Virulence Of Mycobacterium Tuberculosis Research Articles

Suppressing Mycobacterium tuberculosis virulence and drug resistance by targeting Eis protein through computational drug discovery.

Tuberculosis (TB) remains a critical health threat, particularly with the emergence of multidrug-resistant strains. This demands attention from scientific communities and healthcare professionals worldwide to develop effective treatments. The enhanced intracellular survival (Eis) protein is an acetyltransferase enzyme of Mycobacterium tuberculosis that functions by adding acetyl groups to aminoglycoside antibiotics, which interferes with their ability to bind to the bacterial ribosome, thereby preventing them from inhibiting protein synthesis and killing the bacterium. Therefore, targeting this protein accelerates the chance of restoring the aminoglycoside drug activity, thereby reducing the emergence of drug-resistant TB. For this, we have screened 406,747 natural compounds from the Coconut database against Eis protein. Based on MM/GBSA rescoring binding energy, the top 5 most prominent natural compounds, viz. CNP0187003 (-96.14kcal/mol), CNP0176690 (-93.79kcal/mol), CNP0136537 (-92.31kcal/mol), CNP0398701 (-91.96kcal/mol), and CNP0043390 (-91.60kcal/mol) were selected. These compounds exhibited the presence of a substantial number of hydrogen bonds and other significant interactions confirming their strong binding affinity with the Eis protein during the docking process. Subsequently, the MD simulation of these compounds exhibited that the Eis-CNP0043390 complex was the most stable, followed by Eis-CNP0187003 and Eis-CNP0176690 complex, further verified by binding free energy calculation, principal component analysis (PCA), and Free energy landscape analysis. These compounds demonstrated the most favourable results in all parameters utilised for this investigation and may have the potential to inhibit the Eis protein. There these findings will leverage computational techniques to identify and develop a natural compound inhibitor as an alternative for drug-resistant TB.

Toxin-antitoxin system gene mutations driving Mycobacterium tuberculosis transmission revealed by whole genome sequencing

BackgroundThe toxin-antitoxin (TA) system plays a vital role in the virulence and pathogenicity of Mycobacterium tuberculosis (M. tuberculosis). However, the regulatory mechanisms and the impact of gene mutations on M. tuberculosis transmission remain poorly understood.ObjectiveTo investigate the influence of gene mutations in the toxin-antitoxin system on M. tuberculosis transmission dynamics.MethodWe performed whole-genome sequencing on the analyzed strains of M. tuberculosis. The genes associated with the toxin-antitoxin system were obtained from the National Center for Biotechnology Information (NCBI) Gene database. Mutations correlating with enhanced transmission within the genes were identified by using random forest, gradient boosting decision tree, and generalized linear mixed models.ResultsA total of 13,518 M. tuberculosis isolates were analyzed, with 42.29% (n = 5,717) found to be part of genomic clusters. Lineage 4 accounted for the majority of isolates (n = 6488, 48%), followed by lineage 2 (n = 5133, 37.97%). 23 single nucleotide polymorphisms (SNPs) showed a positive correlation with clustering, including vapB1 G34A, vapB24 A76C, vapB2 T171C, mazF2 C85T, mazE2 G104A, vapB31 T112C, relB T226A, vapB11 C54T, mazE5 T344C, vapB14 A29G, parE1 (C103T, C88T), and parD1 C134T. Six SNPs, including vapB6 A29C, vapB31 T112C, parD1 C134T, vapB37 G205C, Rv2653c A80C, and vapB22 C167T, were associated with transmission clades across different countries. Notably, our findings highlighted the positive association of vapB6 A29C, vapB31 T112C, parD1 C134T, vapB37 G205C, vapB19 C188T, and Rv2653c A80C with transmission clades across diverse regions. Furthermore, our analysis identified 32 SNPs that exhibited significant associations with clade size.ConclusionOur study presents potential associations between mutations in genes related to the toxin-antitoxin system and the transmission dynamics of M. tuberculosis. However, it is important to acknowledge the presence of confounding factors and limitations in our study. Further research is required to establish causation and assess the functional significance of these mutations. These findings provide a foundation for future investigations and the formulation of strategies aimed at controlling TB transmission.

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.

Current landscape of exosomes in tuberculosis development, diagnosis, and treatment applications

Tuberculosis (TB), caused by the bacterial pathogen Mycobacterium tuberculosis (MTB), remains one of the most prevalent and deadly infectious diseases worldwide. Currently, there are complex interactions between host cells and pathogens in TB. The onset, progression, and regression of TB are correlated not only with the virulence of MTB but also with the immunity of TB patients. Exosomes are cell-secreted membrane-bound nanovesicles with lipid bilayers that contain a variety of biomolecules, such as metabolites, lipids, proteins, and nucleic acids. Exosome-mediated cell−cell communication and interactions with the microenvironment represent crucial mechanisms through which exosomes exert their functional effects. Exosomes harbor a wide range of regulatory roles in physiological and pathological conditions, including MTB infection. Exosomes can regulate the immune response, metabolism, and cellular death to remodel the progression of MTB infection. During MTB infection, exosomes display distinctive profiles and quantities that may act as diagnostic biomarkers, suggesting that exosomes provide a revealing glimpse into the evolving landscape of MTB infections. Furthermore, exosomes derived from MTB and mesenchymal stem cells can be harnessed as vaccine platforms and drug delivery vehicles for the precise targeting and treatment of TB. In this review, we highlight the functions and mechanisms through which exosomes influence the progression of TB. Additionally, we unravel the critical significance of exosomal constituents in the diagnosis and therapeutic applications of TB, aiming to offer novel perspectives and strategies for combating TB.

Molecular insights into ferric-siderophore transport by the putative TonB-dependent transporter in Mycobacterium tuberculosis

Iron acquisition is critical to the virulence of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To acquire iron within the host, Mtb secretes siderophores that chelate iron with high affinity. Siderophores scavenge iron from host cells using TonB-dependent transporters like FecA. We investigated molecular mechanisms of FecA-mediated ferric-siderophore transport in Mtb. Molecular docking and molecular dynamics simulations revealed a series of interactions between ferric siderophores and FecA. The initial binding occurs at a pocket located on the extracellular surface of FecA. The ligand then migrates deeper through the transport tunnel to a subsequent binding site, aided by conformational changes in FecA that expand the tunnel diameter. We observed the key roles of precise positioning of extracellular loops in the outer membrane barrel and plug domains in the optimal ligand binding and transport. Transport of ferric–siderophore complex into Mtb follows an induced fit model, with ligand interaction eliciting 2–10 Å shifts in the barrel and plug regions. By revealing the conformational dynamics enabling iron import, these findings provide molecular-level insights into a metal ion uptake mechanism in Mtb. Iron acquisition is essential for Mtb pathogenesis, so this work may inform novel therapeutic strategies that disrupt siderophore uptake pathways. Communicated by Ramaswamy H. Sarma

The Role of Rv1476 in Regulating Stress Response and Intracellular Survival of Mycobacterium tuberculosis.

The virulence of Mycobacterium tuberculosis (M. tuberculosis) is related to many factors, including intracellular survival, cell wall permeability, and cell envelope proteins. However, the biological function of the M. tuberculosis membrane protein Rv1476 remains unclear. To investigate the potential role played by Rv1476, we constructed an Rv1476 overexpression strain and found that overexpression of Rv1476 enhanced the intracellular survival of M. tuberculosis, while having no impact on the growth rate in vitro. Stress experiments demonstrated that the Rv1476 overexpression strain displayed increased susceptibility to different stresses compared to the wild-type strain. Transcriptome analysis showed that Rv1476 overexpression causes changes in the transcriptome of THP-1 cells, and differential genes are mainly enriched in cell proliferation, fatty acid degradation, cytokine-cytokine receptor interaction, and immune response pathways. Rv1476 overexpression inhibited the expression of some anti-tuberculosis-related genes, such as CCL1, IL15, IL16, ISG15, GBP5, IL23, ATG2A, IFNβ, and CSF3. Altogether, we conclude that Rv1476 may play a critical role for M. tuberculosis in macrophage survival.

The Role of Proline-Proline-Glutamic Acid (PPE) Proteins in Mycobacterium tuberculosis Virulence: Mechanistic Insights and Therapeutic Implications

The Role of Proline-Proline-Glutamic Acid (PPE) Proteins in Mycobacterium tuberculosis Virulence: Mechanistic Insights and Therapeutic Implications

TB impairs DNA repair.

A Mycobacterium tuberculosis virulence factor promotes foam cell formation by inhibiting DNA repair.

Dual functioning by the PhoR sensor is a key determinant to Mycobacterium tuberculosis virulence

PhoP-PhoR, one of the 12 two-component systems (TCSs) that empower M. tuberculosis to sense and adapt to diverse environmental conditions, remains essential for virulence, and therefore, represents a major target to develop novel anti-TB therapies. Although both PhoP and PhoR have been structurally characterized, the signal(s) that this TCS responds to remains unknown. Here, we show that PhoR is a sensor of acidic pH/high salt conditions, which subsequently activate PhoP via phosphorylation. In keeping with this, transcriptomic data uncover that acidic pH- inducible expression of PhoP regulon is significantly inhibited in a PhoR-deleted M. tuberculosis. Strikingly, a set of PhoP regulon genes displayed a low pH-dependent activation even in the absence of PhoR, suggesting the presence of non-canonical mechanism(s) of PhoP activation. Using genome-wide interaction-based screening coupled with phosphorylation assays, we identify a non-canonical mechanism of PhoP phosphorylation by the sensor kinase PrrB. To investigate how level of P~PhoP is regulated, we discovered that in addition to its kinase activity PhoR functions as a phosphatase of P~PhoP. Our subsequent results identify the motif/residues responsible for kinase/phosphatase dual functioning of PhoR. Collectively, these results uncover that contrasting kinase and phosphatase functions of PhoR determine the homeostatic mechanism of regulation of intra-mycobacterial P~PhoP which controls the final output of the PhoP regulon. Together, these results connect PhoR to pH-dependent activation of PhoP with downstream functioning of the regulator. Thus, PhoR plays a central role in mycobacterial adaptation to low pH conditions within the host macrophage phagosome, and a PhoR-deleted M. tuberculosis remains significantly attenuated in macrophages and animal models.

Association between fatty acid metabolism gene mutations and Mycobacterium tuberculosis transmission revealed by whole genome sequencing

BackgroundFatty acid metabolism greatly promotes the virulence and pathogenicity of Mycobacterium tuberculosis (M.tb). However, the regulatory mechanism of fatty acid metabolism in M.tb remains to be elucidated, and limited evidence about the effects of gene mutations in fatty acid metabolism on the transmission of M.tb was reported.ResultsOverall, a total of 3193 M.tb isolates were included in the study, of which 1596 (50%) were genomic clustered isolates. Most of the tuberculosis isolates belonged to lineage2(n = 2744,85.93%), followed by lineage4(n = 439,13.75%) and lineage3(n = 10,0.31%).Regression results showed that the mutations of gca (136,605, 317G > C, Arg106Pro; OR, 22.144; 95% CI, 2.591-189.272), ogt(1,477,346, 286G > C ,Gly96Arg; OR, 3.893; 95%CI, 1.432–10.583), and rpsA (1,834,776, 1235 C > T, Ala412Val; OR, 3.674; 95% CI, 1.217–11.091) were significantly associated with clustering; mutations in gca and rpsA were also significantly associated with clustering of lineage2. Mutation in arsA(3,001,498, 885 C > G, Thr295Thr; OR, 6.278; 95% CI, 2.508–15.711) was significantly associated with cross-regional clusters. We also found that 20 mutation sites were positively correlated with cluster size, while 11 fatty acid mutation sites were negatively correlated with cluster size.ConclusionOur research results suggested that mutations in genes related to fatty acid metabolism were related to the transmission of M.tb. This research could help in the future control of the transmission of M.tb.

Mycobacterium tuberculosis virulence protein ESAT-6 influences M1/M2 polarization and macrophage apoptosis to regulate tuberculosis progression.

Tuberculosis (TB) is an infectious disease caused by infection with Mycobacterium tuberculosis (Mtb), and it remains one of the major threats to human health worldwide. To our knowledge, the polarization of M1/M2 macrophages were critical innate immune cells which play important roles in regulating the immune response during TB progression. We aimed to explore the potential mechanisms of M1/M2 macrophage polarization in TB development. THP-1 macrophages were treated with early secreted antigenic target of 6kDa (ESAT-6) protein for an increasing time. The polarization profiles, apoptosis levels of M1 and M2 macrophages were detected by RT-qPCR, immunofluorescence, Western blot and flow cytometry. ESAT-6 initially promoted the generation of pro-inflammatory M1-polarized macrophages in THP-1 cells within 24h, which were suppressed by further ESAT-6 treatment at 30-42h. Interestingly, ESAT-6 continuously promoted M2 polarization of THP-1 cells, thereby maintaining the anti-inflammatory response in a time-dependent manner. In addition, ESAT-6 promoted apoptotic cell death in M1-polarized macrophages, which had little effects on apoptosis of M2-phenotype of macrophages. Then, the potential underlying mechanisms were uncovered, and we verified that ESAT-6 increased the protein levels of TLR4, MyD88 and NF-κB to activate the TLR4/MyD88/NF-κB pathway within 24h, and this signal pathway was significantly inactivated at 36h post-treatment. Interestingly, the following experiments confirmed that ESAT-6 TLR4/MyD88/NF-κB pathway-dependently regulated M1/M2 polarization and apoptosis of macrophage in THP-1 cells. Our study investigated the detailed effects and mechanisms of M1/M2 macrophages in regulating innate responses during TB development, which provided a new perspective on the development of treatment strategies for this disease.

Non-clinical evaluation of local and systemic immunity induced by different vaccination strategies of the candidate tuberculosis vaccine M72/AS01

A new efficacious tuberculosis vaccine targeting adolescents/adults represents an urgent medical need. The M72/AS01E vaccine candidate protected half of the latently-infected adults against progression to pulmonary tuberculosis in a Phase IIb trial (NCT01755598). We report that three immunizations of mice, two weeks apart, with AS01-adjuvanted M72 induced polyfunctional, Th1-cytokine-expressing M72-specific CD4+/CD8+ T cells in blood and lungs, with the highest frequencies in lungs. Antigen-dose reductions across the three vaccinations skewed pulmonary CD4+ T-cell profiles towards IL-17 expression. In blood, reducing antigen and adjuvant doses of only the third injection (to 1/5th or 1/25th of those of the first injections) did not significantly alter CD4+ T-cell/antibody responses; applying a 10-week delay for the fractional third dose enhanced antibody titers. Delaying a full-dose booster enhanced systemic CD4+ T-cell and antibody responses. Cross-reactivity with PPE and non-PPE proteins was assessed, as Mycobacterium tuberculosis (Mtb) virulence factors and evasion mechanisms are often associated with PE/PPE proteins, to which Mtb39a (contained in M72) belongs. In silico/in vivo analyses revealed that M72/AS01 induced cross-reactive systemic CD4+ T-cell responses to epitopes in a non-vaccine antigen (putative latency-associated Mtb protein PPE24/Rv1753c). These preclinical data describing novel mechanisms of M72/AS01-induced immunity could guide future clinical development of the vaccine.

Development of a novel target-based cell assay, reporter of the activity of Mycobacterium tuberculosis protein-O-mannosyltransferase.

The Protein-O-mannosyltransferase is crucial for the virulence of Mycobacterium tuberculosis, the etiological agent of tuberculosis. This enzyme, called MtPMT (Rv1002c), is responsible for the post-translational O-mannosylation of mycobacterial proteins. It catalyzes the transfer of a single mannose residue from a polyprenol phospho-mannosyl lipidic donor to the hydroxyl groups of selected Ser/Thr residues in acceptor proteins during their translocation across the membrane. Previously, we provided evidence that the loss of MtPMT activity causes the absence of mannoproteins in Mycobacterium tuberculosis, severely impacting its intracellular growth, as well as a strong attenuation of its pathogenicity in immunocompromised mice. Therefore, it is of interest to develop specific inhibitors of this enzyme to better understand mycobacterial infectious diseases. Here we report the development of a "target-based" phenotypic assay for this enzyme, assessing its O-mannosyltransferase activity in bacteria, in the non-pathogenic Mycobacterium smegmatis strain. Robustness of the quantitative contribution of this assay was evaluated by intact protein mass spectrometry, using a panel of control strains, overexpressing the MtPMT gene, carrying different key point-mutations. Then, screening of a limited library of 30 compounds rationally chosen allowed us to identify 2 compounds containing pyrrole analogous rings, as significant inhibitors of MtPMT activity, affecting neither the growth of the mycobacterium nor its secretion of mannoproteins. These molecular cores could therefore serve as scaffold for the design of new pharmaceutical agents that could improve treatment of mycobacterial diseases. We report here the implementation of a miniaturized phenotypic activity assay for a glycosyltransferase of the C superfamily.

Structure elucidation and identification of novel lead molecules against Sulfur import protein cysA of Mycobacterium tuberculosis.

The present work considers the Sulphate import ABC transporter protein (cysA) as a potential drug target for the identification of inhibitors for the protein. The ABC (ATP binding cassette) transporters play a crucial role in the survival and virulence of Mycobacterium tuberculosis by the acquisition of micronutrients from host tissue. The 3D structural features of the cysA protein are built. Molecular scaffolds are identified by implementing active site identification, ADME properties, Virtual Screening, and a few other computational techniques. The theoretical model of cysA is predicted using homology modeling protocols, and the structure is validated by various validation methods. The prediction of partial dimer formation through protein-protein docking methods gave insight into the conformational changes taking place in the cysA protein. The natural substrate ATP is docked with cysA protein that confirms the ATP binding site. To find the drug-like compounds, virtual screening studies were carried out around the active site by several ligand databases. The findings demonstrate the significance of residues SER41, GLY42, ARG50, GLN85, HIS86, LYS91, ARG142, and ASP161 in drug-target interactions. The docking studies of existing TB drugs against cysA were also performed. The result analysis shows that none of the existing drugs inhibits the ATP active site, which confirms cysA as a promising drug target. Using in-silico methods, the ADME parameters of a few chosen ligand molecules are predicted and contrasted with the ADME characteristics of the available TB medications. The results revealed the values of ADME parameters of selected ligand molecules are more permissible than existing TB drugs, which emphasizes the drug-like activity of ligand molecules by inhibition of cysA proteins. The structural data, active site information, and selected ligand molecules help in the identification of new therapeutic scaffolds for Tuberculosis.

Multilayer precision-based screening of potential inhibitors targeting Mycobacterium tuberculosis acetate kinase using in silico approaches

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a major threat to global health, with the emergence of multi-drug and extensively drug-resistant strains posing a serious challenge. Thereby, understanding the molecular basis of MTB virulence and disease pathogenesis is critical for developing effective therapeutic strategies. Targeting proteins involved in central metabolism has been recognized as a promising therapeutic approach to combat MTB. In this regard, the enzyme AckA of the acetate metabolic pathway which produces acetate from acetyl phosphate, is an important drug target for various pathogenic organisms. Therefore, this study aimed to identify potential AckA inhibitors through in silico methods, including molecular modeling, molecular dynamics simulation (MDS), and high-throughput virtual screening (HTVS) followed by ADMETox, MMGBSA, Density Functional Theory (DFT) calculations. HTVS of one million compounds from the ZINC database against AckA resulted in the top five hits (ZINC82048449, ZINC1219737510, ZINC1771921358, ZINC119699567, and ZINC1427100376) with better binding affinity and optimal binding free energy. MDS studies on complexes revealed that key residues, Asn195, Asp266, Phe267, Gly314, and Asn318 played a significant role in stable interactions of the top-ranked compounds to AckA. These outcomes provide insights into the optimal binding of the leads to inhibit the acetate pathway and aid in the rational design of novel therapeutic agents. Thus, the identified leads may act as promising compounds for targeting AckA and may serve as a potential therapeutic modality for treating TB. Our findings offer valuable insights into the inhibition of the acetate pathway, while also serving as a blueprint for rational drug design. The identified leads hold promise as compelling compounds for targeting AckA, thereby offering a potential therapeutic avenue for tackling TB. Thus, our study uncovers a pathway toward promising TB therapeutics by elucidating AckA inhibitors. By leveraging in silico methodologies, potent compounds that hold the potential to thwart AckA's role in MTB's acetate pathway have been unveiled. This breakthrough fosters optimism in the quest for novel and effective TB treatments, addressing a global health challenge with renewed vigor.

Molecular recognition of trehalose and trehalose analogues by Mycobacterium tuberculosis LpqY-SugABC

Trehalose plays a crucial role in the survival and virulence of the deadly human pathogen Mycobacterium tuberculosis (Mtb). The type I ATP-binding cassette (ABC) transporter LpqY-SugABC is the sole pathway for trehalose to enter Mtb. The substrate-binding protein, LpqY, which forms a stable complex with the translocator SugABC, recognizes and captures trehalose and its analogues in the periplasmic space, but the precise molecular mechanism for this process is still not well understood. This study reports a 3.02-Å cryoelectron microscopy structure of trehalose-bound Mtb LpqY-SugABC in the pretranslocation state, a crystal structure of Mtb LpqY in a closed form with trehalose bound and five crystal structures of Mtb LpqY in complex with different trehalose analogues. These structures, accompanied by substrate-stimulated ATPase activity data, reveal how LpqY recognizes and binds trehalose and its analogues, and highlight the flexibility in the substrate binding pocket of LpqY. These data provide critical insights into the design of trehalose analogues that could serve as potential molecular probe tools or as anti-TB drugs.

Mycobacterium tuberculosis PptT Inhibitors Based on Heterocyclic Replacements of Amidinoureas.

4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme for Mycobacterium tuberculosis (Mtb) survival and virulence and therefore an attractive target for a tuberculosis therapeutic. In this work, two modeling-informed approaches toward the isosteric replacement of the amidinourea moiety present in the previously reported PptT inhibitor AU 8918 are reported. Although a designed 3,5-diamino imidazole unexpectedly adopted an undesired tautomeric form and was inactive, replacement of the amidinourea moiety afforded a series of active PptT inhibitors containing 2,6-diaminopyridine scaffolds.

Proximity-dependent biotin identification links cholesterol catabolism with branched-chain amino acid degradation in Mycobacterium smegmatis.

Cholesterol is a crucial component in Mycobacterium tuberculosis virulence as it is required for phagocytosis of mycobacteria by macrophages. In addition, the tubercle bacilli can grow using cholesterol as the sole carbon source. Thus, cholesterol catabolism represents a valuable target for the development of new antitubercular drugs. However, the molecular partners of cholesterol catabolism remain elusive in mycobacteria. Here, we focused on HsaC and HsaD, enzymes involved in two consecutive steps of cholesterol ring degradation and identified putative partners, using a BirA-based proximity-dependent biotin identification (BioID) approach in Mycobacterium smegmatis. In rich medium, the fusion protein BirA-HsaD was able to fish the endogenous cognate HsaC, thus validating this approach to study protein-protein interactions and to infer metabolic channeling of cholesterol ring degradation. In chemically defined medium, both HsaC and HsaD interacted with four proteins, BkdA, BkdB, BkdC, and MSMEG_1634. BkdA, BkdB, and BkdC are enzymes that participate in the degradation of branched-chain amino acids. As cholesterol and branched-chain amino acid catabolism both generate propionyl-CoA, which is a toxic metabolite for mycobacteria, this interconnection suggests a compartmentalization to avoid dissemination of propionyl-CoA into the mycobacterial cytosol. Moreover, the BioID approach allowed us to decipher the interactome of MSMEG_1634 and MSMEG_6518, two proteins of unknown function, which are proximal to the enzymes involved in cholesterol and branched-chain amino acid catabolism. In conclusion, BioID is a powerful tool to characterize protein-protein interactions and to decipher the interconnections between different metabolic pathways, thereby facilitating the identification of new mycobacterial targets.

Novel scaffolds targeting Mycobacterium tuberculosis plasma membrane Ca2+ transporter CtpF by structure-based strategy

CtpF is a Ca2+ transporter P-type ATPase key to the response to stress conditions and to Mycobacterium tuberculosis virulence, therefore, an interesting target for the design of novel anti-Mtb compounds. In this work, molecular dynamics simulations of four previously identified CtpF inhibitors allowed recognizing the key protein–ligand (P-L) interactions, which were then used to perform a pharmacophore-based virtual screening (PBVS) of 22 million compounds from ZINCPharmer. The top-rated compounds were then subjected to molecular docking, and their scores were refined by MM-GBSA calculations. In vitro assays showed that ZINC04030361 (Compound 7) was the best promising candidate, showing a MIC of 25.0 μg/mL, inhibition of Ca2+-ATPase activity (IC50) of 3.3 μM, cytotoxic activity of 27.2 %, and hemolysis of red blood cells lower than 0.2 %. Interestingly, the ctpF gene is upregulated in the presence of compound 7, compared to other alkali/alkaline P-type ATPases coding genes, strongly suggesting that CtpF is a compound 7-specific target.

Nitrogen metabolism in mycobacteria: the key genes and targeted antimicrobials.

Nitrogen metabolism is an important physiological process that affects the survival and virulence of Mycobacterium tuberculosis. M. tuberculosis's utilization of nitrogen in the environment and its adaptation to the harsh environment of acid and low oxygen in macrophages are closely related to nitrogen metabolism. In addition, the dormancy state and drug resistance of M. tuberculosis are closely related to nitrogen metabolism. Although nitrogen metabolism is so important, limited research was performed on nitrogen metabolism as compared with carbon metabolism. M. tuberculosis can use a variety of inorganic or organic nitrogen sources, including ammonium salts, nitrate, glutamine, asparagine, etc. In these metabolic pathways, some enzymes encoded by key genes, such as GlnA1, AnsP2, etc, play important regulatory roles in the pathogenesis of TB. Although various small molecule inhibitors and drugs have been developed for different nitrogen metabolism processes, however, long-term validation is needed before their practical application. Most importantly, with the emergence of multidrug-resistant strains, eradication, and control of M. tuberculosis will still be very challenging.