Role for nucleotide excision repair in virulence of Mycobacterium tuberculosis

Abstract

Mutations in Mycobacterium tuberculosis uvrB result in severe sensitivity to acidified nitrite, a source of nitric oxide (6). In this study, we show that a uvrB mutant is exquisitely sensitive to UV light but not to several sources of reactive oxygen species in vitro. Furthermore, a uvrB mutant was attenuated in mice as judged by an extension of life span. Attenuation in mice was partially reversed by genetic inactivation of nitric oxide synthase 2 (iNOS) and almost completely reversed in mice lacking both iNOS and phagocyte oxidase. Thus, a gene predicted to encode a key element of DNA repair is required for resistance of M. tuberculosis to both reactive nitrogen and reactive oxygen species in mice.

FIG. 1.

Complementation of uvrB mutations for nitrite sensitivity. (A) Two independent uvrB::ΦMycoMarT7 mutants were complemented with the integrative plasmid pMV-uvrB. (B) Nitrite dose-response curves of wt, uvrB, and complemented strains. All strains contain either the empty vector pMV306 or pMV-uvrB integrated at the att site of the chromosome. Each datum point represents the average of triplicates from one experiment and is representative of the results from three independent experiments. Error bars represent standard deviations.

FIG. 2.

uvrB mutants are hypersusceptible to UV light. (A) Growth of M. tuberculosis strains after exposure to 8.5 mJ/m² of UV light. (B) UV dose-response curve. All strains were transformed by either plasmid pMV306 or pMV-uvrB. Each datum point represents the average of duplicates from one experiment and is representative of the results from three independent experiments.

FIG. 3.

A uvrB mutant has a slight growth defect in wt and phox/iNOS^−/− bone marrow-derived macrophages. Macrophages were infected with M. tuberculosis at a multiplicity of infection of 4. Each datum point represents the average of triplicates ± standard deviation from one of two representative experiments. Differences between wt and uvrB strains in unactivated wt or phox/iNOS^−/− macrophages are statistically significant according to Student's unpaired t test (P < 0.05). compl., complemented; γ, IFN-γ.

FIG. 4.

A uvrB mutant does not grow as well as wt M. tuberculosis in either wt or iNOS^−/− mice but returns to virulence in phox/iNOS-deficient mice. (A) CFU from the lungs of wt, iNOS, or phox/iNOS-deficient mice. The difference between wt and uvrB M. tuberculosis in wt mice at day 56 was statistically significant based on Student's unpaired t test (P = 0.006). Values between wt and uvrB M. tuberculosis in phox/iNOS^−/− mice were not considered statistically significant. (B) CFU from spleens of the same mice represented in panel A. Each datum point represents the average of the results from 3 to 4 mice. Error bars represent ±1 standard deviation. *, no mice survived to this time point.

FIG. 5.

A uvrB mutant is attenuated in wt and iNOS^−/− mice but returns to virulence in phox/iNOS-deficient mice. (A) The first experiment shows that the uvrB mutant is attenuated in both wt and iNOS^−/− mice (left panel). The uvrB mutant is attenuated in iNOS^−/− mice but is almost as virulent as wt M. tuberculosis in phox/iNOS^−/− mice (right panel). Groups of 4 to 5 mice were infected for each survival curve. wt mice for the second experiment were all still alive at the time the manuscript was submitted. (B) An fbiC mutant is not attenuated in mice. Groups of 5 wt or iNOS^−/− mice were infected with an aerosol of an fbiC mutant M. tuberculosis strain as described for the uvrB mutants.