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Adenosine triphosphate content of Mycobacterium leprae isolated from armadillo tissue by Percoll buoyant density
Abstract:
A buoyant density centrifugation procedure using Percoll was developed for the isolation and purification of Mycobacterium leprae from experimentally infected armadillo liver tissue. The method separates the bacteria from host adenosine triphosphate (ATP) and tissue debris and recovers 20-25% of the bacteria within 2-2 1/2 hours under controlled conditions. The mean ATP content (585 pg/10(6] of the purified bacteria was similar to cultivable bacteria. The organisms did not leak intracellular ATP when exposed to phosphate buffer. Temperature-dependent ATP synthesis was observed within minutes and could be inhibited by 2,4-dinitrophenol. Freeze-thawing M. leprae as purified suspensions in buffer damaged the organisms, resulting in decreased ATP levels and an accelerated loss of ATP upon incubation under defined conditions. In vitro treatment with the antileprosy drug clofazimine increased the rate of ATP decay directly proportional to drug concentration.
Insights
Researchers developed a Percoll gradient centrifugation method to isolate Mycobacterium leprae from armadillo liver tissue. This technique purifies the bacteria, enabling further study of their metabolic activity and response to drugs like clofazimine.
Area of Science:
- Microbiology
- Biochemistry
Background:
- Mycobacterium leprae is the causative agent of leprosy.
- Efficient isolation of M. leprae is crucial for understanding its biology and developing treatments.
Purpose of the Study:
- To develop a Percoll buoyant density centrifugation method for isolating and purifying Mycobacterium leprae from infected armadillo liver tissue.
- To assess the viability and metabolic activity of purified M. leprae by measuring adenosine triphosphate (ATP) levels.
- To investigate the effects of environmental conditions and drug treatment on M. leprae viability.
Main Methods:
- Isolation of M. leprae from experimentally infected armadillo liver using Percoll buoyant density centrifugation.
- Measurement of adenosine triphosphate (ATP) content in purified M. leprae.
- Assessment of ATP leakage and synthesis under various conditions (phosphate buffer, temperature).
- Evaluation of the impact of freeze-thawing and clofazimine treatment on M. leprae ATP levels.
Main Results:
- The Percoll method successfully separated M. leprae from host tissue and debris, yielding 20-25% recovery within 2-2.5 hours.
- Purified M. leprae exhibited ATP content comparable to cultivable bacteria and did not leak ATP in phosphate buffer.
- Temperature-dependent ATP synthesis was observed and could be inhibited by 2,4-dinitrophenol.
- Freeze-thawing damaged purified M. leprae, leading to reduced ATP levels and accelerated ATP loss.
- In vitro treatment with clofazimine increased the rate of ATP decay in a dose-dependent manner.
Conclusions:
- Percoll buoyant density centrifugation is an effective method for purifying Mycobacterium leprae.
- The purified bacteria maintain metabolic activity, allowing for studies on ATP synthesis and decay.
- Clofazimine treatment directly impacts M. leprae's metabolic activity, indicated by accelerated ATP decay.