Transfer of a point mutation in Mycobacterium tuberculosis inhA resolves the target of isoniazid

Abstract

Isoniazid is one of the most effective antituberculosis drugs, yet its precise mechanism of action is still controversial. Using specialized linkage transduction, a single point mutation allele (S94A) within the putative target gene inhA was transferred in Mycobacterium tuberculosis. The inhA(S94A) allele was sufficient to confer clinically relevant levels of resistance to isoniazid killing and inhibition of mycolic acid biosynthesis. This resistance correlated with the decreased binding of the INH-NAD inhibitor to InhA, as shown by enzymatic and X-ray crystallographic analyses, and establishes InhA as the primary target of isoniazid action in M. tuberculosis.

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Figure 1: Construction and analysis of M.tuberculosis inhA(S94A) (Supplementary Methods).
Figure 2: X-ray crystallographic analysis of the INH-NAD adduct bound to wild-type InhA or InhA(S94A) protein.

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Acknowledgements

We gratefully acknowledge G. Morlock for sending us the DNA from the INH-resistant clinical isolate carrying the inhA(S94A) mutation. L. Kremer is supported by a grant from the Centre National de la Recherche Scientifique (ATIP “Microbiologie Fondamentale”). J.C.S. acknowledges the Robert A. Welch Foundation grant A-0015. We also acknowledge support for this work from US National Institutes of Health grants AI43268 and AI46669, and from the Structural Genomics Project grant 1P50GM6241. We also thank G. Hatfull for careful reading of this manuscript.

Author information

C.V. performed specialized transductions of inhA in M. tuberculosis and BCG and analyzed the transductants for their inserts, INH and ETH resistance and biochemical resistance to INH, and wrote most of the manuscript with W.R.J. F.W. prepared the INH-NAD adduct, performed the InhA enzymological analyses and X-ray crystallography. M.A. constructed the phages for transduction and performed and analyzed the kasA allelic exchanges in M. tuberculosis and M. bovis BCG. T.R.W. sequenced the transductants. M.H.H. and R.C. conducted the hairpin-shaped primer assays and mRNA experiments. L.K. provided the pMV261::kasA constructs and did the western blot analysis. D.A., J.C.S. and W.R.J. contributed to the design of the study, data analysis and data interpretation.

Correspondence to William R Jacobs Jr.

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Supplementary information

Supplementary Fig. 1

Drug susceptibility of M. bovis BCG inhA(S94A). (PDF 1773 kb)

Supplementary Table 1

Strains depictions and respective minimum inhibitory concentrations (MICs) for INH and ETH. (PDF 62 kb)

Supplementary Methods (PDF 198 kb)

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