11 research outputs found
Identification of a repetitive sequence belonging to a PPE gene of Mycobacterium tuberculosis and its use in diagnosis of tuberculosis
A repetitive sequence specific to Mycobacterium tuberculosis was isolated from a gt11 library of M. tuberculosis by DNA-DNA hybridization using genomic DNA of M. tuberculosis as probe followed by subtractive hybridization with a cocktail of other mycobacterial DNA. This led to identification of CD192, a 1291 bp fragment of M. tuberculosis containing repetitive sequences, which produced positive hybridization signals with M. tuberculosis DNA within 30 min. Nucleotide sequencing revealed the presence of several direct and inverted repeats within the 1291 bp fragment that belonged to a PPE family gene (Rv0355) of M. tuberculosis. The use of CD192 as a DNA probe for the identification of M. tuberculosis in culture and clinical samples was investigated. The 1291 bp sequence was present in M. tuberculosis, Mycobacterium bovis and M. bovis BCG, but was not present in many of the other mycobacterial strains tested, including M. tuberculosis H37Ra. More than 300 clinical isolates of M. tuberculosis were probed with CD192, and the presence of the 1291 bp sequence was observed in all the clinical strains, including those lacking IS6110. The sequence displayed RFLP among the clinical isolates. A PCR assay was developed which detected M. tuberculosis with 100 % specificity from specimens of sputum, cerebrospinal fluid and pleural effusion from clinically diagnosed cases of tuberculosis
Fast-growing, non-infectious and intracellularly surviving drug resistant Mycobacterium aurum: a model for high-throughput antituberculosis drug screening
Objectives: Enoyl acyl-carrier-protein reductase (InhA), the primary endogenous target for isoniazid
and ethionamide, is crucial to type-II fatty acid biosynthesis (FAS-II). The objectives of this study were
first to generate InhA mutants of Mycobacterium aurum, secondly to characterize InhA-mediated
isoniazid and ethionamide resistance mechanisms across those mutants and finally to investigate the
interaction of InhA with enzymes in the FAS-II pathway in M. aurum.
Methods: Spontaneous mutants were generated by isoniazid overdose and limited broth dilution, while
for genetically modified mutants sense–antisense DNA technology was used. Southern hybridization
and immunoprecipitation were both used to identify the InhA homologue in M. aurum. The latter
method was further used to compare the level of InhA expression in M. aurum with that in corresponding
mutants. Isoniazid/ethionamide susceptibility modulation was examined in vitro and ex vivo using
a resazurin assay as well as by cfu counting. In addition, circular dichroism and the bacterial twohybrid
system were exploited to investigate the interaction of InhA with other enzymes of the FAS-II
pathway.
Results: A Mycobacterium tuberculosis InhA homologue was detected in M. aurum. Susceptibility to
isoniazid/ethionamide was significantly altered in genetically modified mutants and simultaneously
InhA was overexpressed in both spontaneous and genetically modified mutants. InhA interacts with
other FAS-II enzymes of M. aurum in vivo.
Conclusion: Close resemblance of isoniazid/ethionamide action on InhA between M. tuberculosis
and M. aurum further supports the use of fast-growing and intracellularly surviving drug-resistant
M. aurum to substitute for highly virulent, extremely slow-growing M. tuberculosis strains in the early
stage of antituberculosis inhibitor screening