Limited in vitro multiplication of Mycobacterium leprae.

The inability to cultivate Mycobacterium leprae in vitro has been a major bottleneck in leprosy research. There have been numerous reports on successful in vitro cultivation of this organism, but these reports could not be confirmed by others in the field. Hence, in vitro multiplication of M. leprae was evaluated in various culture media. Only 2 media supported limited multiplication of M. leprae. One medium was used previously by one of the authors (AMD) for in vitro growth of M. lepraemurium and the other was a conditioned medium used for growth of mouse dorsal root ganglion. Growth was evaluated by 3 biochemical parameters: bacterial ATP, DNA and 3H-thymidine uptake. All 3 measurements revealed a 4-6-fold increase in cell biomass after 16 weeks of incubation at 34 degrees C. The harvested bacilli demonstrated a few of the important properties of M. leprae, including growth in mouse footpads. However, subcultures of these in-vitro-grown cells in the respective media could not be achieved. By the end of 12 weeks, the bacilli lost all intracellular ATP and the ability to incorporate 3H-thymidine; they also failed to multiply in mouse footpads.

Biochemical studies on Mycobacterium leprae.

Very little information is available on the basic biology of Mycobacterium leprae. It is not known why the organism fails to grow in bacteriological media or in cell cultures and why it has an unusual predilection for certain tissues in the human host where cells derived from the neural crest occur (e.g. skin, peripheral nerves, adrenal medulla). Biochemical studies have revealed that M. leprae contains an unusual form of the enzyme diphenoloxidase which has not been detected in other mycobacteria. The presence of a specific glutamic acid decarboxylase in the organism has been demonstrated. Although a few enzymes of glycolysis and tricarboxylic acid cycle have been investigated, nothing characteristic of the bacterium has been discovered, and how M. leprae derives energy for its survival and proliferation still remains obscure.

A possible metabolic role for o-diphenoloxidase in Mycobacterium leprae.

Among mycobacteria, Mycobacterium leprae is unique in its ability to oxidize a variety of diphenols to quinones in vitro. What physiologic role o-diphenoloxidase has in the organism remained unknown. Reducing substrates like NADPH, NADH and ascorbic acid reacted with the quinone formed from dopa (3,4-dihydroxyphenylalanine); the substrates were oxidized and the quinone was reduced back to diphenol in the process. Since the quinone undergoes reversible oxidation-reduction, diphenoloxidase might serve as an alternative respiratory mechanism in M. leprae for the utilization of other substrates, as has been reported in plants.

Uptake of 3,4-dihydroxy[3H]phenylalanine by Mycobacterium leprae isolated from frozen (-80 degrees C) armadillo tissue.

Mycobacterium leprae separated from armadillo tissues stored at -80 degrees C is similar to that from human sources in its ability to take up 3H-labelled 3,4-dihydroxyphenylalanine (DOPA). Several inhibitors were studied which showed complete or partial inhibition of [3H]DOPA uptake. These findings suggest that M. leprae isolated from frozen tissue possesses an active uptake system for [3H]DOPA.

Uptake of 3, 4-dihydroxy(3H)phenylalanine by Mycobacterium leprae isolated from frozen (-80 degrees C) armadillo tissue

Mycobacterium leprae separated from armadillo tissues stored at -80 degrees C is similar to that from human sources in its ability to take up 3H-labelled 3,4-dihydroxyphenylalanine (DOPA). Several inhibitors were studied which showed complete or partial inhibition of [3H]DOPA uptake. These findings suggest that M. leprae isolated from frozen tissue possesses an active uptake system for [3H]DOPA.

Metabolic inhibitors of host-tissue origin in Mycobacterium leprae.

It is not clear why host-derived bacteria are metabolically inert, compared to organisms grown in vitro. o-Diphenoloxidase is the only metabolic property proven to be present in Mycobacterium leprae separated from infected human as well as animal (mouse and armadillo) tissues. However, highly concentrated suspensions of M. leprae obtained from the organs of experimentally infected armadillos showed little or extremely low o-diphenoloxidase, while the organisms bound 14C-labeled dopa. When these preparations were diluted, they readily oxidized D-dopa to pigment. The activity remained unaltered by washing the suspensions with dilute alkali or acetone and ether, indicating that it is an intrinsic property of the bacilli. Treatment with different proteases relieved the inhibition, and resulted in a 100% stimulation of O-diphenoloxidase in the bacilli. Evidently, the M. leprae suspensions obtained from infected tissues contain an inhibitory material which is protein in nature, and the metabolic inertness sometimes observed in host-grown bacteria may not be due to loss of enzymes or metabolites from the organisms.

Mycobacterium leprae and phenoloxidase activity.

Our earlier studies indicated that the enzyme o-diphenoloxidase was absent in Mycobacterium leprae separated from depromatous human tissues. At that time the bacilli were not available from any other source. The existence or absence of this enzyme in M. leprae recovered from infected armadillo tissues were reinvestigated. The intact cells which were metabolically active, failed to oxidize DOPA. Likewise, DOPA and its derivatives were not oxidized by the enzymatically active cell-free preparations from M. leprae. Upon incubation of DOPA for more than 2 h with whole cell suspensions or particulate fractions, there was no development of colour with an absorption maximum of 540 nm as has been reported for an intermediate of DOPA oxidation. However, DOPA and several phenolic compounds were very actively oxidized by mushroom tyrosinase. The results suggested that M. leprae is deficient in o-diphenoloxidase, and this enzyme is not an intrinsic characteristic of this mycobacterium.