An Upstream Truncation of thefurA-katGOperon Confers High-Level Isoniazid Resistance in a Mycobacterium tuberculosis Clinical Isolate with No Known Resistance-Associated Mutations

Abstract

ABSTRACTAlthough the major causes of isoniazid (INH) resistance inMycobacterium tuberculosisare confined to structural mutations inkatGand promoter mutations in themabA-inhAoperon, a significant proportion of INH-resistant strains have unknown resistance mechanisms. Recently, we identified a high-level INH-resistantM. tuberculosisclinical isolate, GB005, with no known resistance-associated mutations. A comprehensive study was performed to investigate the molecular basis of drug resistance in this strain. Although no mutations were found throughout thekatGandfurA-katGintergenic region, thekatGexpression and the catalase activity were greatly diminished compared to those in H37Rv (P< 0.01). Northern blotting revealed that thekatGtranscript from the isolate was smaller than that of H37Rv. Sequencing analysis offurAand upstream genes discovered a 7.2-kb truncation extended from the 96th base preceding the initiation codon ofkatG. Complementation of theM. tuberculosisΔ(furA-katG) strain withkatGand different portions of the truncated region identified a 134-bp upstream fragment offurAthat was essential for full catalase activity and INH susceptibility inM. tuberculosis. The promoter activity of this fragment was also shown to be stronger than that of thefurA-katGintergenic region (P< 0.01). Collectively, these findings demonstrate that deletion of the 134-bpfurAupstream fragment is responsible for the reduction inkatGexpression, resulting in INH resistance in GB005. To our knowledge, this is the first report showing that deletion of the upstream region preceding thefurA-katGoperon causes high-level INH resistance in a clinical isolate ofM. tuberculosis.