Cu,Zn superoxide dismutase of Mycobacterium tuberculosis contributes to survival in activated macrophages that are generating an oxidative burst

Abstract

Macrophages produce reactive oxygen species and reactive nitrogen species that have potent antimicrobial activity. Resistance to killing by macrophages is critical to the virulence of Mycobacterium tuberculosis. M. tuberculosis has two genes encoding superoxide dismutase proteins, sodA and sodC. SodC is a Cu,Zn superoxide dismutase responsible for only a minor portion of the superoxide dismutase activity of M. tuberculosis. However, SodC has a lipoprotein binding motif, which suggests that it may be anchored in the membrane to protect M. tuberculosis from reactive oxygen intermediates at the bacterial surface. To examine the role of the Cu,Zn superoxide dismutase in protecting M. tuberculosis from the toxic effects of exogenously generated reactive oxygen species, we constructed a null mutation in the sodC gene. In this report, we show that the M. tuberculosis sodC mutant is readily killed by superoxide generated externally, while the isogenic parental M. tuberculosis is unaffected under these conditions. Furthermore, the sodC mutant has enhanced susceptibility to killing by gamma interferon (IFN-gamma)-activated murine peritoneal macrophages producing oxidative burst products but is unaffected by macrophages not activated by IFN-gamma or by macrophages from respiratory burst-deficient mice. These observations establish that the Cu,Zn superoxide dismutase contributes to the resistance of M. tuberculosis against oxidative burst products generated by activated macrophages.

FIG. 1

(A) Restriction map of the sodC region in parental and sodC mutant strains of M. tuberculosis. (B) Southern blot of chromosomal DNAs from the parental, mutant, and complemented strains. DNA was digested with EcoRV and probed with DNA containing the gene for sodC or for aph. The sodC-hybridizing fragment in mutant Mtb1612 is larger than the unmutated fragment due to insertion of the drug resistance cassette. This fragment also hybridizes with the aph probe, confirming that Mtb1612 contains the kanamycin resistance gene that produced the mutation.

FIG. 2

Growth of the sodC mutant Mtb1612 and parental M. tuberculosis Erdman in 7H9 broth. Growth was monitored by reading the OD₅₈₀ and is expressed as the mean and standard error of the mean for triplicate samples.

FIG. 3

(A) Survival of the sodC mutant Mtb1612 (circles) and the sodC-complemented strain Mtb1623 (triangles) was compared to survival of parental M. tuberculosis (squares) using hypoxanthine/xanthine oxidase to generate superoxide. The number of surviving bacteria was determined at 0, 1, and 3 h after exposure to superoxide by plating dilutions of the bacteria on 7H10 plates. The means from triplicate tubes were calculated, and the data are expressed as percentages of the time zero value. Results of a representative assay from six experiments are shown. (B) Survival of the sodC mutant Mtb1612 (open symbols) was compared to that of parental M. tuberculosis (closed symbols) using hypoxanthine/xanthine oxidase and SPER/NO (Alexis Biochemicals) to generate superoxide (squares), nitric oxide (circles), or a combination of both (triangles). The number of surviving bacteria was determined at 0, 1, and 3 h after exposure to the compounds by plating dilutions on 7H10 medium. The means from triplicate tubes were calculated, and the data are expressed as percentages of the time zero value. Results of a representative assay from three experiments are shown.

FIG. 4

Survival of parental M. tuberculosis (squares), the sodC mutant Mtb1612 (circles), or the sodC-complemented strain Mtb1623 (triangles) in peritoneal macrophages from C57BL/6 mice (A), gp91^phox−/− mice (B), or iNOS^−/− mice (C). Macrophages were activated with IFN-γ (100 U/ml) overnight and were then infected with bacteria at a multiplicity of infection of 10:1. The number of surviving bacteria was determined by plating dilutions of the macrophage lysate on 7H10 plates. The data are expressed as the mean and standard error of the mean from triplicate wells (C57BL/6 and iNOS^−/−) or quadruplicate wells (gp91^phox−/−) at each time point.

FIG. 5

Southern blot of chromosomal DNAs from 15 mycobacterial species. DNA was digested with NotI and probed with the sodC gene from M. tuberculosis. Lane 1, M. tuberculosis; lane 2, M. africanum; lane 3, M. bovis; lane 4, M. microti; lane 5, M. bovis BCG; lane 6, M. fortuitum; lane 7, M. chelonae; lane 8, M. smegmatis; lane 9, M. avium; lane 10, M. intracellulare; lane 11, M. gordonii; lane 12, M. marinum; lane 13, M. scrofulaceum; lane 14, M. kansasii; and lane 15, M. xenopi.