Activity against Mycobacterium tuberculosis of a new class of spirooxindolopyrrolidine embedded chromanone hybrid heterocycles.

A new class of structurally intriguing heterocycles embedded with spiropyrrolidine, oxindole and chromanones was prepared by regio- and stereoselectively in quantitative yields using an intermolecular tandem cycloaddition protocol. The compounds synthesized were assayed for their anti-mycobacterial activity against Mycobacterium tuberculosis (Mtb) H37Rv and isoniazid-resistant (katG and inhA promoter mutations) clinical Mtb isolates. Four compounds exhibited significant antimycobacterial activity against Mtb strains tested. In particular, a compound possessing a fluorine substituted derivative displayed potent activity at 0.39 µg mL-1 against H37Rv, while it showed 0.09 µg mL-1 and 0.19 µg mL-1 activity against inhA promoter and katG mutation isolates, respectively. A molecular docking study was conducted with the potent compound, which showed results that were consistent with the in vitro experiments.

Identification of essential oils with strong activity against stationary phase Mycobacterium abscessus.

Purpose: To identify essential oils (EOs) active against non-growing stationary phase Mycobacterium abscessus and multidrug-resistant M. abscessus strains. Methods: The activity of EOs against both stationary and log phase M. abscessus was evaluated by colony forming unit (CFU) assay and minimum inhibitory concentration (MIC) testing. Results: We assessed the activity of 80 EOs against stationary phase M. abscessus and found 12 EOs (Cinnamon, Satureja montana, Palmarosa, Lemon eucalyptus, Honey myrtle, Combava, Health shield, Mandarin, Thyme, Rosewood, Valerian Root and Basil) at 0.5% concentration to be active against both growing and non-growing stationary phase M. abscessus. Among them, Satureja montana essential oil and Palmarosa essential oil could eliminate all stationary phase M. abscessus at 0.125% and Cinnamon essential oil could eliminate stationary phase bacteria at 0.063% after 1-day treatment. Interestingly, these EOs also exhibited promising activity against multidrug-resistant M. abscessus clinical strains. Conclusions: Our study indicates that some EOs display outstanding effectiveness against both drug susceptible M. abscessus and multidrug-resistant M. abscessus isolates. These findings may be significant for the treatment of persistent M. abscessus infections.

Repurposing Azoles to Resolve Serotogenic Toxicity Associated with Linezolid to Combat Multidrug-Resistant Tuberculosis.

Serotogenic toxicity is a major hurdle associated with Linezolid in the treatment of drug-resistant tuberculosis (TB) due to the inhibition of monoamine oxidase (MAO) enzymes. Azole compounds demonstrate structural similarities to the recognized anti-TB drug Linezolid, making them intriguing candidates for repurposing. Therefore, we have repurposed azoles (Posaconazole, Itraconazole, Miconazole, and Clotrimazole) for the treatment of drug-resistant TB with the anticipation of their selectivity in sparing the MAO enzyme. The results of repurposing revealed that Clotrimazole showed equipotent activity against the Mycobacterium tuberculosis (Mtb) H37Rv strain compared to Linezolid, with a minimal inhibitory concentration (MIC) of 2.26 µM. Additionally, Clotrimazole exhibited reasonable MIC50 values of 0.17 µM, 1.72 µM, 1.53 µM, and 5.07 µM against the inhA promoter+, katG+, rpoB+, and MDR clinical Mtb isolates, respectively, compared to Linezolid. Clotrimazole also exhibited 3.90-fold less inhibition of MAO-A and 50.35-fold less inhibition of MAO-B compared to Linezolid, suggesting a reduced serotonergic toxicity burden.

Novel Quinoline-Based Thiosemicarbazide Derivatives: Synthesis, DFT Calculations, and Investigation of Antitubercular, Antibacterial, and Antifungal Activities.

The discovery of new antimicrobial agents as a means of treating drug-resistant microbial pathogens is of utmost significance to overcome their immense risk to human well-being. The current investigation involves the development, synthesis, and assessment of the antimicrobial efficacy of novel quinoline derivatives incorporating a thiosemicarbazide functionality. To design the target compounds (QST1-QST14), we applied the molecular hybridization approach to link various thiosemicarbazides to the quinoline core with a sulfonyl group. Upon the synthesis and completion of structural characterization via spectroscopic techniques (1H NMR, 13C NMR, 15N NMR, IR, and HRMS), the title molecules were extensively evaluated for their potential antitubercular, antibacterial, and antifungal activities. N-(3-Chlorophenyl)-2-(quinolin-8-ylsulfonyl)hydrazine-1-carbothioamide (QST4), the most effective compound against Mycobacterium tuberculosis H37Rv, was also tested on isoniazid-resistant clinical isolates with katG and inhA promoter mutations. Based on molecular docking studies, QST4 was also likely to demonstrate its antimycobacterial activity through inhibition of the InhA enzyme. Furthermore, three derivatives (QST3, QST4, and QST10) with preferable antimicrobial and drug-like profiles were also shown to be nontoxic against human embryonic kidney (HEK) cells. All compounds were optimized by the density functional theory method using B3LYP with the 6-31+G(d,p) basis set. Structural analysis, natural bond orbital calculations of donor-acceptor interactions, molecular electrostatic potential analysis, and frontier molecular orbital analysis were carried out. Quantum chemical descriptors and charges on the atoms were determined to compare the strengths of the intramolecular hydrogen bonds formed and their stabilities. We determined that the sulfur atom forms a stronger intramolecular hydrogen bond than the nitrogen, oxygen, and fluorine atoms in these sulfonyl thiosemicarbazide derivatives.

Design, Synthesis, and Evaluation of Novel Δ2-Thiazolino 2-Pyridone Derivatives That Potentiate Isoniazid Activity in an Isoniazid-Resistant Mycobacterium tuberculosis Mutant.

Mycobacterium tuberculosis (Mtb) drug resistance poses an alarming threat to global tuberculosis control. We previously reported that C10, a ring-fused thiazolo-2-pyridone, inhibits Mtb respiration, blocks biofilm formation, and restores the activity of the antibiotic isoniazid (INH) in INH-resistant Mtb isolates. This discovery revealed a new strategy to address INH resistance. Expanding upon this strategy, we identified C10 analogues with improved potency and drug-like properties. By exploring three heterocycle spacers (oxadiazole, 1,2,3-triazole, and isoxazole) on the ring-fused thiazolo-2-pyridone scaffold, we identified two novel isoxazoles, 17h and 17j. 17h and 17j inhibited Mtb respiration and biofilm formation more potently with a broader therapeutic window, were better potentiators of INH-mediated inhibition of an INH-resistant Mtb mutant, and more effectively inhibited intracellular Mtb replication than C10. The (-)17j enantiomer showed further enhanced activity compared to its enantiomer and the 17j racemic mixture. Our potent second-generation C10 analogues offer promise for therapeutic development against drug-resistant Mtb.

Isoniazid Linked to Sulfonate Esters via Hydrazone Functionality: Design, Synthesis, and Evaluation of Antitubercular Activity.

Isoniazid (INH) is one of the key molecules employed in the treatment of tuberculosis (TB), the most deadly infectious disease worldwide. However, the efficacy of this cornerstone drug has seriously decreased due to emerging INH-resistant strains of Mycobacterium tuberculosis (Mtb). In the present study, we aimed to chemically tailor INH to overcome this resistance. We obtained thirteen novel compounds by linking INH to in-house synthesized sulfonate esters via a hydrazone bridge (SIH1-SIH13). Following structural characterization by FTIR, 1H NMR, 13C NMR, and HRMS, all compounds were screened for their antitubercular activity against Mtb H37Rv strain and INH-resistant clinical isolates carrying katG and inhA mutations. Additionally, the cytotoxic effects of SIH1-SIH13 were assessed on three different healthy host cell lines; HEK293, IMR-90, and BEAS-2B. Based on the obtained data, the synthesized compounds appeared as attractive antimycobacterial drug candidates with low cytotoxicity. Moreover, the stability of the hydrazone moiety in the chemical structure of the final compounds was confirmed by using UV/Vis spectroscopy in both aqueous medium and DMSO. Subsequently, the compounds were tested for their inhibitory activities against enoyl acyl carrier protein reductase (InhA), the primary target enzyme of INH. Although most of the synthesized compounds are hosted by the InhA binding pocket, SIH1-SIH13 do not primarily show their antitubercular activities by direct InhA inhibition. Finally, in silico determination of important physicochemical parameters of the molecules showed that SIH1-SIH13 adhered to Lipinski's rule of five. Overall, our study revealed a new strategy for modifying INH to cope with the emerging drug-resistant strains of Mtb.

Urea derivatives carrying a thiophenylthiazole moiety: Design, synthesis, and evaluation of antitubercular and InhA inhibitory activities.

The recent emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) has complicated and significantly slowed efforts to eradicate and/or reduce the worldwide incidence of life-threatening acute and chronic cases of tuberculosis. To overcome this setback, researchers have increased the intensity of their work to identify new small-molecule compounds that are expected to remain efficacious antimicrobials against Mtb. Here, we describe our effort to apply the principles of molecular hybridization to synthesize 16 compounds carrying thiophene and thiazole rings beside the core urea functionality (TTU1-TTU16). Following extensive structural characterization, the obtained compounds were initially evaluated for their antimycobacterial activity against Mtb H37Rv. Subsequently, three derivatives standing out with their anti-Mtb activity profiles and low cytotoxicity (TTU5, TTU6, and TTU12) were tested on isoniazid-resistant clinical isolates carrying katG and inhA mutations. Additionally, due to their pharmacophore similarities to the well-known InhA inhibitors, the molecules were screened for their enoyl acyl carrier protein reductase (InhA) inhibitory potentials. Molecular docking studies were performed to support the experimental enzyme inhibition data. Finally, drug-likeness of the selected compounds was established by theoretical calculations of physicochemical descriptors.

Proteome Profiling of Mycobacterium tuberculosis Cells Exposed to Nitrosative Stress.

Reactive nitrogen species (RNS) are secreted by human cells in response to infection by Mycobacterium tuberculosis (Mtb). Although RNS can kill Mtb under some circumstances, Mtb can adapt and survive in the presence of RNS by a process that involves modulation of gene expression. Previous studies focused primarily on stress-related changes in the Mtb transcriptome. This study unveils changes in the Mtb proteome in response to a sub-lethal dose of nitric oxide (NO) over several hours of exposure. Proteins were identified using liquid chromatography coupled with electrospray ionization mass spectrometry (LC-MS/MS). A total of 2911 Mtb proteins were identified, of which 581 were differentially abundant (DA) after exposure to NO in at least one of the four time points (30 min, 2 h, 6 h, and 20 h). The proteomic response to NO was marked by two phases, with few DA proteins in the early phase and a multitude of DA proteins in the later phase. The efflux pump Rv1687 stood out as being the only protein more abundant at all the time points and might play a role in the early protection of Mtb against nitrosative stress. These changes appeared to be compensatory in nature, contributing to iron homeostasis, energy metabolism, and other stress responses. This study thereby provides new insights into the response of Mtb to NO at the level of proteomics.

Serum proteases prevent bacterial biofilm formation: role of kallikrein and plasmin.

Biofilm formation is a general strategy for bacterial pathogens to withstand host defense mechanisms. In this study, we found that serum proteases inhibit biofilm formation by Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, and Bordetella pertussis. Confocal laser-scanning microscopy analysis revealed that these proteins reduce the biomass and alter the architecture of meningococcal biofilms. To understand the underlying mechanism, the serum was fractionated through size-exclusion chromatography and anion-exchange chromatography, and the composition of the fractions that retained anti-biofilm activity against N. meningitidis was analyzed by intensity-based absolute quantification mass spectrometry. Among the identified serum proteins, plasma kallikrein (PKLK), FXIIa, and plasmin were found to cleave neisserial heparin-binding antigen and the α-peptide of IgA protease on the meningococcal cell surface, resulting in the release of positively charged polypeptides implicated in biofilm formation by binding extracellular DNA. Further experiments also revealed that plasmin and PKLK inhibited biofilm formation of B. pertussis by cleaving filamentous hemagglutinin. We conclude that the proteolytic activity of serum proteases toward bacterial adhesins involved in biofilm formation could constitute a defense mechanism for the clearance of pathogens.

Whole Genome Sequencing of Drug Resistant and Drug Susceptible Mycobacterium tuberculosis Isolates From Tigray Region, Ethiopia.

Background: Tuberculosis, mainly caused by Mycobacterium tuberculosis (Mtb), is an ancient human disease that gravely affects millions of people annually. We wanted to explore the genetic diversity and lineage-specific association of Mtb with drug resistance among pulmonary tuberculosis patients. Methods: Sputum samples were collected from pulmonary tuberculosis patients at six different healthcare institutions in Tigray, Ethiopia, between July 2018 and August 2019. DNA was extracted from 74 Mtb complex isolates for whole-genome sequencing (WGS). All genomes were typed and screened for mutations with known associations with antimicrobial resistance using in silico methods, and results were cross-verified with wet lab methods. Results: Lineage (L) 4 (55.8%) was predominant, followed by L3 (41.2%); L1 (1.5%) and L2 (1.5%) occurred rarely. The most frequently detected sublineage was CAS (38.2%), followed by Ural (29.4%), and Haarlem (11.8%). The recent transmission index (RTI) was relatively low. L4 and Ural strains were more resistant than the other strains to any anti-TB drug (P < 0.05). The most frequent mutations to RIF, INH, EMB, SM, PZA, ETH, FLQs, and 2nd-line injectable drugs occurred at rpoB S450L, katG S315T, embB M306I/V, rpsL K43R, pncA V139A, ethA M1R, gyrA D94G, and rrs A1401G, respectively. Disputed rpoB mutations were also shown in four (16%) of RIF-resistant isolates. Conclusion: Our WGS analysis revealed the presence of diverse Mtb genotypes. The presence of a significant proportion of disputed rpoB mutations highlighted the need to establish a WGS facility at the regional level to monitor drug-resistant mutations. This will help control the transmission of DR-TB and ultimately contribute to the attainment of 100% DST coverage for TB patients as per the End TB strategy.

Correction: Characteristics of pulmonary multidrug-resistant tuberculosis patients in Tigray Region, Ethiopia: A cross-sectional study.

[This corrects the article DOI: 10.1371/journal.pone.0236362.].

Lipid droplets and the transcriptome of Mycobacterium tuberculosis from direct sputa: a literature review.

Mycobacterium tuberculosis (Mtb), the main etiology of tuberculosis (TB), is predominantly an intracellular pathogen that has caused infection, disease and death in humans for centuries. Lipid droplets (LDs) are dynamic intracellular organelles that are found across the evolutionary tree of life. This review is an evaluation of the current state of knowledge regarding Mtb-LD formation and associated Mtb transcriptome directly from sputa.Based on the LD content, Mtb in sputum may be classified into three groups: LD positive, LD negative and LD borderline. However, the clinical and evolutionary importance of each state is not well elaborated. Mounting evidence supports the view that the presence of LD positive Mtb bacilli in sputum is a biomarker of slow growth, low energy state, towards lipid degradation, and drug tolerance. In Mtb, LD may serve as a source of chemical energy, scavenger of toxic compounds, prevent destruction of Mtb through autophagy, delay trafficking of lysosomes towards the phagosome, and contribute to Mtb persistence. It is suggest that LD is a key player in the induction of a spectrum of phenotypic and metabolic states of Mtb in the macrophage, granuloma and extracellular sputum microenvironment. Tuberculosis patients with high proportion of LD positive Mtb in pretreatment sputum was associated with higher rate of poor treatment outcome, indicating that LD may have a clinical application in predicting treatment outcome.The propensity for LD formation among Mtb lineages is largely unknown. The role of LD on Mtb transmission and disease phenotype (pulmonary TB vs extra-pulmonary TB) is not well understood. Thus, further studies are needed to understand the relationships between LD positivity and Mtb lineage, Mtb transmission and clinical types.

Frequency and patterns of first- and second-line drug resistance-conferring mutations in Mycobacterium tuberculosis isolated from pulmonary tuberculosis patients in a cross-sectional study in Tigray Region, Ethiopia.

OBJECTIVES: Tuberculosis (TB) is a preventable and treatable infectious disease, but the continuing emergence and spread of multidrug-resistant TB is threatening global TB control efforts. This study aimed to describe the frequency and patterns of drug resistance-conferring mutations of Mycobacterium tuberculosis (MTB) isolates detected from pulmonary TB patients in Tigray Region, Ethiopia. METHODS: A cross-sectional study design was employed to collect sputum samples from pulmonary TB patients between July 2018 to August 2019. Culture and identification tests were done at Tigray Health Research Institute (THRI). Mutations conferring rifampicin (RIF), isoniazid (INH) and fluoroquinolone (FQ) resistance were determined in 227 MTB isolates using GenoType MTBDRplus and GenoType MTBDRsl. RESULTS: Mutations conferring resistance to RIF, INH and FQs were detected in 40/227 (17.6%), 41/227 (18.1%) and 2/38 (5.3%) MTB isolates, respectively. The majority of mutations for RIF, INH and FQs occurred at codons rpoB S531L (70%), katG S315T (78%) and gyrA D94Y/N (100%), respectively. This study revealed a significant number of unknown mutations in the rpoB, katG and inhA genes. CONCLUSION: High rates of mutations conferring resistance to RIF, INH and FQs were observed in this study. A large number of isolates showed unknown mutations, which require further DNA sequencing analysis. Periodic drug resistance surveillance and scaling-up of drug resistance testing facilities are imperative to prevent the transmission of drug-resistant TB in the community.

Lineage-Specific Proteomic Signatures in the Mycobacterium tuberculosis Complex Reveal Differential Abundance of Proteins Involved in Virulence, DNA Repair, CRISPR-Cas, Bioenergetics and Lipid Metabolism.

Despite the discovery of the tubercle bacillus more than 130 years ago, its physiology and the mechanisms of virulence are still not fully understood. A comprehensive analysis of the proteomes of members of the human-adapted Mycobacterium tuberculosis complex (MTBC) lineages 3, 4, 5, and 7 was conducted to better understand the evolution of virulence and other physiological characteristics. Unique and shared proteomic signatures in these modern, pre-modern and ancient MTBC lineages, as deduced from quantitative bioinformatics analyses of high-resolution mass spectrometry data, were delineated. The main proteomic findings were verified by using immunoblotting. In addition, analysis of multiple genome alignment of members of the same lineages was performed. Label-free peptide quantification of whole cells from MTBC lineages 3, 4, 5, and 7 yielded a total of 38,346 unique peptides derived from 3092 proteins, representing 77% coverage of the predicted proteome. MTBC lineage-specific differential expression was observed for 539 proteins. Lineage 7 exhibited a markedly reduced abundance of proteins involved in DNA repair, type VII ESX-3 and ESX-1 secretion systems, lipid metabolism and inorganic phosphate uptake, and an increased abundance of proteins involved in alternative pathways of the TCA cycle and the CRISPR-Cas system as compared to the other lineages. Lineages 3 and 4 exhibited a higher abundance of proteins involved in virulence, DNA repair, drug resistance and other metabolic pathways. The high throughput analysis of the MTBC proteome by super-resolution mass spectrometry provided an insight into the differential expression of proteins between MTBC lineages 3, 4, 5, and 7 that may explain the slow growth and reduced virulence, metabolic flexibility, and the ability to survive under adverse growth conditions of lineage 7.

Spatial Transcriptomics Reveals Genes Associated with Dysregulated Mitochondrial Functions and Stress Signaling in Alzheimer Disease.

Alzheimer disease (AD) is a devastating neurological disease associated with progressive loss of mental skills and cognitive and physical functions whose etiology is not completely understood. Here, our goal was to simultaneously uncover novel and known molecular targets in the structured layers of the hippocampus and olfactory bulbs that may contribute to early hippocampal synaptic deficits and olfactory dysfunction in AD mice. Spatially resolved transcriptomics was used to identify high-confidence genes that were differentially regulated in AD mice relative to controls. A diverse set of genes that modulate stress responses and transcription were predominant in both hippocampi and olfactory bulbs. Notably, we identify Bok, implicated in mitochondrial physiology and cell death, as a spatially downregulated gene in the hippocampus of mouse and human AD brains. In summary, we provide a rich resource of spatially differentially expressed genes, which may contribute to understanding AD pathology.

Characteristics of pulmonary multidrug-resistant tuberculosis patients in Tigray Region, Ethiopia: A cross-sectional study.

BACKGROUND: Tuberculosis (TB) is among the top 10 causes of mortality and the first killer among infectious diseases worldwide. One of the factors fuelling the TB epidemic is the global rise of multidrug resistant TB (MDR-TB). The aim of this study was to determine the magnitude and factors associated with MDR-TB in the Tigray Region, Ethiopia. METHOD: This study employed a facility-based cross-sectional study design, which was conducted between July 2018 and August 2019. The inclusion criteria for the study participants were GeneXpert-positive who were not under treatment for TB, PTB patients' ≥15 years of age and who provided written informed consent. A total of 300 participants were enrolled in the study, with a structured questionnaire used to collect data on clinical, sociodemographic and behavioral factors. Sputum samples were collected and processed for acid-fast bacilli staining, culture and drug susceptibility testing. Drug susceptibility testing was performed using a line probe assay. Logistic regression was used to analyze associations between outcome and predictor variables. RESULTS: The overall proportion of MDR-TB was 16.7% (11.6% and 32.7% for new and previously treated patients, respectively). Of the total MDR-TB isolates, 5.3% were pre-XDR-TB. The proportion of MDR-TB/HIV co-infection was 21.1%. A previous history of TB treatment AOR 3.75; 95% CI (0.7-2.24), cigarette smoking AOR 6.09; CI (1.65-2.50) and patients who had an intermittent fever (AOR = 2.54, 95% CI = 1.21-5.4) were strongly associated with MDR-TB development. CONCLUSIONS: The magnitude of MDR-TB observed among new and previously treated patients is very alarming, which calls for an urgent need for intervention. The high proportion of MDR-TB among newly diagnosed cases indicates ongoing transmission, which suggests the need for enhanced TB control program performance to interrupt transmission. The increased proportion of MDR-TB among previously treated cases indicates a need for better patient management to prevent the evolution of drug resistance. Assessing the TB control program performance gaps and an optimal implementation of the WHO recommended priority actions for the management of drug-resistant TB, is imperative to help reduce the current high MDR-TB burden in the study region.

Antibiotic resistance: turning evolutionary principles into clinical reality.

Antibiotic resistance is one of the major challenges facing modern medicine worldwide. The past few decades have witnessed rapid progress in our understanding of the multiple factors that affect the emergence and spread of antibiotic resistance at the population level and the level of the individual patient. However, the process of translating this progress into health policy and clinical practice has been slow. Here, we attempt to consolidate current knowledge about the evolution and ecology of antibiotic resistance into a roadmap for future research as well as clinical and environmental control of antibiotic resistance. At the population level, we examine emergence, transmission and dissemination of antibiotic resistance, and at the patient level, we examine adaptation involving bacterial physiology and host resilience. Finally, we describe new approaches and technologies for improving diagnosis and treatment and minimizing the spread of resistance.

Molecular epidemiology of M. tuberculosis in Ethiopia: A systematic review and meta-analysis.

The molecular epidemiology of Mycobacterium tuberculosis (M. tuberculosis, Mtb) is poorly documented in Ethiopia. The data that exists has not yet been collected in an overview metadata form. Thus, this review summarizes available literature on the genomic diversity, geospatial distribution and transmission patterns of Mtb lineages (L) and sublineages in Ethiopia. Spoligotyping and Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats (MIRU-VNTR) based articles were identified from MEDLINE via PubMed and Scopus. The last date of article search was done on 12th February 2019. Articles were selected following the PRISMA flow diagram. The proportion of (sub)lineages was summarized at national level and further disaggregated by region. Clustering and recent transmission index (RTI) were determined using metan command and random effect meta-analysis model. The meta-analysis was computed using Stata 14 (Stata Corp. College Station, TX, USA). Among 4371 clinical isolates, 99.5% were Mtb and 0.5% were M. bovis. Proportionally, L4, L3, L1 and L7 made up 62.3%, 21.7%, 7.9% and 3.4% of the total isolates, respectively. Among sublineages, L4.2. ETH/SIT149, L4.10/SIT53, L3. ETH1/SIT25 and L4.6/SIT37 were the leading clustered isolates accounting for 14.4%, 9.7%, 7.2% and 5.5%, respectively. Based on MIRU-VNTR, the rate of clustering was 41% and the secondary case rate from a single source case was estimated at 29%. Clustering and recent transmission index was higher in eastern and southwestern Ethiopia compared with the northwestern part of the country. High level of genetic diversity with a high rate of clustering was noted which collectively mirrored the phenomena of micro-epidemics and super-spreading. The largest set of clustered strains deserves special attention and further characterization using whole genome sequencing (WGS) to better understand the evolution, genomic diversity and transmission dynamics of Mtb.

Ample glycosylation in membrane and cell envelope proteins may explain the phenotypic diversity and virulence in the Mycobacterium tuberculosis complex.

Multiple regulatory mechanisms including post-translational modifications (PTMs) confer complexity to the simpler genomes and proteomes of Mycobacterium tuberculosis (Mtb). PTMs such as glycosylation play a significant role in Mtb adaptive processes. The glycoproteomic patterns of clinical isolates of the Mycobacterium tuberculosis complex (MTBC) representing the lineages 3, 4, 5 and 7 were characterized by mass spectrometry. A total of 2944 glycosylation events were discovered in 1325 proteins. This data set represents the highest number of glycosylated proteins identified in Mtb to date. O-glycosylation constituted 83% of the events identified, while 17% of the sites were N-glycosylated. This is the first report on N-linked protein glycosylation in Mtb and in Gram-positive bacteria. Collectively, the bulk of Mtb glycoproteins are involved in cell envelope biosynthesis, fatty acid and lipid metabolism, two-component systems, and pathogen-host interaction that are either surface exposed or located in the cell wall. Quantitative glycoproteomic analysis revealed that 101 sites on 67 proteins involved in Mtb fitness and survival were differentially glycosylated between the four lineages, among which 64% were cell envelope and membrane proteins. The differential glycosylation pattern may contribute to phenotypic variabilities across Mtb lineages. The study identified several clinically important membrane-associated glycolipoproteins that are relevant for diagnostics as well as for drug and vaccine discovery.