Evaluation of cytokine profiles related to Mycobacterium tuberculosis latent antigens using a whole-blood assay in the Philippines.

Introduction: There is no useful method to discriminate between latent tuberculosis infection (LTBI) and active pulmonary tuberculosis (PTB). This study aimed to investigate the potential of cytokine profiles to discriminate between LTBI and active PTB using whole-blood stimulation with Mycobacterium tuberculosis (MTB) antigens, including latency-associated antigens. Materials and methods: Patients with active PTB, household contacts of active PTB patients and community exposure subjects were recruited in Manila, the Philippines. Peripheral blood was collected from the participants and used for whole-blood stimulation (WBS) with either the early secretory antigenic target and the 10-kDa culture filtrate protein (ESAT-6/CFP-10), Rv3879c or latency-associated MTB antigens, including mycobacterial DNA-binding protein 1 (MDP-1), α-crystallin (Acr) and heparin-binding hemagglutinin (HBHA). Multiple cytokine concentrations were analyzed using the Bio-Plex™ multiplex cytokine assay. Results: A total of 78 participants consisting of 15 active PTB patients, 48 household contacts and 15 community exposure subjects were eligible. The MDP-1-specific IFN-γ level in the active PTB group was significantly lower than that in the household contact group (p < 0.001) and the community exposure group (p < 0.001). The Acr-specific TNF-α and IL-10 levels in the active PTB group were significantly higher than those in the household contact (TNF-α; p = 0.001, IL-10; p = 0.001) and community exposure (TNF-α; p < 0.001, IL-10; p = 0.01) groups. However, there was no significant difference in the ESAT-6/CFP-10-specific IFN-γ levels among the groups. Conclusion: The patterns of cytokine profiles induced by latency-associated MTB antigens using WBS have the potential to discriminate between LTBI and active PTB. In particular, combinations of IFN-γ and MDP-1, TNF-α and Acr, and IL-10 and Acr are promising. This study provides the first demonstration of the utility of MDP-1-specific cytokine responses in WBS.

[Prokaryotic Expression and Bioinformatic Analysis of Rv3432c From Mycobacterium tuberculosis]. / ç»“æ ¸åˆ†æžæ†èŒRv3432cè›‹ç™½çš„åŽŸæ ¸è¡¨è¾¾ä¸Žç”Ÿç‰©ä¿¡æ¯å­¦åˆ†æž.

Objective: To express the protein enconded by the Rv3432c gene of Mycobacterium tuberculosis (M.tb) in vitro by prokaryotic expression, to analyze the structure of the Rv3432c protein by using bioinformatics software, and to explore for new drug targets against M.tb. Methods: The Rv3432c gene was amplified by PCR using the genomic DNA of the inactivated M.tb strain H37Rv as the template and a recombinant plasmid was constructed with the expression vector pET-28a. The expression products were analyzed by SDS-PAGE and purified using affinity chromatography. The biological properties of Rv3432c were analyzed with Protparam, the Pfam online tool, SOMPA, Protscale, TMHMM Signalp 6.0, NetPhos3.1, SUMOsp 2.0, and SWISS-MODEL. Results: pET-28a-Rv3432c recombinant plasmid sequencing results were fully consistent with those of the target gene. SDS-PAGE analysis showed that the fusion protein existed in the form of a soluble protein with a relative molecular mass of about 55×103, which matched the expected size. ProtParam analysis showed that the Rv3432c protein was hydrophilic (showing a GRAVY value of －0.079). Rv3432c was a protein with no transmembrane structural domains or signal peptide. The secondary structure of Rv3432c mainly consisted of random coils (39.78%) and α-helix (39.57%) and was relatively loosely structured. Conclusion: We successfully constructed a prokaryotic expression plasmid of the Rv3432c protein and analyzed its structure using bioinformatics, laying the foundation for further research on the role of Rv3432c in the pathogenesis and progression of tuberculosis as well as the identification of new drug targets against M.tb.

Uncovering the roles of Mycobacterium tuberculosis melH in redox and bioenergetic homeostasis: implications for antitubercular therapy.

Mycobacterium tuberculosis (Mtb), the pathogenic bacterium that causes tuberculosis, has evolved sophisticated defense mechanisms to counteract the cytotoxicity of reactive oxygen species (ROS) generated within host macrophages during infection. The melH gene in Mtb and Mycobacterium marinum (Mm) plays a crucial role in defense mechanisms against ROS generated during infection. We demonstrate that melH encodes an epoxide hydrolase and contributes to ROS detoxification. Deletion of melH in Mm resulted in a mutant with increased sensitivity to oxidative stress, increased accumulation of aldehyde species, and decreased production of mycothiol and ergothioneine. This heightened vulnerability is attributed to the increased expression of whiB3, a universal stress sensor. The absence of melH also resulted in reduced intracellular levels of NAD+, NADH, and ATP. Bacterial growth was impaired, even in the absence of external stressors, and the impairment was carbon source dependent. Initial MelH substrate specificity studies demonstrate a preference for epoxides with a single aromatic substituent. Taken together, these results highlight the role of melH in mycobacterial bioenergetic metabolism and provide new insights into the complex interplay between redox homeostasis and generation of reactive aldehyde species in mycobacteria. IMPORTANCE: This study unveils the pivotal role played by the melH gene in Mycobacterium tuberculosis and in Mycobacterium marinum in combatting the detrimental impact of oxidative conditions during infection. This investigation revealed notable alterations in the level of cytokinin-associated aldehyde, para-hydroxybenzaldehyde, as well as the redox buffer ergothioneine, upon deletion of melH. Moreover, changes in crucial cofactors responsible for electron transfer highlighted melH's crucial function in maintaining a delicate equilibrium of redox and bioenergetic processes. MelH prefers epoxide small substrates with a phenyl substituted substrate. These findings collectively emphasize the potential of melH as an attractive target for the development of novel antitubercular therapies that sensitize mycobacteria to host stress, offering new avenues for combating tuberculosis.

Mincle as a potential intervention target for the prevention of inflammation and fibrosis (Review).

Macrophage­inducible C­type lectin receptor (Mincle) is predominantly found on antigen­presenting cells. It can recognize specific ligands when stimulated by certain pathogens such as fungi and Mycobacterium tuberculosis. This recognition triggers the activation of the nuclear factor­κB pathway, leading to the production of inflammatory factors and contributing to the innate immune response of the host. Moreover, Mincle identifies lipid damage­related molecules discharged by injured cells, such as Sin3­associated protein 130, which triggers aseptic inflammation and ultimately hastens the advancement of renal damage, autoimmune disorders and malignancies by fostering tissue inflammation. Presently, research on the functioning of the Mincle receptor in different inflammatory and fibrosis­associated conditions has emerged as a popular topic. Nevertheless, there remains a lack of research on the impact of Mincle in promoting long­lasting inflammatory reactions and fibrosis. Additional investigation is required into the function of Mincle receptors in chronological inflammatory reactions and fibrosis of organ systems, including the progression from inflammation to fibrosis. Hence, the present study showed an overview of the primary roles and potential mechanism of Mincle in inflammation, fibrosis, as well as the progression of inflammation to fibrosis. The aim of the present study was to clarify the potential mechanism of Mincle in inflammation and fibrosis and to offer perspectives for the development of drugs that target Mincle.

IL-10 and TGF-ß1 may weaken the efficacy of preoperative anti-tuberculosis therapy in older patients with spinal tuberculosis.

Spinal tuberculosis is a common extrapulmonary type that is often secondary to pulmonary or systemic infections. Mycobacterium tuberculosis infection often leads to the balance of immune control and bacterial persistence. In this study, 64 patients were enrolled and the clinicopathological and immunological characteristics of different age groups were analyzed. Anatomically, spinal tuberculosis in each group mostly occurred in the thoracic and lumbar vertebrae. Imaging before preoperative anti-tuberculosis therapy showed that the proportion of abscesses in the older group was significantly lower than that in the younger and middle-aged groups. However, pathological examination of surgical specimens showed that the proportion of abscesses in the older group was significantly higher than that in the other groups, and there was no difference in the granulomatous inflammation, caseous necrosis, inflammatory necrosis, acute inflammation, exudation, granulation tissue formation, and fibrous tissue hyperplasia. B cell number was significantly lower in the middle-aged and older groups compared to the younger group, while the number of T cells, CD4+ T cells, CD8+ T cells, macrophages, lymphocytes, plasma cells, and NK cells did not differ. Meaningfully, we found that the proportion of IL-10 high expression and TGF-ß1 positive in the older group was significantly higher than that in the younger group. TNF-α, CD66b, IFN-γ, and IL-6 expressions were not different among the three groups. In conclusion, there are some differences in imaging, pathological, and immune features of spinal tuberculosis in different age groups. The high expression of IL-10 and TGF-ß1 in older patients may weaken their anti-tuberculosis immunity and treatment effectiveness.

Synthesis of Novel Triazine-Based Chalcones and 8,9-dihydro-7H-pyrimido[4,5-b][1,4]diazepines as Potential Leads in the Search of Anticancer, Antibacterial and Antifungal Agents.

This study presents the synthesis of four series of novel hybrid chalcones (20,21)a-g and (23,24)a-g and six series of 1,3,5-triazine-based pyrimido[4,5-b][1,4]diazepines (28-33)a-g and the evaluation of their anticancer, antibacterial, antifungal, and cytotoxic properties. Chalcones 20b,d, 21a,b,d, 23a,d-g, 24a-g and the pyrimido[4,5-b][1,4]diazepines 29e,g, 30g, 31a,b,e-g, 33a,b,e-g exhibited outstanding anticancer activity against a panel of 60 cancer cell lines with GI50 values between 0.01 and 100 µM and LC50 values in the range of 4.09 µM to >100 µM, several of such derivatives showing higher activity than the standard drug 5-fluorouracil (5-FU). On the other hand, among the synthesized compounds, the best antibacterial properties against N. gonorrhoeae, S. aureus (ATCC 43300), and M. tuberculosis were exhibited by the pyrimido[4,5-b][1,4]diazepines (MICs: 0.25-62.5 µg/mL). The antifungal activity studies showed that triazinylamino-chalcone 29e and triazinyloxy-chalcone 31g were the most active compounds against T. rubrum and T. mentagrophytes and A. fumigatus, respectively (MICs = 62.5 µg/mL). Hemolytic activity studies and in silico toxicity analysis demonstrated that most of the compounds are safe.

Revealing the Interaction Mechanism between Mycobacterium tuberculosis GyrB and Novobiocin, SPR719 through Binding Thermodynamics and Dissociation Kinetics Analysis.

With the rapid emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), various levels of resistance against existing anti-tuberculosis (TB) drugs have developed. Consequently, the identification of new anti-TB targets and drugs is critically urgent. DNA gyrase subunit B (GyrB) has been identified as a potential anti-TB target, with novobiocin and SPR719 proposed as inhibitors targeting GyrB. Therefore, elucidating the molecular interactions between GyrB and its inhibitors is crucial for the discovery and design of efficient GyrB inhibitors for combating multidrug-resistant TB. In this study, we revealed the detailed binding mechanisms and dissociation processes of the representative inhibitors, novobiocin and SPR719, with GyrB using classical molecular dynamics (MD) simulations, tau-random acceleration molecular dynamics (τ-RAMD) simulations, and steered molecular dynamics (SMD) simulations. Our simulation results demonstrate that both electrostatic and van der Waals interactions contribute favorably to the inhibitors' binding to GyrB, with Asn52, Asp79, Arg82, Lys108, Tyr114, and Arg141 being key residues for the inhibitors' attachment to GyrB. The τ-RAMD simulations indicate that the inhibitors primarily dissociate from the ATP channel. The SMD simulation results reveal that both inhibitors follow a similar dissociation mechanism, requiring the overcoming of hydrophobic interactions and hydrogen bonding interactions formed with the ATP active site. The binding and dissociation mechanisms of GyrB with inhibitors novobiocin and SPR719 obtained in our work will provide new insights for the development of promising GyrB inhibitors.

Diagnostic accuracy of tests for tuberculous pericarditis: A network meta-analysis.

Tuberculous pericarditis (TBP) is a relatively uncommon but potentially fatal extrapulmonary manifestation of tuberculosis. Despite its severity, there is no universally accepted gold standard diagnostic test for TBP currently. The objective of this study is to compare the diagnostic accuracy of the most commonly used tests in terms of specificity, sensitivity, negative predictive value (NPV), and positive predictive value (PPV), and provide a summary of their diagnostic accuracies. A comprehensive literature review was performed using Scopus, MEDLINE, and Cochrane central register of controlled trials, encompassing studies published from start to April 2022. Studies that compared Interferon Gamma Release Assay (IGRA), Xpert MTB/RIF, Adenosine Deaminase levels (ADA), and Smear Microscopy (SM) were included in the analysis. Bayesian random-effects model was used for statistical analysis and mean and standard deviation (SD) with 95% confidence intervals were calculated using the absolute risk (AR) and odds ratio (OR). Rank probability and heterogeneity were determined using risk difference and Cochran Q test, respectively. Sensitivity and specificity were evaluated using true negative, true positive, false positive, and false negative rates. Area under the receiver operating characteristic (AUROC) was calculated for mean and standard error. A total of seven studies comprising 16 arms and 618 patients were included in the analysis. IGRA exhibited the highest mean (SD) sensitivity of 0.934 (0.049), with a high rank probability of 87.5% for being the best diagnostic test, and the AUROC was found to be 94.8 (0.36). On the other hand, SM demonstrated the highest mean (SD) specificity of 0.999 (0.011), with a rank probability of 99.5%, but a leave-one-out analysis excluding SM studies revealed that Xpert MTB/RIF ranked highest for specificity, with a mean (SD) of 0.962 (0.064). The diagnostic tests compared in our study exhibited similar high NPV, while ADA was found to have the lowest PPV among the evaluated methods. Further research, including comparative studies, should be conducted using a standardized cutoff value for both ADA levels and IGRA to mitigate the risk of threshold effect and minimize bias and heterogeneity in data analysis.

Tuberculosis and its clinical consequences on Women's health.

Mycobacterium tuberculosis causes tuberculosis, a fatal infection resulting in widespread illness and death. In 2020, approximately 10 million people were diagnosed with tuberculosis. The top 30 tuberculosis-endemic countries accounted for 86% of all estimated occurrence cases worldwide. In this context, eight of these accounted for two-thirds of the global total, with India having a prevalence of 26%. Aside from lung inflammation, the risk factors for tuberculosis in women include extra-pulmonary infection, particularly genital tuberculosis, tuberculous mastitis, and tuberculous in the peritoneum, intestine, and spine. Depending on the epidemiologic context and screening methods, different tuberculosis symptoms and disease diagnoses are more or less common among expectant mothers. The disease is almost certainly going to have a global impact. The social stigma and anxiety associated with tuberculosis may have a much more significant negative impact on women's health behaviors than men. Notably, the abdominal sites of miliary tuberculosis could mimic tumor likely, carcinoma and lymphoma. Also, the results of the diagnostic accuracy tests for the condition demonstrate that extra-pulmonary tuberculosis can be quickly and accurately diagnosed in various sites using both the T-SPOT assay and the GeneXpert/PCR test. Therefore, this review exemplified the prevalence of extra-pulmonary tuberculosis at various points in women's lives. On the contrary, it also illustrated the symptoms and dangers of TB as they relate to women's health.

Effectiveness of Histopathological Examination of Ultrasound-guided Puncture Biopsy Samples for Diagnosis of Extrapulmonary Tuberculosis.

Objective: To evaluate the diagnostic value of histopathological examination of ultrasound-guided puncture biopsy samples in extrapulmonary tuberculosis (EPTB). Methods: This study was conducted at the Shanghai Public Health Clinical Center. A total of 115 patients underwent ultrasound-guided puncture biopsy, followed by MGIT 960 culture (culture), smear, GeneXpert MTB/RIF (Xpert), and histopathological examination. These assays were performed to evaluate their effectiveness in diagnosing EPTB in comparison to two different diagnostic criteria: liquid culture and composite reference standard (CRS). Results: When CRS was used as the reference standard, the sensitivity and specificity of culture, smear, Xpert, and histopathological examination were (44.83%, 89.29%), (51.72%, 89.29%), (70.11%, 96.43%), and (85.06%, 82.14%), respectively. Based on liquid culture tests, the sensitivity and specificity of smear, Xpert, and pathological examination were (66.67%, 72.60%), (83.33%, 63.01%), and (92.86%, 45.21%), respectively. Histopathological examination showed the highest sensitivity but lowest specificity. Further, we found that the combination of Xpert and histopathological examination showed a sensitivity of 90.80% and a specificity of 89.29%. Conclusion: Ultrasound-guided puncture sampling is safe and effective for the diagnosis of EPTB. Compared with culture, smear, and Xpert, histopathological examination showed higher sensitivity but lower specificity. The combination of histopathology with Xpert showed the best performance characteristics.

Bacterial diversity dominates variable macrophage responses of tuberculosis patients in Tanzania.

The Mycobacterium tuberculosis complex (MTBC) comprises nine human-adapted lineages that differ in their geographical distribution. Local adaptation of specific MTBC genotypes to the respective human host population has been invoked in this context. We aimed to assess if bacterial genetics governs MTBC pathogenesis or if local co-adaptation translates into differential susceptibility of human macrophages to infection by different MTBC genotypes. We generated macrophages from cryopreserved blood mononuclear cells of Tanzanian tuberculosis patients, from which the infecting MTBC strains had previously been phylogenetically characterized. We infected these macrophages ex vivo with a phylogenetically similar MTBC strain ("matched infection") or with strains representative of other MTBC lineages ("mismatched infection"). We found that L1 infections resulted in a significantly lower bacterial burden and that the intra-cellular replication rate of L2 strains was significantly higher compared the other MTBC lineages, irrespective of the MTBC lineage originally infecting the patients. Moreover, L4-infected macrophages released significantly greater amounts of TNF-α, IL-6, IL-10, MIP-1ß, and IL-1ß compared to macrophages infected by all other strains. While our results revealed no measurable effect of local adaptation, they further highlight the strong impact of MTBC phylogenetic diversity on the variable outcome of the host-pathogen interaction in human tuberculosis.

Extrapulmonary, Chronic Septic Arthritis From Mycobacterium tuberculosis in the Ankle and Subtalar Joints.

In the United States, rates of Mycobacterium tuberculosis infection have been declining for decades. Osteoarticular tuberculosis of the ankle is rarely observed. We present the case of a 65-year-old man who immigrated to the United States from India 24 years before the onset of symptoms. The patient initially reported atraumatic swelling and pain of the left ankle and foot and was treated for venous insufficiency. Later, the patient was referred to a nonsurgical orthopaedic clinic for additional workup and was found to have elevated inflammatory markers. MRI showed septic arthritis and osteomyelitis of the talus, distal tibia, and calcaneus. Joint aspiration revealed elevated white blood cell counts with predominately PMNs. The patient was then referred to an orthopaedic foot and ankle surgeon and underwent extensive irrigation and débridement. The patient was discharged on empiric antibiotics. Culture results from the original joint aspirate returned 14 days after surgery as positive for acid-fast bacillus, later identified as M tuberculosis by sequencing. Empiric antibiotics were discontinued, and the patient was started on appropriate antituberculotic therapy. This case report illustrates the challenge in the diagnosis of skeletal tuberculosis and the importance of including this condition on the differential for patients with atypical foot and ankle presentations.

Human Macrophages Activate Bystander Neutrophils' Metabolism and Effector Functions When Challenged with Mycobacterium tuberculosis.

Neutrophils are dynamic cells, playing a critical role in pathogen clearance; however, neutrophil infiltration into the tissue can act as a double-edged sword. They are one of the primary sources of excessive inflammation during infection, which has been observed in many infectious diseases including pneumonia and active tuberculosis (TB). Neutrophil function is influenced by interactions with other immune cells within the inflammatory lung milieu; however, how these interactions affect neutrophil function is unclear. Our study examined the macrophage-neutrophil axis by assessing the effects of conditioned medium (MΦ-CM) from primary human monocyte-derived macrophages (hMDMs) stimulated with LPS or a whole bacterium (Mycobacterium tuberculosis) on neutrophil function. Stimulated hMDM-derived MΦ-CM boosts neutrophil activation, heightening oxidative and glycolytic metabolism, but diminishes migratory potential. These neutrophils exhibit increased ROS production, elevated NET formation, and heightened CXCL8, IL-13, and IL-6 compared to untreated or unstimulated hMDM-treated neutrophils. Collectively, these data show that MΦ-CM from stimulated hMDMs activates neutrophils, bolsters their energetic profile, increase effector and inflammatory functions, and sequester them at sites of infection by decreasing their migratory capacity. These data may aid in the design of novel immunotherapies for severe pneumonia, active tuberculosis and other diseases driven by pathological inflammation mediated by the macrophage-neutrophil axis.

Serum cytokine biosignatures for identification of tuberculosis among HIV-positive inpatients.

BACKGROUND: Serum cytokines correlate with tuberculosis (TB) progression and are predictors of TB recurrence in people living with HIV. We investigated whether serum cytokine biosignatures could diagnose TB among HIV-positive inpatients. METHODS: We recruited HIV-positive inpatients with symptoms of TB and measured serum levels of inflammation biomarkers including IL-2, IL-4, IL-6, IL-10, tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). We then built and tested our TB prediction model. RESULTS: 236 HIV-positive inpatients were enrolled in the first cohort and all the inflammation biomarkers were significantly higher in participants with microbiologically confirmed TB than those without TB. A binary support vector machine (SVM) model was built, incorporating the data of four biomarkers (IL-6, IL-10, TNF-α and IFN-γ). Efficacy of the SVM model was assessed in training (n=189) and validation (n=47) sets with area under the curve (AUC) of 0.92 (95% CI 0.88 to 0.96) and 0.85 (95% CI 0.72 to 0.97), respectively. In an independent test set (n=110), the SVM model yielded an AUC of 0.85 (95% CI 0.76 to 0.94) with 78% (95% CI 68% to 87%) specificity and 85% (95% CI 66% to 96%) sensitivity. Moreover, the SVM model outperformed interferon-gamma release assay (IGRA) among advanced HIV-positive inpatients irrespective of CD4+ T-cell counts, which may be an alternative approach for identifying Mycobacterium tuberculosis infection among HIV-positive inpatients with negative IGRA. CONCLUSIONS: The four-cytokine biosignature model successfully identified TB among HIV-positive inpatients. This diagnostic model may be an alternative approach to diagnose TB in advanced HIV-positive inpatients with low CD4+ T-cell counts.

TOLLIP inhibits lipid accumulation and the integrated stress response in alveolar macrophages to control Mycobacterium tuberculosis infection.

A polymorphism causing deficiencies in Toll-interacting protein (TOLLIP), an inhibitory adaptor protein affecting endosomal trafficking, is associated with increased tuberculosis (TB) risk. It is, however, unclear how TOLLIP affects TB pathogenesis. Here we show that TB severity is increased in Tollip-/- mice, characterized by macrophage- and T cell-driven inflammation, foam cell formation and lipid accumulation. Tollip-/- alveolar macrophages (AM) specifically accumulated lipid and underwent necrosis. Transcriptional and protein analyses of Mycobacterium tuberculosis (Mtb)-infected, Tollip-/- AM revealed increased EIF2 signalling and downstream upregulation of the integrated stress response (ISR). These phenotypes were linked, as incubation of the Mtb lipid mycolic acid with Mtb-infected Tollip-/- AM activated the ISR and increased Mtb replication. Correspondingly, the ISR inhibitor, ISRIB, reduced Mtb numbers in AM and improved Mtb control, overcoming the inflammatory phenotype. In conclusion, targeting the ISR offers a promising target for host-directed anti-TB therapy towards improved Mtb control and reduced immunopathology.

Indole Propionic Acid Disturbs the Normal Function of Tryptophanyl-tRNA Synthetase in Mycobacterium tuberculosis.

Tuberculosis (TB) is the leading infectious disease caused by Mycobacterium tuberculosis and the second-most contagious killer after COVID-19. The emergence of drug-resistant TB has caused a great need to identify and develop new anti-TB drugs with novel targets. Indole propionic acid (IPA), a structural analog of tryptophan (Trp), is active against M. tuberculosis in vitro and in vivo. It has been verified that IPA exerts its antimicrobial effect by mimicking Trp as an allosteric inhibitor of TrpE, which is the first enzyme in the Trp synthesis pathway of M. tuberculosis. However, other Trp structural analogs, such as indolmycin, also target tryptophanyl-tRNA synthetase (TrpRS), which has two functions in bacteria: synthesis of tryptophanyl-AMP by catalyzing ATP + Trp and producing Trp-tRNATrp by transferring Trp to tRNATrp. So, we speculate that IPA may also target TrpRS. In this study, we found that IPA can dock into the Trp binding pocket of M. tuberculosis TrpRS (TrpRSMtb), which was further confirmed by isothermal titration calorimetry (ITC) assay. The biochemical analysis proved that TrpRS can catalyze the reaction between IPA and ATP to generate pyrophosphate (PPi) without Trp as a substrate. Overexpression of wild-type trpS in M. tuberculosis increased the MIC of IPA to 32-fold, and knock-down trpS in Mycolicibacterium smegmatis made it more sensitive to IPA. The supplementation of Trp in the medium abrogated the inhibition of M. tuberculosis by IPA. We demonstrated that IPA can interfere with the function of TrpRS by mimicking Trp, thereby impeding protein synthesis and exerting its anti-TB effect.

Case Report: Clinical and Pathological Findings of Tuberculous Gumma: A Case Report and Literature Review.

Tuberculous gumma (TG) is a rare type of cutaneous tuberculosis thought to occur as a result of the hematogenous spread of Mycobacterium tuberculosis, which is more common in immunosuppressed individuals. An 8-year-old boy presented with a 2-month history of multiple indolent enlarging ulcerated nodules on his left upper extremity. He had a past medical history of bacille Calmette-Guerin vaccine induced lupus vulgaris. Skin biopsy of the nodules showed granulomas and neutrophil-dominated purulent inflammation. Ziehl-Neelsen staining was negative, and the cultures were positive for M. tuberculosis. Furthermore, the M. tuberculosis complex was identified using metagenomic next-generation sequencing. Standard antitubercular therapy was started at full doses, and the skin lesions had significantly improved 3 months later. Here we review the literature since 2000 and describe the clinical and pathological features of TG.

Normalizing granuloma vasculature and matrix improves drug delivery and reduces bacterial burden in tuberculosis-infected rabbits.

Host-directed therapies (HDTs) represent an emerging approach for bacterial clearance during tuberculosis (TB) infection. While most HDTs are designed and implemented for immuno-modulation, other host targets-such as nonimmune stromal components found in pulmonary granulomas-may prove equally viable. Building on our previous work characterizing and normalizing the aberrant granuloma-associated vasculature, here we demonstrate that FDA-approved therapies (bevacizumab and losartan, respectively) can be repurposed as HDTs to normalize blood vessels and extracellular matrix (ECM), improve drug delivery, and reduce bacterial loads in TB granulomas. Granulomas feature an overabundance of ECM and compressed blood vessels, both of which are effectively reduced by losartan treatment in the rabbit model of TB. Combining both HDTs promotes secretion of proinflammatory cytokines and improves anti-TB drug delivery. Finally, alone and in combination with second-line antitubercular agents (moxifloxacin or bedaquiline), these HDTs significantly reduce bacterial burden. RNA sequencing analysis of HDT-treated lung and granuloma tissues implicates up-regulated antimicrobial peptide and proinflammatory gene expression by ciliated epithelial airway cells as a putative mechanism of the observed antitubercular benefits in the absence of chemotherapy. These findings demonstrate that bevacizumab and losartan are well-tolerated stroma-targeting HDTs, normalize the granuloma microenvironment, and improve TB outcomes, providing the rationale to clinically test this combination in TB patients.

METTL3-deficiency m6A-dependently degrades MALAT1 to suppress NLRP3-mediated pyroptotic cell death and inflammation in Mycobacterium tuberculosis (H37Ra strain)-infected mouse macrophages.

Mycobacterium tuberculosis (Mtb)-infected macrophages aggravated the development of pulmonary tuberculosis, but its detailed molecular mechanisms are still largely unknown. Here, the mouse primary peritoneal macrophages were infected with the attenuated strain of Mtb H37Ra, and we firstly verified that targeting a novel METTL3/N6-Methyladenosine (m6A)/LncRNA MALAT1/miR-125b/TLR4 axis was effective to suppress pyroptotic cell death in the Mtb-infected macrophages. Specifically, through performing Real-Time qPCR and Western Blot analysis, we validated that METTL3, LncRNA MALAT1 and TLR4 were elevated, whereas miR-125b and the anti-oxidant agents (Nrf2 and HO-1) were downregulated in Mtb-infected mouse macrophages. In addition, functional experiments confirmed that both ROS scavenger NAC and METTL3-ablation downregulated NLRP3, GSDMD-C, cleaved Caspase-1 and ASC to restrain pyroptotic cell death and decreased the expression levels of IL-1ß, IL-18, IL-6 and TNF-α to restrain inflammatory cytokines expression in Mtb-infected macrophages. Next, METTL3-ablation induced m6A-demethylation and instability in LncRNA MALAT1, and low-expressed LncRNA MALAT1 caused TLR4 downregulation through sponging miR-125b, resulting in the inactivation of NLRP3 inflammasome. Finally, silencing of METTL3-induced protective effects in Mtb-infected macrophages were all abrogated by overexpressing LncRNA MALAT1 and downregulating miR-125b. Thus, we concluded that targeting METTL3-mediated m6A modifications suppressed Mtb-induced pyroptotic cell death in mouse macrophages, and the downstream LncRNA MALAT1/miR-125b/TLR4 axis played critical role in this process.

Marked IDO2 expression and activity related to autophagy and apoptosis in brain tissue of fatal tuberculous meningitis.

In about 1% of tuberculosis (TB) patients, Mycobacterium tuberculosis (M. tuberculosis) can disseminate to the meninges, causing tuberculous meningitis (TBM) with mortality rate up to 60%. Chronic granulomatous inflammation (non-necrotizing and necrotizing) in the brain is the histological hallmark of TBM. The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) and the generated kynurenine metabolites exert major effector functions relevant to TB granuloma functioning. Here we have assessed immunohistochemically IDO1 expression and activity and its effector function and that of its isoform, IDO2, in post-mortem brain tissue of patients that demised with neurotuberculosis. We also related these findings to brain tissue of fatal/severe COVID-19. In this study, IDO1 and IDO2 were abundantly expressed and active in tuberculoid granulomas and were associated with the presence of M. tuberculosis as well as markers of autophagy and apoptosis. Like in fatal/severe COVID-19, IDO2 was also prominent in specific brain regions, such as the inferior olivary nucleus of medulla oblongata and cerebellum, but not associated with granulomas or with M. tuberculosis. Spatially associated apoptosis was observed in TBM, whereas in fatal COVID-19 autophagy dominated. Together, our findings highlight IDO2 as a potentially relevant effector enzyme in TBM, which may relate to the symptomology of TBM.