Lipoprotein processing is essential for resistance of Mycobacterium tuberculosis to malachite green

Abstract

Malachite green, a synthetic antimicrobial dye, has been used for over 50 years in mycobacterial culture medium to inhibit the growth of contaminants. The molecular basis of mycobacterial resistance to malachite green is unknown, although the presence of malachite green-reducing enzymes in the cell envelope has been suggested. The objective of this study was to investigate the role of lipoproteins in resistance of Mycobacterium tuberculosis to malachite green. The replication of an M. tuberculosis lipoprotein signal peptidase II (lspA) mutant (DeltalspA::lspAmut) on Middlebrook agar with and without 1 mg/liter malachite green was investigated. The lspA mutant was also compared with wild-type M. tuberculosis in the decolorization rate of malachite green and sensitivity to sodium dodecyl sulfate (SDS) detergent and first-line antituberculosis drugs. The lspA mutant has a 10(4)-fold reduction in CFU-forming efficiency on Middlebrook agar with malachite green. Malachite green is decolorized faster in the presence of the lspA mutant than wild-type bacteria. The lspA mutant is hypersensitive to SDS detergent and shows increased sensitivity to first-line antituberculosis drugs. In summary, lipoprotein processing by LspA is essential for resistance of M. tuberculosis to malachite green. A cell wall permeability defect is likely responsible for the hypersensitivity of lspA mutant to malachite green.

FIG. 1.

Sensitivity of the M. tuberculosis lspA mutant to malachite green. (a) Chemical structure of malachite green; (b to d) growth of the wild type (H37Rv), the ΔlspA::ΔlspA_mut mutant (::lspA_mut), and the ΔlspA::ΔlspA strain (::lspA) on Middlebrook 7H9 agar with (b) or without (c) malachite green (0.001 g/liter) and on Middlebrook 7H11 agar (d). Bacterial dilutions are shown on top. Images were taken after 3 weeks of incubation. (e) Decolorization of malachite green in the presence of live bacteria. OD₆₂₀, optical density at 620 nm. (f) Bacterial viability after exposure to 0.05% SDS. Data points show means ± standard deviations of triplicates. All data are representative of three independent experiments.