; US group: 45.49%), and visual satisfaction of sampling in the CEUS group was higher (χ2: 47.651, P &amp;lt; 0.001). Histopathological examination sensitivity, specificity, positive predictive value, and negative predictive value were higher in the CEUS group than in the US group. The sensitivity of the Xpert MTB/RIF assay was significantly higher in the CEUS group than in the US group.ConclusionThe study results support the clinical use of CEUS for improving the diagnostic performance and positive rate for cervical LN TB.

Novel quinazolinones active against multidrug-resistant Mycobacterium tuberculosis: synthesis, antimicrobial evaluation and in silico exploration of PonA1 as a potential target.

Quinazolinone derivatives have emerged as promising scaffolds in antimicrobial drug discovery. This work focuses on the design, synthesis, and evaluation of novel quinazolinone-based compounds and predicts their potential to interact with mycobacterial penicillin-binding proteins (PBPs). Relying on established structure-activity relationships of antibacterial quinazolinones, a total of 53 compounds belonging to three different structural types were synthesized and biologically evaluated for antimycobacterial, antibacterial, and antifungal activities. Biological evaluations revealed selective efficacy against Mycobacterium tuberculosis with minimum inhibitory concentrations (MICs) as low as 6.25 µg/mL for some derivatives, and this activity was preserved against drug-resistant strains. Molecular docking studies suggested a potential allosteric binding site in mycobacterial Penicillin-binding protein 1A (PonA1, UniProt ID: P71707), and subsequential molecular dynamics confirmed stable binding with key stabilizing interaction between the carbonyl oxygen of the quinazolinone and either ARG399 or ASP474. These findings suggest quinazolinone derivatives as viable candidates for further development as non-β-lactam PBP inhibitors, addressing the urgent need for new antitubercular therapies.

The role of circular RNA in immune response to tuberculosis and its potential as a biomarker and therapeutic target

Circular RNA (circRNA) is a new type of non-coding RNA that has gained significant attention in recent years, especially in tuberculosis research. Tuberculosis poses a major global public health threat. Its complex pathological mechanisms and worsening drug resistance urgently necessitate new research breakthroughs. The role of circRNA in mycobacterium tuberculosis infection is being gradually revealed, highlighting its importance in regulating gene expression, immune response, and inflammation. Additionally, researchers are interested in circRNA because of its potential for early tuberculosis diagnosis and its role as a biomarker. This article systematically analyzes existing literature to provide new insights into early tuberculosis diagnosis and personalized treatment. We also emphasize the need for future research to enhance the application of circRNA in tuberculosis prevention and control.

Proteomic characterization of Mycobacterium tuberculosis subjected to carbon starvation.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), the leading cause of infectious disease-related deaths worldwide. TB infections present on a spectrum from active to latent disease. In the human host, Mtb faces hostile environments, such as nutrient deprivation, hypoxia, and low pH. Under these conditions, Mtb can enter a dormant, but viable, state characterized by a lack of cell replication and increased resistance to antibiotics. Dormant Mtb poses a major challenge to curing infections and eradicating TB globally. We subjected Mtb mc26020 (ΔlysA and ΔpanCD), a double auxotrophic strain, to carbon starvation (CS), a culture condition that induces growth stasis and mimics environmental conditions associated with dormancy in vivo. We provide a detailed analysis of the proteome in CS compared to replicating samples. We observed extensive proteomic reprogramming, with 36% of identified proteins significantly altered in CS. Many enzymes involved in oxidative phosphorylation and lipid metabolism were retained or more abundant in CS. The cell wall biosynthetic machinery was present in CS, although numerous changes in the abundance of peptidoglycan, arabinogalactan, and mycolic acid biosynthetic enzymes likely result in pronounced remodeling of the cell wall. Many clinically approved anti-TB drugs target cell wall biosynthesis, and we found that these enzymes were largely retained in CS. Lastly, we compared our results to those of other dormancy models and propose that CS produces a physiologically distinct state of stasis compared to hypoxia in Mtb.IMPORTANCETuberculosis is a devastating human disease that kills over 1.2 million people a year. This disease is caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb). Mtb excels at surviving in the human host by entering a non-replicating, dormant state. The current work investigated the proteomic changes that Mtb undergoes in response to carbon starvation, a culture condition that models dormancy. The authors found broad effects of carbon starvation on the proteome, with the relative abundance of 37% of proteins significantly altered. Protein changes related to cell wall biosynthesis, metabolism, and drug susceptibility are discussed. Proteins associated with a carbon starvation phenotype are identified, and results are compared to other dormancy models, including hypoxia.

Comprehensive Whole Genome Sequencing Dataset of Mycobacterium tuberculosis Strains Collected Across Italy

Tuberculosis (TB), caused by the Mycobacterium tuberculosis complex (MTBC), remains a major global health challenge. Whole genome sequencing (WGS) offers an invaluable tool for understanding the genetic diversity and drug resistance profiles of MTBC. This study provides a comprehensive WGS dataset of 2,520 MTBC isolates collected from four Italian regions—Lombardy, Piedmont, Emilia-Romagna, and Lazio—between 2017 and 2020. The dataset includes genomic data along with associated metadata, such as geographic location and drug susceptibility profiles, providing a robust resource for studying TB epidemiology and transmission dynamics. This collection represents the largest publicly available MTBC WGS dataset from Italy and has been validated to ensure accuracy and completeness. By making this dataset accessible, we aim to support collaborative research, facilitate the exploration of MTBC evolution and drug resistance, and enhance TB surveillance efforts.

Live-attenuated Mycobacterium tuberculosis vaccine, MTBVAC, in adults with or without M tuberculosis sensitisation: a single-centre, phase 1b-2a, double-blind, dose-escalation, randomised controlled trial.

Live-attenuated Mycobacterium tuberculosis vaccine, MTBVAC, in adults with or without M tuberculosis sensitisation: a single-centre, phase 1b-2a, double-blind, dose-escalation, randomised controlled trial.

Combating TB Pathology: Advanced Diagnostics, Innovative Therapies, and Public Health Strategies: A Review.

Tuberculosis (TB) continues to be a major global health challenge, largely due to the complex nature of Mycobacterium tuberculosis. Its early detection and effective management are heavily reliant on advanced diagnostic methods. New drug delivery systems and repurposing existing drugs show great promise in improving TB treatment. This study explores the progress and hurdles in developing anti-TB drugs, focusing on those currently in clinical trials. Additionally, innovative approaches like immunotherapy, combination therapy, and adjunct therapy, which include the use of phytochemicals, are examined for their potential to enhance treatment outcomes and tackle drug resistance. These innovative approaches could be the key to the future of the fight against TB. It also highlights how these strategies could accelerate TB treatment. It incorporates public health strategies for preventing TB transmission and ensuring patients adhere to treatments. By addressing these key areas, this work aims to contribute to the global fight against TB and improve the lives of those affected by the disease.

Establishment of minimum protein standards for Mycobacterium tuberculosis-derived extracellular vesicles through comparison of EV enrichment methods

Mycobacterium tuberculosis extracellular vesicles (MEV) have been described as having potent immunological activities that are both beneficial and harmful to the host. Key to understanding this conflicting information is the proteomic characterization of MEVs. However, there is neither a standard for a purification method nor markers to assess relative purity and quality of MEVs. In this study, we purified MEVs by four different methods (simple ultracentrifugation, differential density gradient-based ultracentrifugation, qEV size exclusion chromatography, and Capto™Core size exclusion chromatography) and assessed the variability of MEV characteristics (size, concentration, appearance, purity, and protein content) amongst isolation methods. The vesicle appearance and size were consistent across all methods; however variability was found between and within all methods, with simple ultracentrifugation demonstrating the most variability both in reproducibility and purity. Protein concentration and content, and particle yield and purity, varied amongst all methods. The two size exclusion chromatography-based methods were more technically reproducible than either ultracentrifugation-based method, while qEV size exclusion chromatography and differential density gradient ultracentrifugation afforded MEV samples of the highest purity. Nonetheless, all methods had 7 proteins in common, the Sec-independent membrane bound twin-arginine translocase TatA (Rv2094c), the periplasmic phosphate-binding lipoprotein PstS3 (Rv0928), the heparin binding hemagglutinin HBHA (Rv0475), lipoprotein antigens LprG (Rv1411c) and LpqH (Rv3763), a member of the conserved 13E12 repeat protein family P95201 (Rv0393), and the tuberculin related peptide Rv0431 (P96277), suggesting the use of these proteins as qualitative markers of MEVs versus contaminants, in addition to size and appearance criteria, to benefit reproducibility and consensus for ongoing MEV studies.

Bird's Eye View on Mycobacterium tuberculosis-HIV Coinfection: Understanding the Molecular Synergism, Challenges, and New Approaches to Therapeutics.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), is the most common secondary infection in the Human Immunodeficiency Virus (HIV) infected population, accounting for more than one-fourth of deaths in people living with HIV (PLWH). Reciprocally, HIV infection increases the susceptibility to primary TB or reactivation of latent TB by several folds. The synergistic interactions between M.tb and HIV not only potentiate their deleterious impact but also complicate the clinical management of both the diseases. M.tb-HIV coinfected patients have a high risk of failure of accurate diagnosis, treatment inefficiency for both TB and HIV, concurrent nontuberculous mycobacterial infections, other comorbidities such as diabetes mellitus, severe cytotoxicity due to drug overburden, and immune reconstitution inflammatory syndrome (IRIS). The need of the hour is to understand M.tb-HIV coinfection biology and their collective impact on the host immunocompetence and to think of out-of-the-box treatment perspectives, including host-directed therapy under the rising view of homeostatic medicines. This review aims to highlight the molecular players, both from the pathogens and host, that facilitate the synergistic interactions and host-associated proteins/enzymes regulating immunometabolism, underlining potential targets for designing and screening chemical inhibitors to reduce the burden of both pathogens concomitantly during M.tb-HIV coinfection. To appreciate the necessity of revisiting therapeutic approaches and research priorities, we provide a glimpse of anti-TB and antiretroviral drug-drug interactions, project the gaps in our understanding of coinfection biology, and also enlist some key research initiatives that will help us deal with the synergistic epidemic of M.tb-HIV coinfection.

A Photoactivatable Free Mycolic Acid Probe to Investigate Mycobacteria-Host Interactions.

Mycolic acids are long-chain, α-branched, β-hydroxylated fatty acid lipids that populate the outer mycomembrane of mycobacteria, including the pathogen Mycobacterium tuberculosis. Mycolic acids predominantly occur in the form of glycolipids, but nonglycosylated free mycolic acids (fMA), which are generated during mycomembrane remodeling, are major constituents of the M. tuberculosis biofilm extracellular matrix and promote host immune evasion during M. tuberculosis infection. However, our understanding of these processes is nascent, and there is limited information about the fMA-protein interactions involved. To facilitate such studies, we synthesized a fMA analogue probe (x-Alk-MA) containing a photo-cross-linking diazirine and a clickable alkyne to enable live-cell capture and analysis of protein interactors. The synthetic strategy featured asymmetric hydrogenation to establish the β-hydroxy group, diastereoselective alkylation to establish the α-branch, and late-stage modification to install the functional tags. In macrophages, x-Alk-MA recapitulated the cytokine response of native MA and selectively photolabeled TREM2, a host cell receptor for fMAs that suppresses macrophage activation and has been implicated in M. tuberculosis immune evasion. The synthetic strategy, chemical probes, and photolabeling methods disclosed herein should facilitate future studies aimed at understanding the roles of fMA in mycobacterial physiology and pathogenesis.

New Advances in the Development and Design of Mycobacterium tuberculosis Vaccines: Construction and Validation of Multi-Epitope Vaccines for Tuberculosis Prevention

Mycobacterium tuberculosis (Mtb) vaccines are designed to prevent infection, prevent reactivation of latent infection, and/or provide adjuvant therapy to standard TB treatment for active Mtb. Emerging vaccine technologies include reverse vaccinology, DNA and RNA vaccines, subunit vaccines, and multi-epitope vaccines. Currently, many different types of vaccine candidates are in clinical trials, though, to date, BCG remains the only approved Mtb vaccine. Mtb has a complex genome with numerous antigens, but not all are equally effective in eliciting immunity, so a critical challenge is the selection of antigens and epitopes that are most likely to induce a long-term, broad-spectrum protective immune response. Multi-epitope vaccines (MEVs) represent a new event horizon in vaccine development. Bioinformatic computer modeling is being used to maximize efficacy and minimalize adverse effects. Although no multi-epitope vaccines have proceeded to in vivo clinical trials, three candidate MEVs have made it through in silico tests. Multi-epitope vaccine candidate PP13138R, containing 13 HTL epitopes, 13 CTL epitopes, and 8 B cell epitopes in addition to both TLR2 and TLR4 agonists, aims to elicit a broad immune response that could address both active and latent Mtb infection. Similarly, immunoinformatic data were used to design and validate another MEV candidate based on the biomarker PE_PGRS17 with four B cell, nine HTL, and six CTL linked epitopes, with a griselimycin sequence as the adjuvant. A third novel prophylactic and therapeutic MEV was developed that targets Ag85A, AG85B, ESAT-6, and CFP-10 proteins with 12 CTL, 25 HTL, and 21 LBL epitopes with a CpG adjuvant.

Serine protease Rv2569c inhibits inflammatory response and promotes intracellular survival of Mycobacterium tuberculosis by targeting the RhoG-NF-κB-NLRP3 pathway.

Serine protease Rv2569c inhibits inflammatory response and promotes intracellular survival of Mycobacterium tuberculosis by targeting the RhoG-NF-κB-NLRP3 pathway.

Linezolid administration to lactating Wistar rats affects the health of their offspring.

Lactational exposure to antibacterial medications may affect the normal development of newborns during this crucial stage and later in adult life. Linezolid (LNZ) is an oxazolidinone antibacterial drug that is effective against drug-resistant Gram-positive bacteria and multidrug-resistant Mycobacterium tuberculosis. Although it is relatively toxic, there is insufficient data about LNZ use during lactation. This study aimed to elucidate the impact of linezolid administration during lactation on Wistar rats' offspring. Eighteen lactating Wistar female rats were separated into three groups (n = 6): control, therapeutic, and low dose groups. The therapeutic dose group received 61.66 mg/kg of LNZ (equivalent to the human dose), while the low dose group received 15.41 mg/kg of LNZ (1/4 of the human therapeutic dose) by gavage twice daily. All lactating dams and their offspring died fourdays after receiving a therapeutic dose. In the low dose group, LNZ significantly reduced the body weight of lactating females and their pups. The liver tissue of the pups showed a considerable increase in malondialdehyde levels, along with a decrease in the catalase, glutathione, and superoxide dismutase activities accompanied by moderate histological alterations like congestion, and infiltration, and DNA fragmentation as indicated by comet assay. Microscopic examination of renal tissue revealed glomeruli deterioration, cellular infiltration, and intratubular protein deposits. In conclusion, this study highlights the potential risks linezolid may pose to infants during postpartum. Therefore, there is a need for preweaning monitoring and caution should be taken during breastfeeding.