Cell Wall Of Mycobacterium Tuberculosis Research Articles

Prediction of Mycobacterium tuberculosis cell wall permeability using machine learning methods.

Tuberculosis (TB) caused by the bacteria Mycobacterium tuberculosis (M. tb), continues to pose a significant worldwide health threat. The advent of drug-resistant strains of the disease highlights the critical need for novel treatments. The unique cell wall of M. tb provides an extra layer of protection for the bacteria and hence only compounds that can penetrate this barrier can reach their targets within the bacterial cell wall. The creation of a reliable machine learning (ML) model to predict the mycobacterial cell wall permeability of small molecules is presented in this work and four ML algorithms, including Random Forest, Support Vector Machines (SVM), k-nearest Neighbour (k-NN) and Logistic Regression were trained on a dataset of 5368 compounds. RDKit and Mordred toolkits were used to calculate features. To determine the most effective model, various performance metrics were used such as accuracy, precision, recall, F1 score, and area under the receiver operating characteristic curve. The best-performing model was further refined with hyperparameter tuning and tenfold cross-validation. The SVM model with filtering outperformed the other machine learning models and demonstrated 80.26% and 81.13% accuracy on the test and validation datasets, respectively. The study also provided insights into the molecular descriptors that play the most important role in predicting the ability of a molecule to pass the M. tb cell wall, which could guide future compound design. The model is available at https://github.com/PGlab-NIPER/MTB_Permeability .

Molecular cloning, expression, purification and functional characterization of Rv1588c of Mycobacteriumtuberculosis

Biotin is an essential cofactor for biotin dependent enzymes necessary for synthesis of fatty acids including mycolic acids, an essential component of cell wall of Mycobacterium tuberculosis. Biotin is synthesized by the genes located in bio operons which are fairly conserved in mycobacteria. Biotin synthesis is controlled intracellularly in Mycobacterium smegmatis by the gene bioQ, located opposite to bioB. Such locations of biotin regulators are found to be conserved among various mycobacteria species. In M. tuberculosis, rv1588c is such an uncharacterized gene, located opposite to bioB which shares limited similarity with DNA-binding region of BioQ of M. smegmatis, a TetR-type protein. A highly similar M. smegmatis-like BioQ binding site is also present within the bioB promoter of M. tuberculosis. This suggests that Rv1588c might be the BioQ for M. tuberculosis. The present study reports in-silico and functional characterization of Rv1588c. The structure of Rv1588c has been partially modelled and validated computationally. The N-terminal domain of Rv1588c is found to be structurally similar with DNA-binding domains of several transcriptional regulators. rv1588c gene has been cloned, expressed, and purified. Limited proteolysis study revealed presence of C-terminal domain in Rv1588c which indicates presence of a two-domain structure, like those of canonical transcriptional regulators. Rv1588c forms significant amount of dimers in solution, mediated by cysteine residues. Purified Rv1588c binds with bioB promoter region in-vitro. This suggests that Rv1588c is probably the transcriptional regulator that might control the expression of bioB of M. tuberculosis.

Design, Synthesis, Characterization and Antitubercular Activity of Novel Benzimidazole Mannich Base Derivatives

In present work, the newly synthesized benzimidazole Mannich base derivatives were design, synthesized and evaluated the in silico and in vitro antitubercular activity. These compounds were synthesized by condensation reaction between 1-(1H-benzo[d]imidazol-1-yl)ethanone and aliphatic/aromatic amines. The synthesized compound structures were identified by FTIR, 13C NMR, 1H NMR and mass spectroscopies. The results indicated that these derivatives have significant antitubercular activity against Mycobacterium tuberculosis (M.tb) cell wall enzyme enoyl acyl carrier protein reductase (InhA), EthR regulatory protein in H73Rv strain. The results found in the in vitro study are firmly similar to the in silico study. Among the synthesized compounds, 3d and 3e exhibited the highest activity due to the connection of the electron-donating group to the Mannich base. Therefore, these compounds deserve the development of new antitubercular agents.

Docking Studies, Synthesis, SAR and Anti-TB Activity of Glycinamido Analogues

Mycolic acid is a crucial component of the Mycobacterium tuberculosis cell wall and mycolic acid methyltransferases (MAMTs) are essential for mycolic acids to mature. In the present study, an inhouse library of 330 ligands was designed taking glycinamido moiety as scaffold. Virtual screening was carried out with this library of compounds against MmaA1 as the target protein. About 55 hits were identified through docking, ADMET studies and these molecules were synthesized by the Schotten-Baumann reaction followed by a nucleophilic substitution reaction. All the compounds were subjected to in vitro anti-Tb screening by microplate alamar blue assay (MABA). The Mdb1, Mdb4 & Meb1 exhibited excellent activity against M. tuberculosis H37Rv bacilli strain with an MIC of 1.56 µg/mL. The SAR studies shows that the aryl ring attached directly to the nitrogen atom as present in 2(-N-substituted glycinamido) derivatives is essential for the compound to exhibit potent anti-TB activity.

3D-QSAR and ADMET studies of morpholino-pyrimidine inhibitors of DprE1 from Mycobacterium tuberculosis

DprE1 is involved in the synthesis of Mycobacterium tuberculosis cell wall and is a potent drug target for Tuberculosis (TB) treatment. The structure and dynamics of the loops L-I and L-II flanking the inhibitor binding site was studied using molecular dynamics (MD) simulation and MMPBSA in Amber v18. Docking and three-dimensional quantitative structure–activity relationship (3D-QSAR) of 55 Morpholino-pyrimidine (MP) inhibitors was carried out using Autodock v1.2.0 and Forge v10. ADMET analysis was done using SwissADME and pkCSM. All MP inhibitors docked in the DprE1 binding pocket, making contacts with L-II residues. MD studies showed that L-I and L-II unfold in the absence of the inhibitor but fold stably structure with reduced protein motions in the presence of MP-38, the highest affinity inhibitor. This was confirmed by k-means clustering and secondary structure analysis. L-II residues, L317, F320 and R325 contributed most towards the MMPBSA binding free energy of MP-38. A robust field-based 3D-QSAR model showed values of r2 train = 0.982, r2 test = 0.702 and q2 = 0.516. The MP inhibitor field points were broadly divided into negative electrostatics near the A, B rings and hydrophobic electrostatics near the D, E rings. Addition of negative groups at methanone position and ring B as well as addition of hydrophobic and bulky groups at ring E will improve activity. Highly active compounds 47, 49 and 50 of MP series exhibited highly favourable drug-like properties. SAR and ADMET insights attained from this model will help in the development of active DprE1 inhibitors in future. Communicated by Ramaswamy H. Sarma

Identification of potential molecular targets and repurposed drugs for tuberculosis using network-based screening approach, molecular docking, and simulation

The spread of drug-resistant strains of tuberculosis has hampered efforts to control the disease worldwide. The Mycobacterium tuberculosis cell wall envelope is dynamic, with complex features that protect it from the host immunological response. As a result, the bacterial cell wall components represent a potential target for drug discovery. Protein-protein interaction networks (PPIN) are critical for understanding disease conditions and identifying precise therapeutic targets. We used a rational theoretical approach by constructing a PPIN with the proteins involved in cell wall biosynthesis. The PPIN was constructed through the STRING database and embB was identified as a key protein by using four topological measures, betweenness, closeness, degree, and eigenvector, in the CytoNCA tool in Cytoscape. The ‘Drug repurposing’ approach was employed to find suitable inhibitors against embB. We used the Schrödinger suites for molecular docking, molecular dynamics simulation, and binding free energy calculations to validate the binding of protein with the ligand. FDA-approved drugs from the ZINC database and DrugBank were screened against embB (PDB ID: 7BVF) using high-throughput virtual screening, standard precision, and extra precision docking. The drugs were screened based on the XP docking score of the standard drug ethambutol. Accordingly, from the top five hits, azilsartan and dihydroergotamine were selected based on the binding free energy values and were further subjected to Molecular Dynamics Simulation studies for 100 ns. Our study confirms that Azilsartan and Dihydroergotamine form stable complexes with embB and can be used as potential lead molecules based on further in vitro and in vivo experimental validation. Communicated by Ramaswamy H. Sarma

Drug distribution and efficacy of the DprE1 inhibitor BTZ-043 in the C3HeB/FeJ mouse tuberculosis model.

BTZ-043, a suicide inhibitor of the Mycobacterium tuberculosis cell wall synthesis decaprenylphosphoryl-beta-D-ribose 2' epimerase, is under clinical development as a potential new anti-tuberculosis agent. BTZ-043 is potent and bactericidal in vitro but has limited activity against non-growing bacilli in rabbit caseum. To better understand its behavior in vivo, BTZ-043 was evaluated for efficacy and spatial drug distribution as a single agent in the C3HeB/FeJ mouse model presenting with caseous necrotic pulmonary lesions upon Mycobacterium tuberculosis infection. BTZ-043 promoted significant reductions in lung and spleen bacterial burdens in the C3HeB/FeJ mouse model after 2 months of therapy. BTZ-043 penetrates cellular and necrotic lesions and was retained at levels above the serum-shifted minimal inhibitory concentration in caseum. The calculated rate of kill was found to be highest and dose-dependent during the second month of treatment. BTZ-043 treatment was associated with improved histology scores of pulmonary lesions, especially compared to control mice, which experienced advanced fulminant neutrophilic alveolitis in the absence of treatment. These positive treatment responses to BTZ-043 monotherapy in a mouse model of advanced pulmonary disease can be attributed to favorable distribution in tissues and lesions, retention in the caseum, and its high potency and bactericidal nature at drug concentrations achieved in necrotic lesions.

Discovery of novel and potent InhA direct inhibitors by ensemble docking-based virtual screening and biological assays.

Multidrug-resistant tuberculosis (MDR-TB) continues to spread worldwide and remains one of the leading causes of death among infectious diseases. The enoyl-acyl carrier protein reductase (InhA) belongs to FAS-II family and is essential for the formation of the Mycobacterium tuberculosis cell wall. Recent years, InhA direct inhibitors have been extensively studied to overcome MDR-TB. However, there are still no inhibitors that have entered clinical research. Here, the ensemble docking-based virtual screening along with biological assay were used to identify potent InhA direct inhibitors from Chembridge, Chemdiv, and Specs. Ultimately, 34 compounds were purchased and first assayed for the binding affinity, of which four compounds can bind InhA well with KD values ranging from 48.4 to 56.2 µM. Among them, compound 9,222,034 has the best inhibitory activity against InhA enzyme with an IC50 value of 18.05 µM. In addition, the molecular dynamic simulation and binding free energy calculation indicate that the identified compounds bind to InhA with "extended" conformation. Residue energy decomposition shows that residues such as Tyr158, Met161, and Met191 have higher energy contributions in the binding of compounds. By analyzing the binding modes, we found that these compounds can bind to a hydrophobic sub-pocket formed by residues Tyr158, Phe149, Ile215, Leu218, etc., resulting in extensive van der Waals interactions. In summary, this study proposed an efficient strategy for discovering InhA direct inhibitors through ensemble docking-based virtual screening, and finally identified four active compounds with new skeletons, which can provide valuable information for the discovery and optimization of InhA direct inhibitors.

Synthesis, Characterization Gd(Iii) Complex with Heptilmethyl- Ditiocarbamate and 2,9-Dimethyl Phenanthroline as Anti-Tuberculosis

Mycobacterium tuberculosis is a bacterium that causes tuberculosis infection disease. The aim of this study was to produce complex compounds that have the ability to damage the cell wall of Mycobacterium tuberculosis. Gd(III)Heptylmethyldithiocarbamate 2,9-dimethyl phenanthroline complex was synthesised and characterised by reacting N-heptylmethylamine, carbondisulphide, 2,9-dimethyl phenanthroline and lanthanide group metal ion (Gadolinium) using in situ method. Characterisation was carried out using electrothermal melting point, conductometer, UV-Vis spectrophotometer, and FT-IR spectrophotometer. The resulting Gd(III)Heptylmethyldithiocarbamate 2,9-dimethyl phenanthroline complex was white in colour as much as 7.01%, conductivity 20 Ω-1 and melting point 208-210o C. Testing of complex compounds gave effective results as antibacterial Mycobacterium tuberculosis.

Structural basis for the development of potential inhibitors targeting FadD23 from Mycobacterium tuberculosis.

Sulfolipid-1 (SL-1) is a lipid that is abundantly found in the cell wall of Mycobacterium tuberculosis (Mtb). MtbFadD23 is crucial in the SL-1 synthesis pathway. Previously, 5'-O-[N-(11-phenoxyundecanoyl)sulfamoyl]adenosine (PhU-AMS) has been shown to be a general inhibitor of fatty-acid-adenylating enzymes (FadDs) in Mtb. However, the fatty acyl-AMP ligase (FAAL) class of FadDs, which includes MtbFadD23, appears to be functionally nonredundant in the production of multiple fatty acids. In this study, the ability of PhU-AMS to bind to MtbFadD23 was examined under in vitro conditions. The crystal structure of the MtbFadD23-PhU-AMS complex was determined at a resolution of 2.64 Å. Novel features were identified by structural analysis and comparison. Although PhU-AMS could bind to MtbFadD23, it did not inhibit the FAAL adenylation activity of MtbFadD23. However, PhU-AMS improved the main Tm value in a differential scanning fluorimetry assay, and a structural comparison of MtbFadD23-PhU-AMS with FadD32 and PA1221 suggested that PhU-AMS blocks the loading of the acyl chain onto Pks2. This study sheds light on the structure-based design of specific inhibitors of MtbFadD23 and general inhibitors of FAALs.

Prospective Subunit Nanovaccine against Mycobacterium tuberculosis Infection─Cubosome Lipid Nanocarriers of Cord Factor, Trehalose 6,6' Dimycolate.

An improved vaccine is urgently needed to replace the now more than 100-year-old Bacillus Calmette-Guérin (BCG) vaccine against tuberculosis (TB) disease, which represents a significant burden on global public health. Mycolic acid, or cord factor trehalose 6,6' dimycolate (TDM), a lipid component abundant in the cell wall of the pathogen Mycobacterium tuberculosis (MTB), has been shown to have strong immunostimulatory activity but remains underexplored due to its high toxicity and poor solubility. Herein, we employed a novel strategy to encapsulate TDM within a cubosome lipid nanocarrier as a potential subunit nanovaccine candidate against TB. This strategy not only increased the solubility and reduced the toxicity of TDM but also elicited a protective immune response to control MTB growth in macrophages. Both pre-treatment and concurrent treatment of the TDM encapsulated in lipid monoolein (MO) cubosomes (MO-TDM) (1 mol %) induced a strong proinflammatory cytokine response in MTB-infected macrophages, due to epigenetic changes at the promoters of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in comparison to the untreated control. Furthermore, treatment with MO-TDM (1 mol %) cubosomes significantly improved antigen processing and presentation capabilities of MTB-infected macrophages to CD4 T cells. The ability of MO-TDM (1 mol %) cubosomes to induce a robust innate and adaptive response in vitro was further supported by a mathematical modeling study predicting the vaccine efficacy in vivo. Overall, these results indicate a strong immunostimulatory effect of TDM when delivered through the lipid nanocarrier, suggesting its potential as a novel TB vaccine.

Mycobacterium tuberculosis cell-wall and antimicrobial peptides: a mission impossible?

Mycobacterium tuberculosis (Mtb) is one of the most important infectious agents worldwide and causes more than 1.5 million deaths annually. To make matters worse, the drug resistance among Mtb strains has risen substantially in the last few decades. Nowadays, it is not uncommon to find patients infected with Mtb strains that are virtually resistant to all antibiotics, which has led to the urgent search for new molecules and therapies. Over previous decades, several studies have demonstrated the efficiency of antimicrobial peptides to eliminate even multidrug-resistant bacteria, making them outstanding candidates to counterattack this growing health problem. Nevertheless, the complexity of the Mtb cell wall makes us wonder whether antimicrobial peptides can effectively kill this persistent Mycobacterium. In the present review, we explore the complexity of the Mtb cell wall and analyze the effectiveness of antimicrobial peptides to eliminate the bacilli.

Identification of d-arabinan-degrading enzymes in mycobacteria

Bacterial cell growth and division require the coordinated action of enzymes that synthesize and degrade cell wall polymers. Here, we identify enzymes that cleave the d-arabinan core of arabinogalactan, an unusual component of the cell wall of Mycobacterium tuberculosis and other mycobacteria. We screened 14 human gut-derived Bacteroidetes for arabinogalactan-degrading activities and identified four families of glycoside hydrolases with activity against the d-arabinan or d-galactan components of arabinogalactan. Using one of these isolates with exo-d-galactofuranosidase activity, we generated enriched d-arabinan and used it to identify a strain of Dysgonomonas gadei as a d-arabinan degrader. This enabled the discovery of endo- and exo-acting enzymes that cleave d-arabinan, including members of the DUF2961 family (GH172) and a family of glycoside hydrolases (DUF4185/GH183) that display endo-d-arabinofuranase activity and are conserved in mycobacteria and other microbes. Mycobacterial genomes encode two conserved endo-d-arabinanases with different preferences for the d-arabinan-containing cell wall components arabinogalactan and lipoarabinomannan, suggesting they are important for cell wall modification and/or degradation. The discovery of these enzymes will support future studies into the structure and function of the mycobacterial cell wall.

In SilicoIdentification of Triclosan Derivatives as Potential Inhibitors of MutantMycobacterium tuberculosisInhA

Enoyl acyl carrier protein reductase (InhA) is a crucial enzyme for the biosynthesis of mycolic acids which are major compartments of the Mycobacterium tuberculosis (Mtb) cell wall. Direct inhibition of InhA without activation by drug-NADH adduct has clinical utility to overcome drug resistance. We aimed at the in silico identification of triclosan derivatives with the potential inhibitory effect of S94A-InhA as a clinically important mutant form. Caver Web 1.0 server was used to analyze the ligand transport through access tunnels. Two macrocyclic triclosan derivatives ( 4 and 6) could be identified with more energy-favorable transfer routes toward the enzyme active site. Molecular dynamics (MD) simulations (50 ns) of the best-scored compounds revealed the stability of enzyme structure upon binding to 4 and 6. Compound 4 could better retain enzyme stability upon target binding. Results of intermolecular H-bond analysis indicated that both complexes were mediated through hydrophobic contacts. Declined solvent accessible surface area (SASA) for the apo and bound enzyme states indicated non-destabilizing behavior and no structural relaxation. Electrostatic and van der Waals interactions between triclosan derivatives and their surroundings were used to acquire binding free energies through the linear interaction energy (LIE) method based on MD simulations (Average [Formula: see text], [Formula: see text] kcal/mol and [Formula: see text] kcal/mol). Both of the triclosan derivatives showed relatively stable energy variations and their steady accommodation inside enzyme active site could be confirmed during 50 ns. These results may be implicated in further structure-guided approaches against drug-resistant Mtb.

The Key Roles of Mycobacterium tuberculosis FadD23 C-terminal Domain in Catalytic Mechanisms.

Sulfolipid-1 (SL-1) is located in the Mycobacterium tuberculosis (M. tb) cell wall, and is essential for pathogen virulence and intracellular growth. Multiple proteins (e.g., Pks2, FadD23, PapA1, and MmpL8) in the SL-1 synthesis pathway can be treated as drug targets, but, to date, their structures have not been solved. The crystal structures of FadD23 bound to ATP or hexadecanoyl adenylate was determined in this study. We have also investigated long-chain saturated fatty acids as biological substrates of FadD23 through structural, biological, and chemical analyses. The mutation at the active site of FadD23 greatly influences enzymatic activity. Meanwhile, the FadD23 N-terminal domain alone cannot bind palmitic acid without C-terminal domain facilitation since it is almost inactive after removing the C-terminal domain. FadD23 is the first protein in the SL-1 synthesis pathway whose structure has been solved. These results reveal the importance of the C-terminal domain in the catalytic mechanism.

Structure and dynamics of the essential endogenous mycobacterial polyketide synthase Pks13.

The mycolic acid layer of the Mycobacterium tuberculosis cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are targets for antimycobacterial drug development. Polyketide synthase 13 (Pks13) is a module encoding several enzymatic and transport functions that carries out the condensation of two different long-chain fatty acids to produce mycolic acids. We determined structures by cryogenic-electron microscopy of dimeric multi-enzyme Pks13 purified from mycobacteria under normal growth conditions, captured with native substrates. Structures define the ketosynthase (KS), linker and acyl transferase (AT) domains at 1.8 Å resolution and two alternative locations of the N-terminal acyl carrier protein. These structures suggest intermediate states on the pathway for substrate delivery to the KS domain. Other domains, visible at lower resolution, are flexible relative to the KS-AT core. The chemical structures of three bound endogenous long-chain fatty acid substrates were determined by electrospray ionization mass spectrometry.

Mycobacterium tuberculosis Cell Wall Antigens Induce the Formation of Immune Complexes and the Development of Vasculitis in an Experimental Murine Model.

Tuberculosis (TB) of the central nervous system (CNS) presents high mortality due to brain damage and inflammation events. The formation and deposition of immune complexes (ICs) in the brain microvasculature during Mycobacterium tuberculosis (Mtb) infection are crucial for its pathobiology. The relevance of ICs to Mtb antigens in the pathogenesis of CNS-TB has been poorly explored. Here, we aimed to establish a murine experimental model of ICs-mediated brain vasculitis induced by cell wall antigens of Mtb. We administered a cell wall extract of the prototype pathogenic Mtb strain H37Rv to male BALB/c mice by subcutaneous and intravenous routes. Serum concentration and deposition of ICs onto blood vessels were determined by polyethylene glycol precipitation, ELISA, and immunofluorescence. Histopathological changes in the brain, lung, spleen, liver, and kidney were evaluated by hematoxylin and eosin staining. Our results evidenced that vasculitis developed in the studied tissues. High serum levels of ICs and vascular deposition were evident in the brain, lung, and kidneys early after the last cell wall antigen administration. Cell wall Mtb antigens induce strong type III hypersensitivity reactions and the development of systemic vasculitis with brain vascular changes and meningitis, supporting a role for ICs in the pathogenesis of TB.

Machine Learning Prediction of Mycobacterial Cell Wall Permeability of Drugs and Drug-like Compounds.

The cell wall of Mycobacterium tuberculosis and related organisms has a very complex and unusual organization that makes it much less permeable to nutrients and antibiotics, leading to the low activity of many potential antimycobacterial drugs against whole-cell mycobacteria compared to their isolated molecular biotargets. The ability to predict and optimize the cell wall permeability could greatly enhance the development of novel antitubercular agents. Using an extensive structure-permeability dataset for organic compounds derived from published experimental big data (5371 compounds including 2671 penetrating and 2700 non-penetrating compounds), we have created a predictive classification model based on fragmental descriptors and an artificial neural network of a novel architecture that provides better accuracy (cross-validated balanced accuracy 0.768, sensitivity 0.768, specificity 0.769, area under ROC curve 0.911) and applicability domain compared with the previously published results.

A recombinant Mycobacterium smegmatis-based surface display system for developing the T cell-based COVID-19 vaccine

ABSTRACT The immune escape mutations of SARS-CoV-2 variants emerged frequently, posing a new challenge to weaken the protective efficacy of current vaccines. Thus, the development of novel SARS-CoV-2 vaccines is of great significance for future epidemic prevention and control. We herein reported constructing the attenuated Mycobacterium smegmatis (M. smegmatis) as a bacterial surface display system to carry the spike (S) and nucleocapsid (N) of SARS-CoV-2. To mimic the native localization on the surface of viral particles, the S or N antigen was fused with truncated PE_PGRS33 protein, which is a transportation component onto the cell wall of Mycobacterium tuberculosis (M.tb). The sub-cellular fraction analysis demonstrated that S or N protein was exactly expressed onto the surface (cell wall) of the recombinant M. smegmatis. After the immunization of the M. smegmatis-based COVID-19 vaccine candidate in mice, S or N antigen-specific T cell immune responses were effectively elicited, and the subsets of central memory CD4+ T cells and CD8+ T cells were significantly induced. Further analysis showed that there were some potential cross-reactive CTL epitopes between SARS-CoV-2 and M.smegmatis. Overall, our data provided insights that M. smegmatis-based bacterial surface display system could be a suitable vector for developing T cell-based vaccines against SARS-CoV-2 and other infectious diseases.

An Integrated approach for Proteomic and Immunological analysis of Cell wall and Cell membrane proteins of Mycobacterium tuberculosis

An Integrated approach for Proteomic and Immunological analysis of Cell wall and Cell membrane proteins of Mycobacterium tuberculosis