Pathways In Mycobacterium Tuberculosis Research Articles

Genome mining of Mycobacterium tuberculosis: targeting SufD as a novel drug candidate through in silico characterization and inhibitor screening.

Tuberculosis (TB) stands as the second most fatal infectious disease globally, causing 1.3 million deaths in 2022. The resurgence of TB and the alarming rise of antibiotic resistance demand urgent call to develop novel antituberculosis drugs. Despite concerted efforts to control TB, the disease persists and spreads rapidly on a global scale. Targeting stress response pathways in Mycobacterium tuberculosis (Mtb) has become imperative to achieve complete eradication. This study employs subtractive genomics to identify and prioritize potential drug targets among the hypothetical proteins of Mtb, focusing on indispensable pathways. Amongst 177 essential hypothetical proteins, 152 were nonhomologous to human. These proteins participated in 34 pathways, and a 20-fold enrichment of SUF pathway genes led to its selection as a target pathway. Fe-S clusters are fundamental, widely distributed protein cofactors involved in vital cellular processes. The survival of Mtb in a hypoxic environment relies on the iron-sulfur (Fe-S) cluster biogenesis pathway for the repair of damaged Fe-S clusters. It also protects pathogen against drugs, ensuring controlled iron utilization and contributing to drug resistance. In Mtb, six proteins of Fe-S cluster assembly pathway are encoded by the suf operon. The present study was focused on SufD because of its role in iron acquisition and prevention of Fenton reaction. The research further delves into the in silico characterization of SufD, utilizing bioinformatics tools for sequence and structure based analysis. The protein's structural features, including the identification of conserved regions, motifs, and 3D structure prediction enhanced functional annotation. Target based virtual screening of compounds from the ChEMBL database resulted in 12 inhibitors with best binding affinities. Drug likeness and ADMET profiling of potential inhibitors identified promising compounds with favorable drug-like properties. The study also involved cloning in SUMO-pRSF-Duet1 expression vector, overexpression, and purification of recombinant SufD from E. coli BL21 (DE3) cells. Optimization of expression conditions resulted in soluble production, and subsequent purification highlighting the efficacy of the SUMO fusion system for challenging Mtb proteins in E. coli. These findings provide valuable insights into pharmacological targets for future experimental studies, holding promise for the development of targeted therapy against Mtb.

Efflux pump Rv1258c activates novel functions of the oxidative stress and via the VII secretion system ESX-3-mediated iron metabolic pathway in Mycobacterium tuberculosis

Oxidative stress and iron metabolism are essential for Mycobacterium tuberculosis (M.tb) survival in host cells. The efflux pump Rv1258c belongs to the major facilitator superfamily (MFS) and can actively pump drugs to promote certain drug resistance in M.tb. In this study, we compared H37RvΔRv1258c with wild-type (WT) M.tb H37Rv. The qRT-PCR results suggested that Rv1258c is potentially involved in the iron metabolic pathway by regulating the expression of ESX-3, which is required for iron uptake. Protein–Protein Affinity Predictor (PPA-Pred2) and the artificial intelligence program AlphaFold 2 were used for prediction and showed that Rv1258c has direct interactions with PPE4 and EccD3 but weak interactions with EccB3. This was further determined via protein–protein interaction analysis of the yeast two-hybrid expression system. By comparing mutant H37RvΔRv1258c strains with WT strains, we discovered that the absence of Rv1258c led to elevated intracellular H+ potential and NAD+/NADH ratios in M.tb, thereby resulting in oxidative stress. We hypothesize that the efflux pump Rv1258c not only has the function of regulating drug resistance in M.tb but also has a novel function in activating oxidative stress and regulating ESX-3-associated iron metabolism in M.tb.

Exploring the dark side of tertiary and quaternary structure dynamics in MtbFBPaseII

Tuberculosis (TB) is a major global cause of mortality, primarily stemming from latent tuberculosis infection (LTBI). Failure to fully treat LTBI can result in drug-resistant forms of TB. Therefore, it is essential to develop novel drugs with unique mechanisms of action to combat TB effectively. One crucial metabolic pathway in Mycobacterium tuberculosis (Mtb), which contributes to TB infection and persistence, is gluconeogenesis. Within this pathway, the enzyme fructose bisphosphatase (FBPase) plays a significant role and is considered a promising target for drug development. By targeting MtbFBPaseII, a specific class of FBPase, researchers have employed molecular dynamics simulations to identify regions capable of binding new drugs, thereby inhibiting the enzyme’s activity and potentially paving the way for the development of effective treatments. Communicated by Ramaswamy H. Sarma

Dual-Target Mycobacterium tuberculosis Inhibition: Insights into the Molecular Mechanism of Antifolate Drugs.

The escalating prevalence of drug-resistant strains of Mycobacterium tuberculosis has posed a significant challenge to global efforts in combating tuberculosis. To address this issue, innovative therapeutic strategies are required that target essential biochemical pathways while minimizing the potential for resistance development. The concept of dual targeting has gained prominence in drug discovery against resistance bacteria. Dual targeting recognizes the complexity of cellular processes and disrupts more than one vital pathway, simultaneously. By inhibiting more than one essential process required for bacterial growth and survival, the chances of developing resistance are substantially reduced. A previously reported study investigated the dual-targeting potential of a series of novel compounds against the folate pathway in Mycobacterium tuberculosis. Expanding on this study, we investigated the predictive pharmacokinetic profiling and the structural mechanism of inhibition of UCP1172, UCP1175, and UCP1063 on key enzymes, dihydrofolate reductase (DHFR) and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate reductase (RV2671), involved in the folate pathway. Our findings indicate that the compounds demonstrate lipophilic physiochemical properties that promote gastrointestinal absorption, and may also inhibit the drug-metabolizing enzyme, cytochrome P450 3A4, thus enhancing their biological half-life. Furthermore, key catalytic residues (Serine, Threonine, and Aspartate), conserved in both enzymes, were found to participate in vital molecular interactions with UCP1172, which demonstrated the most favorable free binding energies to both DHFR and RV2671 (-41.63 kcal/mol, -48.04 kcal/mol, respectively). The presence of characteristic loop shifts, which are similar in both enzymes, also indicates a common inhibitory mechanism by UCP1172. This elucidation advances the understanding of UCP1172's dual inhibition mechanism against Mycobacterium tuberculosis.

Screening of marine natural products for potential inhibitors targeting biotin biosynthesis pathway in Mycobacterium tuberculosis

Tuberculosis (TB) remains as one of the major public health concerns worldwide. A successful TB control and treatment is very challenging, due to continuing emergence of Mycobacterium tuberculosis strains resistant to known drugs. Therefore, the development of new drugs with different chemical and biological approaches is necessary to obtain more efficient anti-tubercular therapeutics. Biotin is an essential cofactor for lipid biosynthesis and gluconeogenesis in M. tuberculosis. M. tuberculosis relies on de novo biotin biosynthesis to obtain this vital cofactor since it cannot scavenge sufficient biotin from a mammalian host. In this study, comprehensive in silico methods including structure-based virtual screening, molecular docking, and molecular dynamic simulation analysis for ∼8000 marine natural products were performed against two essential enzymes involved in biotin synthesis and ligation of M. tuberculosis namely, pyridoxal 5′-phosphate-dependent transaminase (BioA) and mycobacterial biotin protein ligase (MtBPL). Two compounds; CMNPD10112 and CMNPD10113 are unveiled to bind the enzymes consistently and with high affinities. The binding pattern of compounds is further noticed in very stable binding modes as analyzed by molecular dynamics simulation and the mean RMSD of the complexes is within 4 Å. The intermolecular binding free energies validated complexes are less than −40 kcal/mol, which demonstrates strong and stable complexes formation. The identified hit compounds could be seeds for design of effective anti-mycobacterium therapeutics by inhibition of bacterial growth through blocking the biotin biosynthesis. Communicated by Ramaswamy H. Sarma

Compromised base excision repair pathway in Mycobacterium tuberculosis imparts superior adaptability in the host.

Tuberculosis caused by Mycobacterium tuberculosis (Mtb) is a significant public health concern, exacerbated by the emergence of drug-resistant TB. To combat the host's dynamic environment, Mtb encodes multiple DNA repair enzymes that play a critical role in maintaining genomic integrity. Mtb possesses a GC-rich genome, rendering it highly susceptible to cytosine deaminations, resulting in the occurrence of uracils in the DNA. UDGs encoded by ung and udgB initiate the repair; hence we investigated the biological impact of deleting UDGs in the adaptation of pathogen. We generated gene replacement mutants of uracil DNA glycosylases, individually (RvΔung, RvΔudgB) or together (RvΔdKO). The double KO mutant, RvΔdKO exhibited remarkably higher spontaneous mutation rate, in the presence of antibiotics. Interestingly, RvΔdKO showed higher survival rates in guinea pigs and accumulated large number of SNPs as revealed by whole-genome sequence analysis. Competition assays revealed the superior fitness of RvΔdKO over Rv, both in ex vivo and in vivo conditions. We propose that compromised DNA repair results in the accumulation of mutations, and a subset of these drives adaptation in the host. Importantly, this property allowed us to utilize RvΔdKO for the facile identification of drug targets.

Structural characterization of glycinamide-RNase-transformylase T from Mycobacterium tuberculosis

ABSTRACT Enzymes from the purine salvage pathway in Mycobacterium tuberculosis (Mtb) have been regarded as an attractive target for the development of anti-bacterial drugs. Although this pathway has not been extensively studied in Mtb, it has been identified as essential for growth and survival. Glycinamide-RNase-transformylase T (PurT) is found only in some specific bacteria including Mtb and utilizes ATP-dependent ligation to catalyze the formylation of 5′-phosphoribosyl-glycinamide (GAR) in the third reaction of the de novo purine salvage pathway. In the study, we determined the crystal structure of MtbPurT at a resolution of 2.79 Å. In contrast to Pyrococcus horikoshii OT3 PurT (phBCCPPurT), MtbPurT exhibits an “open” conformation, which results in a broader ATP-binding pocket and thus might facilitate the entry and exit of the cofactor. Additionally, active site superposition with E.coli PurT (EcPurT) showed that residues involved in the ATP-binding site in MtbPurT exhibited structural similarity but had notable difference in the GAR-binding site. The loop 383-389 in MtbPurT was much shorter and shifted 5.7 Å away from the phosphate of the GAR substrate. The different GAR-binding mode might result in a large conformational change in MtbPurT, and would provide a possible opportunity for anti-TB drug development.

Analysis of mutations leading to para-aminosalicylic acid resistance in Mycobacterium tuberculosis

Thymidylate synthase A (ThyA) is the key enzyme involved in the folate pathway in Mycobacterium tuberculosis. Mutation of key residues of ThyA enzyme which are involved in interaction with substrate 2′-deoxyuridine-5′-monophosphate (dUMP), cofactor 5,10-methylenetetrahydrofolate (MTHF), and catalytic site have caused para-aminosalicylic acid (PAS) resistance in TB patients. Focusing on R127L, L143P, C146R, L172P, A182P, and V261G mutations, including wild-type, we performed long molecular dynamics (MD) simulations in explicit solvent to investigate the molecular principles underlying PAS resistance due to missense mutations. We found that these mutations lead to (i) extensive changes in the dUMP and MTHF binding sites, (ii) weak interaction of ThyA enzyme with dUMP and MTHF by inducing conformational changes in the structure, (iii) loss of the hydrogen bond and other atomic interactions and (iv) enhanced movement of protein atoms indicated by principal component analysis (PCA). In this study, MD simulations framework has provided considerable insight into mutation induced conformational changes in the ThyA enzyme of Mycobacterium.

Homology modeling and in silico toxicity assessment of potential inhibitors of cytidylate kinase from Mycobacterium tuberculosis

Cytidylate kinase (CMK) plays a central role in the phosphorylation of ATP to nucleoside diphosphates in the purine and pyrimidine pathway in Mycobacterium tuberculosis (MTB). The enzyme is specific to bacteria, fungi, and plants and it shares the same pathway with a DNA-directed RNA polymerase subunit beta, the known target of rifampicin (RIF). Therefore, it serves as an attractive alternative target to inhibit the purine and pyrimidine pathway in MTB. In this study, the novel inhibitors of CMK were identified using in silico approach. Homology modeling was used to build the three-dimensional (3D) structure of CMK using program implemented in Modeller 9.16 and based on the template (3R20) obtained from Mycobacterium smegmatis. Structural analysis of CMK showed that it has P-loop or core in the ATP binding domain, nucleoside monophosphate binding (NMP) site, and a LID domain. Inhibition of any of the residues that formed the nucleoside binding site or ATP binding site blocks the catalytic activity of the CMK. Eleven thousand four hundred and fifty-four compounds that had an affinity to bind to the CMK with minimum binding energies were obtained through virtual screening from Zinc and PubChem databases using PyRx 8.0. These compounds were further filtered for Lipinski rule of five, molecular docking analysis, and ADMET properties (absorption distribution, metabolism, excretion, and toxicity). Four compounds (ZINC95485891 = − 8.80 kcal/mol, ZINC95485880 = − 8.44 kcal/mol, ZINC95485900 = − 8.24 kcal/mol, and ZINC95486231 = − 8.20 kcal/mol) with good binding energies and desirable pharmacokinetic properties were selected. These compounds were further subjected to molecular dynamic (MD) simulation and molecular mechanics generalized Born and surface area (MM-GBSA) analyses. The results of the analyses showed that all the four ligands formed a stable complex and had good free binding energy after 50 ns MD simulation. Therefore, these compounds were considered as possible inhibitors of MTB after experimental validation.

Combination of Repurposed Drug Diosmin with Amoxicillin-Clavulanic acid Causes Synergistic Inhibition of Mycobacterial Growth

Effective therapeutic regimens for the treatment of tuberculosis (TB) are limited. They are comprised of multiple drugs that inhibit the essential cellular pathways in Mycobacterium tuberculosis (Mtb). The present study investigates an approach which enables a combination of Amoxicillin-Clavulanic acid (AMC) and a repurposed drug for its synergistic effect towards TB treatment. We identified Diosmin (DIO), by targeting the active site residues of L,D-transpeptidase (Ldt) enzymes involved in Mtb cell wall biosynthesis by using a structure-based drug design method. DIO is rapidly converted into aglycone form Diosmetin (DMT) after oral administration. Binding of DIO or DMT towards Ldt enzymes was studied using molecular docking and bioassay techniques. Combination of DIO (or DMT) and AMC exhibited higher mycobactericidal activity against Mycobacterium marinum as compared to individual drugs. Scanning electron microscopy study of M. marinum treated with AMC-DIO and AMC-DMT showed marked cellular leakage. M. marinum infected Drosophila melanogaster fly model showed an increased fly survival of ~60% upon treatment with a combination of AMC and DIO (or DMT). Finally, the enhanced in vitro antimicrobial activity of AMC-DIO was validated against Mtb H37Ra and a MDR clinical isolate. Our results demonstrate the potential for AMC and DIO (or DMT) as a synergistic combination for the treatment of TB.

Drugging the Folate Pathway in Mycobacterium tuberculosis: The Role of Multi-targeting Agents

The folate biosynthetic pathway offers many druggable targets that have yet to be exploited in tuberculosis therapy. Herein, we have identified a series of small molecules that interrupt Mycobacterium tuberculosis (Mtb) folate metabolism by dual targeting ofdihydrofolate reductase (DHFR), a key enzyme inthe folate pathway, and its functional analog, Rv2671. We have also compared the antifolate activity of these compounds with that of para-aminosalicylic acid (PAS). We found that the bioactive metabolite of PAS, in addition to previously reported activity against DHFR, inhibits flavin-dependent thymidylate synthase in Mtb, suggesting a multi-targeted mechanism of action for this drug. Finally, we have shown that antifolate treatment in Mtb decreases the production of mycolic acids, most likely due to perturbation of the activated methyl cycle. We conclude that multi-targeting of the folate pathway in Mtb is associated with highly potent anti-mycobacterial activity.

Computer-aided drug design based on comparative modeling, molecular docking and molecular dynamic simulation of Polyphosphate kinase (PPK) from Mycobacterium tuberculosis

This study aimed to identify compounds that are capable of inhibiting polyphosphate kinase (PPK), the protein that shares a similar pathway with the target of isoniazid (INH) in the oxidative phosphorylation pathway of Mycobacterium tuberculosis (MTB). The three-dimensional structure of the PPK was predicted based on the homology modeling principle via Modeller9.16. Structural analysis revealed that the PPK has three active sites—metal ion-binding site (Arg431 and Arg461), ATP-binding site (Asn91, Tyr524, Arg624, and His652), and phosphohistidine intermediate active site (His491). The amino acids mentioned earlier play an essential role in the activity of the PPK, and their inhibition would block the function of the PPK. Ten thousand one hundred and six (10,106) ligands were obtained through virtual screening against Zinc database. These compounds were filtered by Lipinski rule of five and molecular docking analysis. A total of ten compounds with good AutoDock binding energy that varied between − 9.92 and − 8.01 kcal/mol which was lower than the binding energy of − 1.13 kcal/mol of Mg2+ (metallic cofactor) were selected. These compounds were further filtered for their pharmacokinetic and toxicity properties to further remove the compounds with unwanted properties. Three ligands—ZINC41125011, ZINC20318248, and ZINC20321877 with desired pharmacokinetic and toxicity properties were selected for molecular dynamic (MD) simulation and molecular generalized Born surface area (MM-GBSA) analysis. The result of the studies shows that all the three complexes are relatively stable in the binding site of the PPK, after 50 ns MD simulation. Therefore, these identified compounds are regarded as prospective inhibitors of MTB after positive experimental validation.

The Biotin Biosynthetic Pathway in Mycobacterium tuberculosis is a Validated Target for the Development of Antibacterial Agents.

Mycobacterium tuberculosis, responsible for Tuberculosis (TB), remains the leading cause of mortality among infectious diseases worldwide from a single infectious agent, with an estimated 1.7 million deaths in 2016. Biotin is an essential cofactor in M. tuberculosis that is required for lipid biosynthesis and gluconeogenesis. M. tuberculosis relies on de novo biotin biosynthesis to obtain this vital cofactor since it cannot scavenge sufficient biotin from a mammalian host. The biotin biosynthetic pathway in M. tuberculosis has been well studied and rigorously genetically validated providing a solid foundation for medicinal chemistry efforts. This review examines the mechanism and structure of the enzymes involved in biotin biosynthesis and ligation, summarizes the reported genetic validation studies of the pathway, and then analyzes the most promising inhibitors and natural products obtained from structure-based drug design and phenotypic screening.

Correction: A Novel Role of the PrpR as a Transcription Factor Involved in the Regulation of Methylcitrate Pathway in Mycobacterium tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0043651.].

Steroid Degradation in Comamonas testosteroni TA441: Identification of Metabolites and the Genes Involved in the Reactions Necessary before D-Ring Cleavage.

Bacterial steroid degradation has been studied mainly with Rhodococcus equi (Nocardia restrictus) and Comamonas testosteroni as representative steroid degradation bacteria for more than 50 years. The primary purpose was to obtain materials for steroid drugs, but recent studies showed that many genera of bacteria (Mycobacterium, Rhodococcus, Pseudomonas, etc.) degrade steroids and that steroid-degrading bacteria are globally distributed and found particularly in wastewater treatment plants, the soil, plant rhizospheres, and the marine environment. The role of bacterial steroid degradation in the environment is, however, yet to be revealed. To uncover the whole steroid degradation process in a representative steroid-degrading bacterium, C. testosteroni, to provide basic information for further studies on the role of bacterial steroid degradation, we elucidated the two indispensable oxidative reactions and hydration before D-ring cleavage in C. testosteroni TA441. In bacterial oxidative steroid degradation, A- and B-rings of steroids are cleaved to produce 2-hydroxyhexa-2,4-dienoic acid and 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid. The latter compound was revealed to be degraded to the coenzyme A (CoA) ester of 9α-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid, which is converted to the CoA ester of 9,17-dioxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid by ORF31-encoded hydroxylacyl dehydrogenase (ScdG), followed by conversion to the CoA ester of 9,17-dioxo-1,2,3,4,5,6,10,19-octanorandrost-8(14)-en-7-oic acid by ORF4-encoded acyl-CoA dehydrogenase (ScdK). Then, a water molecule is added by the ORF5-encoded enoyl-CoA hydratase (ScdY), which leads to the cleavage of the D-ring. The conversion by ScdG is presumed to be a reversible reaction. The elucidated pathway in C. testosteroni TA441 is different from the corresponding pathways in Mycobacterium tuberculosis H37Rv.IMPORTANCE Studies on representative steroid degradation bacteria Rhodococcus equi (Nocardia restrictus) and Comamonas testosteroni were initiated more than 50 years ago primarily to obtain materials for steroid drugs. A recent study showed that steroid-degrading bacteria are globally distributed and found particularly in wastewater treatment plants, the soil, plant rhizospheres, and the marine environment, but the role of bacterial steroid degradation in the environment is yet to be revealed. This study aimed to uncover the whole steroid degradation process in C. testosteroni TA441, in which major enzymes for steroidal A- and B-ring cleavage were elucidated, to provide basic information for further studies on bacterial steroid degradation. C. testosteroni is suitable for exploring the degradation pathway because the involvement of degradation-related genes can be determined by gene disruption. We elucidated the two indispensable oxidative reactions and hydration before D-ring cleavage, which appeared to differ from those present in Mycobacterium tuberculosis H37Rv.

Arginine-deprivation–induced oxidative damage sterilizes Mycobacterium tuberculosis

Reactive oxygen species (ROS)-mediated oxidative stress and DNA damage have recently been recognized as contributing to the efficacy of most bactericidal antibiotics, irrespective of their primary macromolecular targets. Inhibitors of targets involved in both combating oxidative stress as well as being required for in vivo survival may exhibit powerful synergistic action. This study demonstrates that the de novo arginine biosynthetic pathway in Mycobacterium tuberculosis (Mtb) is up-regulated in the early response to the oxidative stress-elevating agent isoniazid or vitamin C. Arginine deprivation rapidly sterilizes the Mtb de novo arginine biosynthesis pathway mutants ΔargB and ΔargF without the emergence of suppressor mutants in vitro as well as in vivo. Transcriptomic and flow cytometry studies of arginine-deprived Mtb have indicated accumulation of ROS and extensive DNA damage. Metabolomics studies following arginine deprivation have revealed that these cells experienced depletion of antioxidant thiols and accumulation of the upstream metabolite substrate of ArgB or ArgF enzymes. ΔargB and ΔargF were unable to scavenge host arginine and were quickly cleared from both immunocompetent and immunocompromised mice. In summary, our investigation revealed in vivo essentiality of the de novo arginine biosynthesis pathway for Mtb and a promising drug target space for combating tuberculosis.

In silico docking and molecular dynamics simulation of 3-dehydroquinate synthase (DHQS) from Mycobacterium tuberculosis.

The shikimate pathway is as an attractive target because it is present in bacteria, algae, fungi, and plants but does not occur in mammals. In Mycobacterium tuberculosis (MTB), the shikimate pathway is integral to the biosynthesis of naphthoquinones, menaquinones, and mycobactin. In these study, novel inhibitors of 3-dehydroquinate synthase (DHQS), an enzyme that catalyzes the second step of the shikimate pathway in MTB, were determined. 12,165 compounds were selected from two public databases through virtual screening and molecular docking analysis using PyRx 8.0 and Autodock 4.2, respectively. A total of 18 compounds with the best binding energies (-13.23 to -8.22kcal/mol) were then selected and screened for absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis, and nine of those compounds were found to satisfy all of the ADME and toxicity criteria. Among those nine, the three compounds-ZINC633887 (binding energy = -10.29kcal/mol), ZINC08983432 (-9.34kcal/mol), and PubChem73393 (-8.61kcal/mol)-with the best binding energies were further selected for molecular dynamics (MD) simulation analysis. The results of the 50-ns MD simulations showed that the two compounds ZINC633887 and PubChem73393 formed stable complexes with DHQS and that the structures of those two ligands remained largely unchanged at the ligand-binding site during the simulations. These two compounds identified through docking and MD simulation are potential candidates for the treatment of TB, and should undergo validation in vivo and in vitro.

Drugging the Folate Pathway in Mycobacterium Tuberculosis: The Role of Multi-Targeting Agents

The folate biosynthetic pathway, responsible for the generation of reduced folate cofactors, offers many druggable targets that have not been exploited in TB therapy. Here, we have identified a series of novel small molecules that interrupt Mycobacterium tuberculosis folate metabolism by dual targeting of dihydrofolate reductase (DHFR), a key enzyme in the folate pathway, and its functional analog, Rv2671. We have also compared the antifolate activity of these compounds to that of para-aminosalicylic acid (PAS). We found the bioactive metabolite of PAS, in addition to previously reported activity against DHFR, inhibits flavin-dependent thymidylate synthase (FDTS) in Mtb, suggesting a multi-targeted mechanism of action for this drug. Finally, we have shown that antifolate treatment in Mtb decreases the production of mycolic acids, most likely due to perturbation of the activated methyl cycle. We conclude that multi-targeting of the folate pathway in Mtb is a promising strategy to develop novel anti-TB agents.

Construction of Mycobacterium tuberculosis cdd knockout and evaluation of invasion and growth in macrophages.

Cytidine deaminase (MtCDA), encoded by cdd gene (Rv3315c), is the only enzyme identified in nucleotide biosynthesis pathway of Mycobacterium tuberculosis that is able to recycle cytidine and deoxycytidine. An M. tuberculosis knockout strain for cdd gene was obtained by allelic replacement. Evaluation of mRNA expression validated cdd deletion and showed the absence of polar effect. MudPIT LC-MS/MS data indicated thymidine phosphorylase expression was decreased in knockout and complemented strains. The cdd disruption does not affect M. tuberculosis growth both in Mid- dlebrook 7H9 and in RAW 264.7 cells, which indicates that cdd is not important for macrophage invasion and virulence.

Role of ESAT-6 in renal injury by regulating microRNA-155 expression via TLR4/MyD88 signaling pathway in mice with Mycobacterium tuberculosis infection.

The study aims to investigate the underlying mechanism involved in the early secretory antigenic target-6 (ESAT-6) in renal injury through regulation of the expression of miR-155 through the oll-like receptor (TLR)-4 (TLR4)/myeloid differentiation factor 88 (MyD88) signaling pathway in Mycobacterium tuberculosis (MTB)-infected mice. Sixty C57BL/6 mice with MTB-induced renal injury were randomly assigned into control, MTB, mimic, inhibitor, inhibitor + ESAT6, and inhibitor + ESAT6 + TAK242 groups. Body weight, the ratio of kidney weight to body weight (Kw/Bw), blood urea nitrogen (BUN), and serum creatinine (Scr) of mice were measured. Flow cytometry was used to detect renal activation in mice. Expressions of miR-155 and ESAT6 were detected by quantitative real-time PCR (qRT-PCR), and Western blotting was used to examine the expressions of ESAT6, TLR4, and MyD88. Expressions of tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), and interferon-γ (IFN-γ) were measured by qRT-PCR and ELISA. Compared with the control group, the BUN and Scr levels as well as the expression levels of miR-155, TLR4, MyD88, TNF-α, IL-17, and IFN-γ increased, while Kw/Bw decreased in the MTB and mimic groups. In comparison with the MTB group, the above indexes except Kw/Bw were elevated in the mimic group, but were reduced in the inhibitor group, while the Kw/Bw dropped in the mimic group but increased in the inhibitor group. Compared with the inhibitor group, the Kw/Bw decreased while the rest of the indexes increased in the inhibitor + ESAT6 group. ESAT6 may induce renal injury by promoting miR-155 expression through the TLR-4/MyD88 signaling pathway in MTB-infected mice.