Sanjib Bhakta

Mycobacterium tuberculosis: Immune evasion, latency and reactivation

One-third of the global human population harbours Mycobacterium tuberculosis in dormant form. This dormant or latent infection presents a major challenge for global efforts to eradicate tuberculosis, because it is a vast reservoir of potential reactivation and transmission. This article explains how the pathogen evades the host immune response to establish a latent infection, and how it emerges from a state of latency to cause reactivation disease. This review highlights the key factors responsible for immune evasion and reactivation. It concludes by identifying interesting candidates for drug or vaccine development, as well as identifying unresolved questions for the future research.

Arylamine N-Acetyltransferase Is Required for Synthesis of Mycolic Acids and Complex Lipids in Mycobacterium bovis BCG and Represents a Novel Drug Target

Mycolic acids represent a major component of the unique cell wall of mycobacteria. Mycolic acid biosynthesis is inhibited by isoniazid, a key frontline antitubercular drug that is inactivated by mycobacterial and human arylamine N-acetyltransferase (NAT). We show that an in-frame deletion of Mycobacterium bovis BCG nat results in delayed entry into log phase, altered morphology, altered cell wall lipid composition, and increased intracellular killing by macrophages. In particular, deletion of nat perturbs biosynthesis of mycolic acids and their derivatives and increases susceptibility of M. bovis BCG to antibiotics that permeate the cell wall. Phenotypic traits are fully complemented by introduction of Mycobacterium tuberculosis nat. We infer from our findings that NAT is critical to normal mycolic acid synthesis and hence other derivative cell wall components and represents a novel target for antituberculosis therapy. In addition, this is the first report of an endogenous role for NAT in mycobacteria.

Anti-tubercular screening of natural products from Colombian plants: 3-methoxynordomesticine, an inhibitor of MurE ligase of Mycobacterium tuberculosis

OBJECTIVES New anti-mycobacterial entities with novel mechanisms of action are clinically needed for treating resistant forms of tuberculosis. The purpose of this study was to evaluate anti-tubercular activity and selectivity of seven recently isolated natural products from Colombian plants. METHODS MICs were determined using a liquid medium growth inhibition assay for Mycobacterium tuberculosis H(37)Rv and both solid and liquid media growth inhibition assays for Mycobacterium bovis BCG. Escherichia coli growth inhibition and mammalian macrophage cell toxicity were evaluated to establish the degree of selectivity of the natural product against whole cell organisms. Enzymatic inhibition of ATP-dependent MurE ligase from M. tuberculosis was assayed using a colorimetric phosphate detection method. The most active compound, 3-methoxynordomesticine hydrochloride, was further investigated on M. bovis BCG for its inhibition of sigmoidal growth, acid-fast staining and viability counting analysis. RESULTS Aporphine alkaloids were found to be potent inhibitors of slow-growing mycobacterial pathogens showing favourable selectivity and cytotoxicity. In terms of their endogenous action, the aporphine alkaloids were found inhibitory to M. tuberculosis ATP-dependent MurE ligase at micromolar concentrations. A significantly low MIC was detected for 3-methoxynordomesticine hydrochloride against both M. bovis BCG and M. tuberculosis H(37)Rv. CONCLUSIONS Considering all the data, 3-methoxynordomesticine hydrochloride was found to be a potent anti-tubercular compound with a favourable specificity profile. The alkaloid showed MurE inhibition and is considered an initial hit for exploring related chemical space.

Antitubercular specific activity of ibuprofen and the other 2-arylpropanoic acids using the HT-SPOTi whole-cell phenotypic assay

Objectives Lead antituberculosis (anti-TB) molecules with novel mechanisms of action are urgently required to fuel the anti-TB drug discovery pipeline. The aim of this study was to validate the use of the high-throughput spot culture growth inhibition (HT-SPOTi) assay for screening libraries of compounds against Mycobacterium tuberculosis and to study the inhibitory effect of ibuprofen (IBP) and the other 2-arylpropanoic acids on the growth inhibition of M tuberculosis and other mycobacterial species. Methods The HT-SPOTi method was validated not only with known drugs but also with a library of 47 confirmed anti-TB active compounds published in the ChEMBL database. Three over-the-counter non-steroidal anti-inflammatory drugs were also included in the screening. The 2-arylpropanoic acids, including IBP, were comprehensively evaluated against phenotypically and physiologically different strains of mycobacteria, and their cytotoxicity was determined against murine RAW264.7 macrophages. Furthermore, a comparative bioinformatic analysis was employed to propose a potential mycobacterial target. Results IBP showed antitubercular properties while carprofen was the most potent among the 2-arylpropanoic class. A 3,5-dinitro-IBP derivative was found to be more potent than IBP but equally selective. Other synthetic derivatives of IBP were less active, and the free carboxylic acid of IBP seems to be essential for its anti-TB activity. IBP, carprofen and the 3,5-dinitro-IBP derivative exhibited activity against multidrug-resistant isolates and stationary phase bacilli. On the basis of the human targets of the 2-arylpropanoic analgesics, the protein initiation factor infB (Rv2839c) of M tuberculosis was proposed as a potential molecular target. Conclusions The HT-SPOTi method can be employed reliably and reproducibly to screen the antimicrobial potency of different compounds. IBP demonstrated specific antitubercular activity, while carprofen was the most selective agent among the 2-arylpropanoic class. Activity against stationary phase bacilli and multidrug-resistant isolates permits us to speculate a novel mechanism of antimycobacterial action. Further medicinal chemistry and target elucidation studies could potentially lead to new therapies against TB.

Characterisation of ATP-Dependent Mur Ligases Involved in the Biogenesis of Cell Wall Peptidoglycan in Mycobacterium tuberculosis

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.

Characterization of the putative operon containing arylamine N-acetyltransferase (nat) in Mycobacterium bovis BCG

Mycobacterium bovis BCG and Mycobacterium tuberculosis possess a single arylamine N‐acetyltransferase whose gene is predicted to occur within a six‐gene operon. Deletion of the nat gene caused an extended lag phase in M. bovis BCG and a cell morphology associated with an altered pattern of cell wall mycolates. Analysis of cDNA from M. bovis BCG shows that during in vitro growth all the genes in the putative nat operon are expressed and the open reading frames are contiguous, supporting the existence of an operon. Two genes in the operon, Mb3599c and Mb3600c, are predicted to encode homologues of enzymes annotated as a 2,3‐dihydroxybiphenyl 1,2‐dioxygenase (bphC5) and a 2‐hydroxy‐6‐oxo‐6‐phenylhexa‐2,4‐dienoate hydrolase (bphD2), respectively, in Rhodococcus RHA1. As predicted, M. bovis BCG cell lysates metabolized the BphC substrate 2,3‐dihydroxybiphenyl (2,3‐DHB) to 2‐hydroxy‐6‐oxo‐6‐phenylhexa‐2,4‐dienoic acid (HOPDA), a BphD substrate, which was subsequently hydrolysed. Immunoprecipitation of the BphD homologue from these lysates led to an accumulation of HOPDA. M. bovis BCG growth on both solid and liquid media was inhibited with either 2,3‐DHB or an inhibitor of BphC, 3‐chlorocatechol (3‐CC). In addition, incubation with 2,3‐DHB affects the lipid composition of the cell wall resulting in a diminished level of mycolates and an altered cell morphology similar to the Δnat strain. We propose the enzymes encoded by the putative operon have a similar endogenous role to that of the NAT enzyme and are part of a pathway important for cell wall synthesis.

Identification of arylamine N-acetyltransferase inhibitors as an approach towards novel anti-tuberculars

New anti-tubercular drugs and drug targets are urgently needed to reduce the time for treatment and also to identify agents that will be effective against Mycobacterium tuberculosis persisting intracellularly. Mycobacteria have a unique cell wall. Deletion of the gene for arylamine N-acetyltransferase (NAT) decreases mycobacterial cell wall lipids, particularly the distinctive mycolates, and also increases antibiotic susceptibility and killing within macrophage of Mycobacterium bovis BCG. The nat gene and its associated gene cluster are almost identical in sequence in M. bovis BCG and M. tuberculosis. The gene cluster is essential for intracellular survival of mycobacteria. We have therefore used pure NAT protein for high-throughput screening to identify several classes of small molecules that inhibit NAT activity. Here, we characterize one class of such molecules—triazoles—in relation to its effects on the target enzyme and on both M. bovis BCG and M. tuberculosis. The most potent triazole mimics the effects of deletion of the nat gene on growth, lipid disruption and intracellular survival. We also present the structure-activity relationship between NAT inhibition and effects on mycobacterial growth, and use ligand-protein analysis to give further insight into the structure-activity relationships. We conclude that screening a chemical library with NAT protein yields compounds that have high potential as anti-tubercular agents and that the inhibitors will allow further exploration of the biochemical pathway in which NAT is involved.

ATP-Dependent Mure Ligase in Mycobacterium Tuberculosis: Biochemical and Structural Characterisation.

New therapies are required against Mycobacterium tuberculosis and its cell wall peptidoglycan biosynthesis is a potential therapeutic target. UDP-MurNAc-tripeptide ligase (MurE) is a member of the ATP-dependent ligase family, which incorporate amino acids including meso-diaminopimelic acid (m-DAP) into peptidoglycan during synthesis in a species-specific manner. In the present study, we have cloned, over-expressed, and characterised MurE from M. tuberculosis (Mtb-MurE). The crystal structure has been determined at 3.0A resolution in the presence of the substrate UDP-MurNAc-l-Ala-d-Glu (UAG). The activity of the enzyme was measured through estimating inorganic phosphate released in a non-radioactive high-throughput colourimetric assay. UDP-MurNAc-l-Ala-d-Glu-m-DAP (UMT) formation coupled to inorganic phosphate release was confirmed by HPLC and mass spectrometric analyses. Kinetic constants were determined for a range of natural substrates using optimised conditions. From our findings, it is evident that Mtb-MurE is highly specific in adding m-DAP to UDP-MurNAc-dipeptide and ATP-hydrolysis is an absolute requirement for its activity.

An integrated surrogate model for screening of drugs against Mycobacterium tuberculosis

OBJECTIVES The intracellularly surviving and slow-growing pathogen, Mycobacterium tuberculosis, adapts the host cell environment for its active and dormant life cycle. It is evident that the lack of appropriate high-throughput screening of inhibitors within host cells is an impediment for the early stages of anti-tubercular drug discovery. We aimed to develop an integrated surrogate model that enhances the screening of large inhibitor libraries. METHODS Different mycobacterial species were compared for their growth, drug susceptibility and intracellular uptake. A 6-well plate solid agar-based spot culture growth inhibition (SPOTi) assay was developed into a higher throughput format. The uptake and intracellular survival of Mycobacterium aurum within mouse macrophage cells (RAW 264.7) were optimized using 24/96-well plate formats. RESULTS Fast-growing, non-pathogenic M. aurum was found to have an antibiotic-susceptibility profile similar to that of M. tuberculosis. The sensitivity to an acidic pH environment and the ability to multiply inside RAW 264.7 macrophages provided additional advantages for employing M. aurum in intracellular drug screening methods. A selection of anti-tubercular drugs inhibited the growth and viability of M. aurum inside the macrophages at different levels. CONCLUSIONS We present a rapid, convenient, high-throughput surrogate model, which provides a comprehensive evaluation platform for new chemical scaffolds against different physiological stages of mycobacteria within the primary cell environment of the host. The results using anti-tubercular drugs validate this model for screening libraries of existing and novel chemical entities.

Arylamine N-Acetyltransferases in Mycobacteria

Polymorphic Human arylamine N-acetyltransferase (NAT2) inactivates the anti-tubercular drug isoniazid by acetyltransfer from acetylCoA. There are active NAT proteins encoded by homologous genes in mycobacteria including M. tuberculosis, M. bovis BCG, M. smegmatis and M. marinum. Crystallographic structures of NATs from M. smegmatis and M. marinum, as native enzymes and with isoniazid bound share a similar fold with the first NAT structure, Salmonella typhimurium NAT. There are three approximately equal domains and an active site essential catalytic triad of cysteine, histidine and aspartate in the first two domains. An acetyl group from acetylCoA is transferred to cysteine and then to the acetyl acceptor e.g. isoniazid. M. marinum NAT binds CoA in a more open mode compared with CoA binding to human NAT2. The structure of mycobacterial NAT may promote its role in synthesis of cell wall lipids, identified through gene deletion studies. NAT protein is essential for survival of M. bovis BCG in macrophage as are the proteins encoded by other genes in the same gene cluster (hsaA-D). HsaA-D degrade cholesterol, essential for mycobacterial survival inside macrophage. Nat expression remains to be fully understood but is co-ordinated with hsaA-D and other stress response genes in mycobacteria. Amide synthase genes in the streptomyces are also nat homologues. The amide synthases are predicted to catalyse intramolecular amide bond formation and creation of cyclic molecules, e.g. geldanamycin. Lack of conservation of the CoA binding cleft residues of M. marinum NAT suggests the amide synthase reaction mechanism does not involve a soluble CoA intermediate during amide formation and ring closure.