Identification of katG mutations associated with high-level isoniazid resistance in Mycobacterium tuberculosis

Abstract

Isoniazid (INH) is an effective first-line antituberculosis drug. KatG, a catalase-peroxidase, converts INH to an active form in Mycobacterium tuberculosis, and katG mutations are major causes of INH resistance. In the present study, we sequenced katG of 108 INH-resistant M. tuberculosis clinical isolates. Consequently, 9 novel KatG mutants with a single-amino-acid substitution were found. All of these mutants had significantly lower INH oxidase activities than the wild type, and each mutant showed various levels of activity. Isolates having mutations with relatively low activities showed high-level INH resistance. On the basis of our results and known mutations associated with INH resistance, we developed a new hybridization-based line probe assay for rapid detection of INH-resistant M. tuberculosis isolates.

FIG. 1.

Maps of large-scale deleted regions adjacent to katG in six Inh^r M. tuberculosis isolates. Bold arrows indicate the open reading frames annotated in the H37Rv genome sequence (http://genolist.pasteur.fr/TubercuList/). The dotted lines correspond to the deleted regions, with the end sequences and H37Rv genome coordinates given below. Underlined sequences are possible substrates for recombination. The box labeled “IS” represents the 750-bp fragment of IS6110. Numbers 1 to 6 represent the names of the isolates and correspond to the numbers shown in Table S1 in the supplemental material. A nucleotide shown in lowercase in region 5 indicates a mutation.

FIG. 2.

Western blot of whole-cell extracts from katG-deficient E. coli strain UM262 transformed with the empty vector, pTrcHis2-TOPO, or recombinant plasmids expressing various KatG mutations as follows: lanes 1 and 15, WT; lanes 2 and 16, empty vector; lane 3, R463L and D542H; lane 4, S315T and R463L; lane 5, Q127E and R463L; lane 6, P232S and R463L; lane 7, G123E, G299S, and R463L; lane 8, frame shift mutation from position 160; lane 9, S315T and D387H; lane 10, R463L and R489S; lane 11, S315R; lane 12, M420T and R463L; lane 13, A65A, M176T, and R463L; lane 14, H97R and R463L; lane 17, Δ(191W-192E) and R463L; lane 18, N133T; lane 19, R463L; lane 20, R463L and R632C; lane 21, S315T; lane 22, D419H and R463L; lane 23, S383P and R463L; lane 24, frame shift mutation from position 124; lane 25, in-frame insertion and deletion and R463L. The positions of molecular mass markers are shown on the left.