Effect of katG mutations on the virulence of Mycobacterium tuberculosis and the implication for transmission in humans

Abstract

The usefulness of isoniazid (INH), a key component of short-course chemotherapy of tuberculosis, is threatened by the emergence of drug-resistant strains of Mycobacterium tuberculosis with mutations in the katG gene. It is shown here that the most commonly occurring KatG mutation, where Ser 315 is replaced by Thr (S315T), is associated with clinically significant levels of INH resistance. In contrast to another resistant isolate, in which Pro replaces Thr 275, the S315T mutant produces active catalase-peroxidase and is virulent in the mouse model of the disease, indicating that a significant loss of bacterial fitness does not result from this frequent mutation. The implications of this finding for the transmission and reactivation of multidrug-resistant strains of M. tuberculosis are severe.

FIG. 1.

Schematic representation of the cloning strategy used to construct plasmids for complementing a ΔfurA-katG strain of M. tuberculosis with different katG alleles. Site-directed mutagenesis of katG was carried out with plasmid pKATII. An NdeI-MluI fragment spanning the point mutations was subsequently transferred to the equivalent sites in pPD28 to create pAP21 to pAP23. pPD28 is based on pKINT, a Bluescript-based mycobacterial integration vector. The boldface arrow marked P denotes the site of the main katG promoter in M. tuberculosis.

FIG. 2.

Western blot analysis of whole-cell protein fractions extracted from M. tuberculosis strain INH34 (ΔfurA-katG) complemented with a control plasmid (pAP01) or plasmids carrying the katG alleles T275P (pAP21), A139V (pAP22), and S315T (pAP23) or wild-type katG (pPD28) and also extracted from M. tuberculosis strain H37Rv. Extracts were immunoblotted with an anti-KatG polyclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to a nitrocellulose membrane. The positions of molecular mass markers are shown.

FIG. 3.

Catalase and peroxidase activity gel analysis of whole-cell protein fractions from M. tuberculosis strain INH34 (ΔfurA-katG) complemented with a control plasmid (pAP01) or plasmids carrying the katG alleles T275P (pAP21), A139V (pAP22), and S315T (pAP23) or wild-type katG (pPD28) and also from M. tuberculosis strain H37Rv.

FIG. 4.

(A and B) Growth in lungs (A) and spleen (B) of M. tuberculosis strain INH34 (ΔfurA-katG) complemented with control plasmid pAP01 (diamonds) or plasmids carrying the katG allele T275P (triangles) or S315T (circles) or wild-type katG (pPD28) (squares) in BALB/c mice following intravenous infection with 10⁶ CFU. (C and D) Results of an experiment in which we monitored the growth in BALB/c mice of strain INH34 complemented with the plasmid (pAP22) carrying the katG allele A139V compared to that of INH34 complemented with the other plasmids, pAP01, pAP21, pAP23, and pPD28. The white bars correspond to the CFU after 1 day, and the gray bars correspond to the CFU after 40 days, in the lungs (C) and spleen (D). Each time point represents the mean result of three or four mice, and the error bars correspond to the standard deviation.