Characterization of the MurT/GatD complex in Mycobacterium tuberculosis towards validating a novel anti-tubercular drug target.

OBJECTIVES: Identification and validation of novel therapeutic targets is imperative to tackle the rise of drug resistance in tuberculosis. An essential Mur ligase-like gene (Rv3712), expected to be involved in cell-wall peptidoglycan (PG) biogenesis and conserved across mycobacteria, including the genetically depleted Mycobacterium leprae, was the primary focus of this study. METHODS: Biochemical analysis of Rv3712 was performed using inorganic phosphate release assays. The operon structure was identified using reverse-transcriptase PCR and a transcription/translation fusion vector. In vivo mycobacterial protein fragment complementation assays helped generate the interactome. RESULTS: Rv3712 was found to be an ATPase. Characterization of its operon revealed a mycobacteria-specific promoter driving the co-transcription of Rv3712 and Rv3713. The two gene products were found to interact with each other in vivo. Sequence-based functional assignments reveal that Rv3712 and Rv3713 are likely to be the mycobacterial PG precursor-modifying enzymes MurT and GatD, respectively. An in vivo network involving Mtb-MurT, regulatory proteins and cell division proteins was also identified. CONCLUSIONS: Understanding the role of the enzyme complex in the context of PG metabolism and cell division, and the implications for antimicrobial resistance and host immune responses will facilitate the design of therapeutics that are targeted specifically to M. tuberculosis.

The draft genome of Mycobacterium aurum, a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae.

Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two embB homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum, this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae.

ProTides of N-(3-(5-(2'-deoxyuridine))prop-2-ynyl)octanamide as potential anti-tubercular and anti-viral agents.

The flavin-dependent thymidylate synthase X (ThyX), rare in eukaryotes and completely absent in humans, is crucial in the metabolism of thymidine (a DNA precursor) in many microorganisms including several human pathogens. Conserved in mycobacteria, including Mycobacterium leprae, and Mycobacterium tuberculosis, it represents a prospective anti-mycobacterial therapeutic target. In a M. tuberculosis ThyX-enzyme inhibition assay, N-(3-(5-(2'-deoxyuridine-5'-phosphate))prop-2-ynyl)octanamide was reported to be the most potent and selective 5-substituted 2'-deoxyuridine monophosphate analogue. In this study, we masked the two charges at the phosphate moiety of this compound using our ProTide technology in order to increase its lipophilicity and then allow permeation through the complex mycobacterial cell wall. A series of N-(3-(5-(2'-deoxyuridine))prop-2-ynyl)octanamide phosphoroamidates were chemically synthesized and their biological activity as potential anti-tuberculars was evaluated. In addition to mycobacteria, several DNA viruses depend on ThyX for their DNA biosynthesis, thus these prodrugs were also screened for their antiviral properties.