Studies will be carried out with inocula of Mycobacterium leprae, freshly harvested, from the infected foot pads of nu/nu mice and enriched for viable organisms, to explore optimal biophysical and nutritional parameters for the metabolic maintenance of the leprosy bacillus using the following combination of sensitive metabolic assays: a) quantitation of intracellular adenosine triphosphate (ATP), b) incorporation of 14C-palmitate into PGL-1, c) measurement of the rate of 14CO2 released from 14C- palmitate. The combined use of these indicators of net energy status, anabolic and catabolic activity will overcome the limitations inherent in using a single metabolic assay. Preliminary studies indicate an acidic pH optimum (5.5-6.5) and a requirement for continuous maintenance of a gaseous environment with an oxygen concentration below ambient (2.5-10%) Metabolic maintenance and in vitro drug susceptibility testing under these conditions are markedly superior to closed systems employing neutral pH. This approach should lead to the step-wise development of a medium which will support long-term maintenance and subsequently genuine growth of M. leprae. Less than total success should still allow significant improvement in an in vitro medium in which drug activity against M. leprae could be assessed via changes in metabolic activity.
