Devising Isolation Forest-Based Method to Investigate the sRNAome of Mycobacterium tuberculosis Using sRNA-seq Data.

Small non-coding RNAs (sRNAs) regulate the synthesis of virulence factors and other pathogenic traits, which enables the bacteria to survive and proliferate after host infection. While high-throughput sequencing data have proved useful in identifying sRNAs from the intergenic regions (IGRs) of the genome, it remains a challenge to present a complete genome-wide map of the expression of the sRNAs. Moreover, existing methodologies necessitate multiple dependencies for executing their algorithm and also lack a targeted approach for the de novo sRNA identification. We developed an Isolation Forest algorithm-based method and the tool Prediction Of sRNAs using Isolation Forest for the de novo identification of sRNAs from available bacterial sRNA-seq data (http://posif.ibab.ac.in/). Using this framework, we predicted 1120 sRNAs and 46 small proteins in Mycobacterium tuberculosis. Besides, we highlight the context-dependent expression of novel sRNAs, their probable synthesis, and their potential relevance in stress response mechanisms manifested by M. tuberculosis.

Detection of Mycobacterium tuberculosis complex in saliva by quantitative PCR: A potential alternative specimen for pulmonary tuberculosis diagnosis.

BACKGROUND: Current tuberculosis (TB) diagnostic tests primarily rely on sputum samples, yet many TB patients cannot produce sputum. This study explored whether saliva could be used instead of sputum to diagnose pulmonary TB (PTB). METHOD: The study included 32 patients with confirmed PTB and 30 patients with other respiratory diseases (ORD). Saliva from all study participants was subjected to quantitative (qPCR) assays targeting the IS1081 gene for detection of M. tuberculosis complex DNA. RESULTS: The sensitivity of saliva IS1081 qPCR was 65.6 % (95 % CI 48.4-80.2 %) with positive results for 21/32 PTB cases, while the specificity was 96.7 % (95 % CI 85.9-99.6 %) with negative results for 29/30 participants with ORD. Sensitivity improved to 72.4 % (95 % CI 54.6-86.0 %) when sputum-Xpert was used as the reference standard, while remaining similar at 65.5 % (95 % CI 47.4-80.7 %) when culture was used as the reference standard. In receiver operating characteristic (ROC) curve analysis, the area under the curve (AUC) for saliva IS1081 qPCR was 82.5 % (95 % CI 71.7-93.3 %). CONCLUSION: Saliva testing offers a promising alternative to sputum for TB diagnosis among confirmed PTB cases. Larger multicenter studies, encompassing diverse clinical TB characteristics, are needed to provide improved estimates of diagnostic sensitivity and specificity.

Analysis of unsuccessful tests and the effect of prolonged clinical sample preprocessing in the GeneXpert MTB/RIF assay.

BACKGROUND: The GeneXpert MTB/RIF (Xpert) assay is a widely used technology for detecting Mycobacterium tuberculosis (MTB) in clinical samples. However, the study on the failure of the Xpert assay during routine implementation and its potential solutions is limited. METHODS: We retrospectively analyzed the records of unsuccessful tests in the Xpert and the GeneXpert MTB/RIF Ultra (Ultra) assays between April 2017 and April 2021 at the Shanghai Public Health Clinical Center. To further investigate the effect of prolonged preprocessing on clinical sputum, an additional 120 sputum samples were collected for Xpert testing after 15 min, 3 h, and 6 h preprocessing. The analysis was performed by SPSS version 19.0 software. RESULTS: A total of 11,314 test records were analyzed, of which 268 (2.37%) had unsuccessful test results. Among these, 221 (1.95%) were reported as "Error", 43 (0.38%) as "Invalid", and 4 (0.04%) as "No result". The most common clinical specimen for Xpert tests was sputum, accounting for 114 (2.17%) unsuccessful tests. The failure rate of urine specimens was lower than that of sputum (OR = 0.12, 95% CI: 0.02-0.88, χ2 = 6.22, p = 0.021). In contrast, the failure rate of stool specimens was approximately twice as high as that of sputum (OR = 1.93, 95% CI: 1.09-3.40, χ2 = 5.35, p = 0.014). In the prolonged preprocessing experiment, 102 cases (85%) yielded consistent results in Xpert tests. Furthermore, 7 cases (5.83%) detected an increase in MTB load, 8 cases (6.67%) detected a decrease in MTB load, and 3 cases (2.5%) yielded incongruent results in MTB and rifampicin resistance detection. CONCLUSIONS: The primary cause of unsuccessful tests in the Xpert assay was reported as "Error". Despite varying failure rates depending on the samples, the Xpert assay can be applied to extrapulmonary samples. For paucibacillary specimens, retesting the remaining preprocessed mixture should be carefully considered.

Assay for interferon gamma release as a novel marker in pediatric patients with systemic lupus erythematosus.

BACKGROUND: The interferon-gamma (IFN-γ) release assay (IGRA) is an important laboratory diagnosis for latent Mycobacterium tuberculosis (TB) infection. The TB-IGRA measures the release of IFN-γ from peripheral blood cells, who are exposed to TB antigen (Ag), mitogen (MT), or negative/nil control (NL) in vitro. While, an exceptional higher TB Ag-NL level will reflect an elevation of peripheral lymphocytes released IFN-γ in a same condition. Therefore, we found that the elevated levels of TB Ag-NL could become a new biomarker for the diagnosis and treatment of pediatric systemic lupus erythematosus (SLE) patients. METHODS: We have analyzed the clinical data of 776 children who are underwent TB-IGRA testing in the Department of Allergy and Rheumatology of Guangzhou Women and Children's Medical Center from 2018 to 2020. To investigate the association between TB Ag-NL and SLE, we have analyzed the clinical data of 47 SLE patients and TB Ag-NL testing results, and then evaluated the association between TB Ag-NL and SLE disease activity. RESULTS: The TB Ag-NL levels were significantly higher in patients with active SLE than those in inactive SLE (p = 0.0002). The TB Ag-NL levels were positively correlated with the SLE disease activity index (SLEDAI) and laboratory diagnosis parameters. The mean value of TB Ag-NL in SLE patients (0.04191 ± 0.07955, IU/mL) were significantly higher than those in patients with juvenile dermatomyositis (JDM) (0.0158 ± 0.0337, IU/mL, p = 0.036), juvenile idiopathic arthritis (JIA) (0.0162 ± 0.0388, IU/mL, p = 0.001), and healthy controls (HC) (0.0001 ± 0.0027, IU/mL, p = 0.0003). Therefore, the elevated TB Ag-NL levels could serve as a potential diagnostic biomarker of SLE, especially for the active SLE. CONCLUSION: The detection of IFN-γ release levels by the TB-IGRA may be useful to assess SLE disease activity in pediatric patients with active SLE.

[Evaluation of molecular diagnosis of tuberculosis and resistance to rifampicin with GeneXpert® MTB/RIF in Algeria]. / Évaluation du GeneXpert® MTB/RIF dans le diagnostic moléculaire de la tuberculose et de la résistance à la rifampicine en Algérie.

Objective: 1) To evaluate the contribution of the GeneXpert® MTB/RIF (GX) test in the diagnosis of pulmonary and extra-pulmonary tuberculosis compared to culture. 2) To compare the rifampicin results resistance obtained by GX with the phenotypic sensitivity test. Materials and methods: Retrospective study carried out over a period of five years, from May 2017 to June 2022 at the microbiology laboratory of the Central army Hospital Mohamed Seghir Nekkache, Algiers (Algeria). The pulmonary and extrapulmonary clinical specimens were collected, cultivated, tested by GX PCR and direct examination by Ziehl-Neelsen staining. The study of sensitivity to antituberculosis drugs was performed according to the proportion method on liquid medium Bactec MGIT 960 (or on solid medium Lowenstein-Jensen at the Algerian Pasteur Institute). Results: 310 samples were included in the final analysis of the study, of which 156 were of pulmonary origin and 154 of extrapulmonary origin. Mycobacterium tuberculosis complex (MTBC) was detected in 95 samples from 88 tuberculosis patients (sex ratio 2,03 and middle age 37 years) with 49 cases of pulmonary tuberculosis and 39 cases of extra-pulmonary tuberculosis. For 2 cases, the GX was positive while the culture was negative and for 11 cases, the GX was negative while the culture was positive. Thus, in our study and compared to culture, GX showed an overall sensitivity of 88.2%, a specificity of 98.6%, a positive predictive value (PPV) of 96.4% and a negative predictive value (NPV) of 95.2%. The analysis of the data according to the type of samples, the sensitivity, specificity, PPV and NPV of GX for the pulmonary and extrapulmonary samples were 96.3% vs. 77.0%, 98.0% vs. 99.1%, 96.2% vs. 96.5% and 98.0% vs. 92.7% respectively. The sensitivity of GX for disco-vertebral, lymph node, meningeal and pleural tuberculosis were 100%, 90.0%, 71.4% and 57.1% respectively. The sensitivity of GX for pulmonary tuberculosis compared to microscopy was 96% vs. 68%. The comparison of the results of detection of resistance to rifampicin by GX and by phenotypic methods showed perfect agreement. Discussion and conclusion: A good sensitivity of GX compared to microscopy was revealed. The GX is a useful tool for the diagnosis of pulmonary tuberculosis, especially in smear-negative cases. The sensitivity of GX in extrapulmonary tuberculosis varied depending on the location of the infection. A negative result by GX does not exclude tuberculosis and cases of resistance to RIF detected by GX must be confirmed by phenotypic method.

USP25 Promotes the Antimycobacterial Response of Macrophages Through Stabilizing B-Raf and C-Raf.

Ubiquitin-specific peptidase 25 (USP25) is one of the best-characterized deubiquitinating enzymes and plays a vital regulatory role in various biological processes, especially in cancer development and immune regulation. However, the exact role of USP25 and its underlying mechanisms in macrophage activation and immunogenicity during Mycobacterium tuberculosis infection remain unclear. In this study, we found that M tuberculosis infection induced USP25 expression in human and mouse macrophages. In particular, USP25 expression is elevated in multiple cell types, especially monocytes, in patients with tuberculosis. Additionally, USP25 deficiency in macrophages and mice resulted in compromised immunity against M tuberculosis infection, accompanied by reduced expressions of various proinflammatory cytokines and chemokines. Mechanistically, USP25 in macrophages promoted the activation of the ERK signaling pathway through deubiquitination and stabilization of B-Raf and C-Raf. These findings collectively suggest the critical roles of USP25 in M tuberculosis infection and its potential as a therapeutic target.

Physiologic medium renders human iPSC-derived macrophages permissive for M. tuberculosis by rewiring organelle function and metabolism.

In vitro studies are crucial for our understanding of the human macrophage immune functions. However, traditional in vitro culture media poorly reflect the metabolic composition of blood, potentially affecting the outcomes of these studies. Here, we analyzed the impact of a physiological medium on human induced pluripotent stem cell (iPSC)-derived macrophages (iPSDM) function. Macrophages cultured in a human plasma-like medium (HPLM) were more permissive to Mycobacterium tuberculosis (Mtb) replication and showed decreased lipid metabolism with increased metabolic polarization. Functionally, we discovered that HPLM-differentiated macrophages showed different metabolic organelle content and activity. Specifically, HPLM-differentiated macrophages displayed reduced lipid droplet and peroxisome content, increased lysosomal proteolytic activity, and increased mitochondrial activity and dynamics. Inhibiting or inducing lipid droplet formation revealed that lipid droplet content is a key factor influencing macrophage permissiveness to Mtb. These findings underscore the importance of using physiologically relevant media in vitro for accurately studying human macrophage function. IMPORTANCE: This work compellingly demonstrates that the choice of culture medium significantly influences M. tuberculosis replication outcomes, thus emphasizing the importance of employing physiologically relevant media for accurate in vitro host-pathogen interaction studies. We anticipate that our work will set a precedent for future research with clinical relevance, particularly in evaluating antibiotic efficacy and resistance in cellulo.

Gingiva as the primary site of extrapulmonary tuberculosis: A rare case report with brief review of literature.

Multiple strains of Mycobacteria cause tuberculosis (TB), a chronic, specific infectious granulomatous disease. It mainly occurs with pulmonary involvement when compared to extrapulmonary involvement. Primary oral occurrence is uncommon and oral lesions are usually secondary to pulmonary involvement. When there are no active pulmonary clinical manifestations of TB, the diagnosis of the very rare entity of primary gingival TB poses a great challenge to clinicians. In this case report, we discuss a case of primary gingival TB in a 24-year-old lactating mother. This article briefs the onset and course of the lesion during pregnancy and postpartum, elaborates the pathway to diagnosis, various investigations performed and the regimen of antitubercular therapy for 6 months, followed by complete resolution of the lesion without recurrence. This report also describes the significance of considering TB as a differential diagnosis in oral lesions and the various diagnostic methods available. It also emphasizes the sole importance of histopathology in the early detection of the lesion and its management.

Mycobacterium tuberculosis-Human Immunodeficiency Virus Infection and the Role of T Cells in Protection.

Tuberculosis (TB), primarily caused by Mycobacterium tuberculosis (M. tb), remains a widespread fatal health issue that becomes significantly detrimental when coupled with HIV. This study explores the host's innate and adaptive immune system response to TB in HIV immunocompromised patients, highlighting the significant role of CD8+ T cells. While the crucial role of macrophages and cytokines, like TNF-α and IFN-γ, in managing the host's immune response to M. tb is examined, the emphasis is on the changes that occur as a result of HIV coinfection. With the progression of HIV infection, the primary source of IFN-γ changes from CD4+ to CD8+ T cells, especially when latent TB advances to an active state. This study sheds light on the necessity of developing new preventative measures such as vaccines and new treatment approaches to TB, especially for immunocompromised patients, who are at a higher risk of life-threatening complications due to TB-HIV coinfection.

Unveiling the Significance of LysE in Survival and Virulence of Mycobacterium tuberculosis: A Review Reveals It as a Potential Drug Target, Diagnostic Marker, and a Vaccine Candidate.

Tuberculosis (TB) remains a global health threat, necessitating innovative strategies for control and prevention. This comprehensive review explores the Mycobacterium tuberculosis Lysine Exporter (LysE) gene, unveiling its multifaceted roles and potential uses in controlling and preventing tuberculosis (TB). As a pivotal player in eliminating excess L-lysine and L-arginine, LysE contributes to the survival and virulence of M. tuberculosis. This review synthesizes findings from different electronic databases and includes 13 studies focused on the LysE of M. tuberculosis. The research unveils that LysE can be a potential drug target, a diagnostic marker for TB, and a promising candidate for vaccine development. The absence of LysE in the widely used BCG vaccine underscores its uniqueness and positions it as a novel area for TB prevention. In conclusion, this review underscores the significance of LysE in TB pathogenesis and its potential as a drug target, diagnostic marker, and vaccine candidate. The multifaceted nature of LysE positions it at the forefront of innovative approaches to combat TB, calling for sustained research efforts to harness its full potential in the global fight against this infectious disease.

From nature's bounty to drug discovery: Leveraging phytochemicals and molecular approaches to combat multi-drug-resistant (MDR) tuberculosis.

A large number of people annually lose their lives to tuberculosis (TB), which is an age-old disease caused by the Mycobacterium tuberculosis. The global spread of TB is a concern for all regions. The south-east Asian region recorded 46% of all new TB cases in 2021, followed by the African and western Pacific regions with 23% and 18%, respectively. Researchers are always searching at natural substances for potential alternative therapeutics to tackle the worrisome growth in multi-drug-resistant (MDR) tuberculosis due to the high costs associated with developing new treatments and unfavourable side effects of currently used synthetic pharmaceuticals. Phytochemicals show promising results as a future health aid due to their multi-targeting ability on pathogen cells. In the search for new drug leads, the Ayurvedic and Siddha medical systems have made an extensive use of ethnomedicinal tools, including the use of plants like Amalaki (Emblica officinalis Gaertn.), Guduchi (Tinospora cordifolia willd.), Sariva (Hemidesmus indicus R.Br.), Kustha (Saussurea lappa Falc.), turmeric (Curcuma longa Mal.) and Green tea (Camellia sinensis Linn.). These sources are high in flavonoids, polyphenols, tannins and catechins, has been shown to reduce the risk of TB. In this overview, we look at how natural sources like plants, algae and mushrooms have helped researchers to find new drug leads, and how to back these natural sources through mapping the molecular approaches and other approaches has helped them to defeat MDR.

Strain identification of Mycobacterium tuberculosis in multidrug-resistant tuberculosis (MDR-TB) patients at Undata Hospital, Palu, Central Sulawesi.

MDR-TB is a tuberculosis disease resistant to the two most effective anti-TB drugs, rifampin and isoniazid. MDR-TB is a threat to TB control. This study aims to identify the Mycobacterium tuberculosis strain in MDR-TB patients at Undata Hospital in Palu, Central Sulawesi. This type of research is descriptive and observational with a cross-sectional design. The study was conducted in Palu City, Donggala Regency, and Sigi Regency from April-June 2021. The sample in this study consisted of 22 patients who had undergone MDR-TB treatment from 2019 to 2020. The results showed that 55% of the patients with MDR-TB lived in Palu City, 27% in Donggala Regency, and 18% in Sigi Regency. Out of the 22 patients, 13 were men (59%) and nine were women (41%). Based on the results of the examination of Mycobacterium tuberculosis culture, three samples from patients with MDR-TB were positive for M. tuberculosis. The results of the spoligotyping examination showed that the strain belonged to the Beijing family. Identifying the type of M. tuberculosis strain through spoligotyping examination should be carried out in TB patients who have not undergone MDR-TB treatment or those who have failed treatment and are still found to be positive for M. tuberculosis bacteria.

Study of adverse drug reactions during the treatment of drug resistant tuberculosis.

BACKGROUND: Pharmacovigilance entails monitoring of patients for timely detection of ADR and reporting them so that more information about drug safety can be obtained. This may help in the future for dose modification or alteration of regimen. In NTEP, ADSm (Active Drug Safety monitoring) is part of pharmacovigilance. In this study we shall be studying ADRs to Anti TB drugs in DRTB. METHODOLOGY: This study is observational, retrospective and record based, of patients admitted from 2021 to 2023 in the DOTS ward of Respiratory Medicine Department of a tertiary care hospital in Goa. Data such as age, sex, regimen, date of AKT initiation and adverse effects documented has been noted and compiled. RESULTS: ADRs have been tabulated in the form of tables. Statistical analysis is done to find out the commonest ADR, time when they are likely to occur, which age and gender are most likely affected and if there are any other associated risk factors for ADRs. CONCLUSION: This study will enable in future to better monitor patients with regard to particular adverse drug reaction, patient safety and if needed to alter the regimen as early as possible.

In-silico transcriptome analysis of antibiotic-treated Mycobacterium tuberculosis identifies novel antibiotic resistance factors.

The emergence of drug resistant Mycobacterium tuberculosis strains increases the burden on the treatment of tuberculosis. In this study, through in-silico transcriptome analysis of drug-treated M. tuberculosis samples, novel drug targets for the treatment of drug resistance in tuberculosis were identified. Gene expression datasets of tuberculosis patients samples treated with different antibiotics (Isoniazid, Rifampicin, Pyrazinamide, Bedaquiline and Linezolid) were considered in this study. DESeq2 was used to identify the differentially regulated genes. Novel genes which were up-regulated during antibiotic treatment were identified which could be antibiotic resistance factors. Further, to understand the resistance mechanism of the novel genes, we performed gene ontology and gene network analysis for the differentially up-regulated genes. Thus, the in-silico transcriptome analysis paves way for a deeper understanding of the antibiotic resistance in M. tuberculosis.

CD137 expression and signal function drive pleiotropic γδ T-cell effector functions that inhibit intracellular M. tuberculosis growth.

Co-activation signal that induces/sustains pleiotropic effector functions of antigen-specific γδ T cells remains unknown. Here, Mycobacteria tuberculosis (Mtb) tuberculin administration during tuberculosis (TB) skin test resulted in rapid expression of co-activation signal molecules CD137 and CD107a by fast-acting Vγ2Vδ2 T cells in TB-resistant subjects (Resisters), but not patients with active TB. And, anti-CD137 agonistic antibody treatment experiments showed that CD137 signaling enabled Vγ2Vδ2 T cells to produce more effector cytokines and inhibit intracellular Mtb growth in macrophages (Mɸ). Consistently, Mtb antigen (Ag) HMBPP stimulation induced sustainable high-level CD137 expression in fresh and activated Vγ2Vδ2 T cells from uninfected subjects, but not TB patients. CD137+Vγ2Vδ2 T-cell subtype predominantly displayed central memory phenotype and mounted better proliferative responses than CD137-Vγ2Vδ2 T-cells. In response to HMBPP, CD137+Vγ2Vδ2 T-cell subtype rapidly differentiated into greater numbers of pleiotropic effector cells producing anti-Mtb cytokines compared to CD137-Vγ2Vδ2 T subtype, with the non-canonical NF-κB pathway involved. CD137 expression in Vγ2Vδ2 T cells appeared to signal anti-Mtb effector functions leading to intracellular Mtb growth inhibition in Mɸ, and active TB disrupted such CD137-driven anti-Mtb effector functions. CD137+Vγ2Vδ2 T-cells subtype exhibited an epigenetic-driven high-level expression of GM-CSF and de novo production of GM-CSF critical for Vγ2Vδ2 T-cell controlling of Mtb growth in Mϕ. Concurrently, exosomes produced by CD137+Vγ2Vδ2 T cells potently inhibited intracellular mycobacterial growth. Furthermore, adoptive transfer of human CD137+Vγ2Vδ2 T cells to Mtb-infected SCID mice conferred protective immunity against Mtb infection. Thus, our data suggest that CD137 expression/signaling drives pleiotropic γδ T-cell effector functions that inhibit intracellular Mtb growth.

Understanding the Spectrum and Management of Post-Tuberculosis Lung Disease: A Comprehensive Review.

Post-tuberculosis lung disease (PTLD) poses a significant clinical challenge in regions with a high burden of tuberculosis (TB). This review provides a comprehensive overview of PTLD, encompassing its pathogenesis, clinical manifestations, diagnostic modalities, management strategies, long-term outcomes, and public health implications. PTLD arises from residual lung damage following TB treatment and is characterized by a spectrum of pathological changes, including fibrosis, bronchiectasis, and cavitation. Clinical presentation varies widely, from chronic cough and hemoptysis to recurrent respiratory infections, which are oftentimes a diagnostic dilemma. Radiological imaging, pulmonary function tests, and careful consideration of patient history play pivotal roles in diagnosis. Management strategies involve pharmacological interventions to alleviate symptoms and prevent disease progression, which are influenced by the extent of lung damage, comorbidities, and access to healthcare. Rehabilitation programs and surgical options are available for select cases. Prognosis is influenced by the extent of lung damage, comorbidities, and access to healthcare. Prevention efforts through a TB control program and early detection are crucial in reducing the burden of PTLD. This review stresses the importance of understanding and addressing PTLD to mitigate its impact on individuals and public health systems worldwide.

Occurrence of multidrug-resistant Mycobacterium tuberculosis in upper Southern Thailand.

Background and Aim: Mycobacterium tuberculosis causes global concern with tuberculosis (TB). Multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) pose additional challenges, as they resist to multiple first-line drugs. This study investigated the occurrence of TB, antibiotic resistance due to inhA and katG gene mutations, and multidrug resistance in M. tuberculosis during fiscal years 2020-2022. Materials and Methods: Samples were gathered from hospitals in seven provinces of upper Southern Thailand. The study investigated the correlation between inhA and katG gene mutations in M. tuberculosis and the development of antimicrobial resistance and isoniazid resistance. Results: A total of 19,186 samples were sent to the Office of Disease Prevention and Control Region 11st, Nakhon Si Thammarat, Thailand. The results showed that 51% of the samples were obtained from patients located in Nakhon Si Thammarat, followed by Surat Thani provinces. Regarding the spatial distribution of TB-infected cases, the incidence of TB was high in the province, which has a moderate to high population density. The highest average occurrence of TB in this study was found in Phuket province (9.75/100,000 risk person-year). The detected isoniazid resistance was 394, 255, and 179 cases in 2020, 2021, and 2022, respectively. A total of 99 isolates were MDR, whereas four isolates were XDR. The antimicrobial resistance associated with the inhA mutation was 192, 142, and 105 isolates, respectively, whereas the resistance associated with the katG mutation was 249, 182, and 120 cases in 2020, 2021, and 2022, respectively. Conclusion: These findings contribute to the understanding of the occurrence of antibiotic-resistant TB that could lead to use as data for preventing MDR-TB.

Differential Abundance of Protein Acylation in Mycobacterium tuberculosis Under Exposure to Nitrosative Stress.

BACKGROUND: Human macrophages generate antimicrobial reactive nitrogen species in response to infection by Mycobacterium tuberculosis (Mtb). Exposure to these redox-reactive compounds induces stress response in Mtb, which can affect posttranslational modifications (PTM). METHODS: Here, we present the global analysis of the PTM acylation of Mtb proteins in response to a sublethal dose of nitrosative stress in the form of nitric oxide (NO) using label free quantification. RESULTS: A total of 6437 acylation events were identified on 1496 Mtb proteins, and O-acylation accounted for 92.2% of the events identified, while 7.8% were N-acylation events. About 22% of the sites identified were found to be acylated by more than one acyl-group. Furthermore, the abundance of each acyl-group decreased as their molecular weight increased. Quantitative PTM analysis revealed differential abundance of acylation in proteins involved in stress response, iron ion homeostasis, growth, energy metabolism, and antimicrobial resistance (AMR) induced by nitrosative stress over time. CONCLUSIONS: The results reveal a potential role of Mtb protein acylation in the bacterial stress responses and AMR. To our knowledge, this is the first report on global O-acylation profile of Mtb in response to NO. This will significantly improve our understanding of the changes in Mtb acylation under nitrosative stress, highly relevant for global health.

Shifting Mycobacterial Serine Hydrolase Activity Visualized Using Multi-Layer In-Gel Activity Assays.

The ability of Mycobacterium tuberculosis to derive lipids from the host, store them intracellularly, and then break them down into energy requires a battery of serine hydrolases. Serine hydrolases are a large, diverse enzyme family with functional roles in dormant, active, and reactivating mycobacterial cultures. To rapidly measure substrate-dependent shifts in mycobacterial serine hydrolase activity, we combined a robust mycobacterial growth system of nitrogen limitation and variable carbon availability with nimble in-gel fluorogenic enzyme measurements. Using this methodology, we rapidly analyzed a range of ester substrates, identified multiple hydrolases concurrently, observed functional enzyme shifts, and measured global substrate preferences. Within every growth condition, mycobacterial hydrolases displayed the full, dynamic range of upregulated, downregulated, and constitutively active hydrolases independent of the ester substrate. Increasing the alkyl chain length of the ester substrate also allowed visualization of distinct hydrolase activity likely corresponding with lipases most responsible for lipid breakdown. The most robust expression of hydrolase activity was observed under the highest stress growth conditions, reflecting the induction of multiple metabolic pathways scavenging for energy to survive under this high stress. The unique hydrolases present under these high-stress conditions could represent novel drug targets for combination treatment with current front-line therapeutics. Combining diverse fluorogenic esters with in-gel activity measurements provides a rapid, customizable, and sensitive detection method for mycobacterial serine hydrolase activity.

Universal Lineage-Independent Markers of Multidrug Resistance in Mycobacterium tuberculosis.

(1) Background: This study was aimed to identify universal genetic markers of multidrug resistance (MDR) in Mycobacterium tuberculosis (Mtb) and establish statistical associations among identified mutations to enhance understanding of MDR in Mtb and inform diagnostic and treatment development. (2) Methods: GWAS analysis and the statistical evaluation of identified polymorphic sites within protein-coding genes of Mtb were performed. Statistical associations between specific mutations and antibiotic resistance were established using attributable risk statistics. (3) Results: Sixty-four polymorphic sites were identified as universal markers of drug resistance, with forty-seven in PE/PPE regions and seventeen in functional genes. Mutations in genes such as cyp123, fadE36, gidB, and ethA showed significant associations with resistance to various antibiotics. Notably, mutations in cyp123 at codon position 279 were linked to resistance to ten antibiotics. The study highlighted the role of PE/PPE and PE_PGRS genes in Mtb's evolution towards a 'mutator phenotype'. The pathways of acquisition of mutations forming the epistatic landscape of MDR were discussed. (4) Conclusions: This research identifies marker mutations across the Mtb genome associated with MDR. The findings provide new insights into the molecular basis of MDR acquisition in Mtb, aiding in the development of more effective diagnostics and treatments targeting these mutations to combat MDR tuberculosis.