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Temperature-induced changes in viability, diphenoloxidase and permeability of Mycobacterium leprae
Abstract:
Among mycobacteria secretion of the enzyme diphenoloxidase has been established as a property of Mycobacterium leprae. The antileprosy drug dapsone (DDS), which completely inhibits the enzyme from plant and mammalian sources, does not readily penetrate intact M. leprae. When the drug is complexed with polylysine, it easily permeates the bacteria and produces 100% inhibition of its diphenoloxidase, suggesting a permeability barrier of the cytoplasmic membrane of M. leprae to dapsone. In this study: (1) when the organisms, purified from fresh tissues of experimentally infected armadillos, were treated with dilute alkali or exposed to warmer temperatures, DDS penetrated the bacteria and inhibited the diphenoloxidase. Washing with trypsin had no effect. Dapsone easily permeated the bacilli, purified from tissues stored at 0 degrees C or at -80 degrees C. (2) Diphenoloxidase of freshly-prepared M. leprae was stimulated when the bacteria were exposed to 50 degrees C for 10 min; at 60 degrees C the activity decreased, and at 100 degrees C the enzyme was completely inactivated. When the enzyme was assayed at temperatures below 37 degrees C, the activity was considerably lower, indicating that M. leprae may not be a psychrophilic organism in this respect. (3) The bacteria exposed to 50 degrees C failed to multiply in mouse footpads. M. leprae remained viable in tissues stored at 0 degrees C or -80 degrees C; but when the bacteria purified from these tissues were frozen, they lost their viability. On the other hand, the organisms separated from fresh tissues remained viable when frozen at -80 degrees C. The inhibition of diphenoloxidase of M. leprae by dapsone could serve as an indirect method to assess the integrity of the bacterial cell membrane and to predict whether the bacteria would retain their viability on freezing.
Insights
The antileprosy drug dapsone (DDS) can inhibit Mycobacterium leprae diphenoloxidase if the bacterial membrane is compromised by heat or alkali. This inhibition can assess cell integrity and predict viability after freezing.
Area of Science:
- Microbiology
- Biochemistry
- Drug Discovery
Background:
- Mycobacterium leprae secretes diphenoloxidase, an enzyme targeted by the antileprosy drug dapsone (DDS).
- Intact M. leprae exhibits a cytoplasmic membrane barrier that prevents dapsone penetration.
- Polylysine complexation facilitates dapsone entry into M. leprae, inhibiting diphenoloxidase.
Purpose of the Study:
- To investigate methods for overcoming the dapsone permeability barrier in M. leprae.
- To characterize the thermal stability and activity of M. leprae diphenoloxidase.
- To assess the viability of M. leprae under different storage and freezing conditions.
Main Methods:
- Treatment of M. leprae with dilute alkali, varying temperatures, trypsin, and dapsone.
- Purification of M. leprae from fresh and stored armadillo tissues.
- Assay of diphenoloxidase activity at different temperatures and assessment of bacterial multiplication in mouse footpads.
Main Results:
- Alkali or heat treatment enabled dapsone penetration and diphenoloxidase inhibition in M. leprae.
- Diphenoloxidase activity was optimal around 50°C, with inactivation at higher temperatures.
- M. leprae exposed to 50°C lost infectivity; viability was retained in stored tissues but lost upon freezing post-storage.
Conclusions:
- Dapsone inhibition of M. leprae diphenoloxidase can be achieved by disrupting the cell membrane.
- M. leprae diphenoloxidase activity suggests it is not a psychrophilic organism.
- Dapsone inhibition serves as a potential indicator of M. leprae cell membrane integrity and viability post-freezing.