Fast-growing, non-infectious and intracellularly surviving drug-resistant Mycobacterium aurum: a model for high-throughput antituberculosis drug screening.
OBJECTIVE
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 two-hybrid 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.


CONCLUSIONS
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.
Gupta
Antima
A
Microbiology Division, Central Drug Research Institute, Lucknow 226001, Uttar Pradesh, India. a.gupta@sbc.bbk.ac.uk
Bhakta
Sanjib
S
Kundu
Subir
S
Gupta
Manish
M
Srivastava
Brahm S
BS
Srivastava
Ranjana
R
D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D015169:Colony Count, Microbial; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D024881:Drug Resistance, Bacterial; D005000:Ethionamide / Q000494:pharmacology; D005227:Fatty Acids / Q000096:biosynthesis; D020869:Gene Expression Profiling; D007538:Isoniazid / Q000494:pharmacology; D050296:Microbial Viability; D009154:Mutation; D009161:Mycobacterium / Q000187:drug effects*; D010078:Oxazines / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping; D020798:Two-Hybrid System Techniques; D014966:Xanthenes / Q000378:metabolism
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D005227:Fatty Acids; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin; D010088:Oxidoreductases; C085257:InhA protein, Mycobacterium; D005000:Ethionamide; D007538:Isoniazid