Secretion of ATP-utilizing enzymes, nucleoside diphosphate kinase and ATPase, by Mycobacterium bovis BCG: sequestration of ATP from macrophage P2Z receptors?

Mycobacterium bovis BCG secretes two ATP-scavenging enzymes, nucleoside diphosphate kinase (Ndk) and ATPase, during growth in Middlebrook 7H9 medium. In synthetic Sauton medium without any protein supplements, there is less secretion of these two enzymes unless proteins such as bovine serum albumin (BSA), ovalbumin or extracts of macrophages are added to the medium. There is a gradient of activity among various proteins in triggering the induction of secretion of these two enzymes. Other mycobacteria, such as M. smegmatis, primarily secrete Ndk, while M. chelonae does not appear to secrete either of these two enzymes. Purification of the enzymes from the culture filtrate of 7H9-grown M. bovis BCG cells and determination of the N-terminal amino-acid sequence have demonstrated a high level of sequence identity of one of the ATPases with DnaK, a heat shock chaperone, of M. tuberculosis and M. leprae, while that of Ndk shows significant identity with the Ndk of Myxococcus xanthus. As both Ndk and ATPase use ATP as a substrate, the physiological significance of the secretion of these two ATP-utilizing enzymes was explored. External ATP is important in the activation of macrophage surface-associated P2Z receptors, whose activation has been postulated to allow phagosome-lysosome fusion and macrophage cell death. We demonstrate that the presence of the filtrate containing these enzymes prevents ATP-induced macrophage cell death, as measured by the release of an intracellular enzyme, lactate dehydrogenase. In vitro complexation studies with purified Ndk/ATPase and hyperproduced P2Z receptor protein will demonstrate whether these enzymes may be used by mycobacteria to sequester ATP from the macrophage P2Z receptors, thereby preventing phagosome-lysosome fusion or macrophage apoptotic death.

LDH isozymes with anomalous bands in semen of leprosy patients.

Activity of LDH isozymes was evaluated electrophoretically on 7% acrylamide gel in semen of 37 leprosy patients (15 with borderline, 12 with borderline tuberculoid and ten with lepromatous leprosy) and ten fertile men of 30-45 years of age. Significantly lower activities were recorded of LDH1 in all categories of leprosy patients. Similarly, lowering of LDH2 activity was noticed in borderline and lepromatous cases only, lowering of LDH4 activity in lepromatous cases only and LDH5 activity was lowered in borderline leprosy patients. Lowest activity of LDH3 and absence of LDHx were found in lepromatous leprosy. However, in borderline tuberculoid patients, LDH3 and LDHx were significantly higher. This exceptional increase in activity was found to be due to presence of additional (anomalous) isozymes bands of LDH3, LDHx and LDH4 in 25% of borderline tuberculoid patients. Additional bands of LDH3 have also been located in 40% of the borderline leprosy patients.

LDH isoenzyme as a tool for the identification of mycobacteria.

Using Polyacrylamide gel electrophoretic technique, the lactate dehydrogenase (LDH) isoenzyme patterns have been studied in four slow growing mycobacteria viz. Mycobacterium tuberculosis, M. avium, M. microti, and M. bovis and four rapid growing mycobacteria viz. M. Fortuitum, M. parafortuitum, M. thermoresistible and M. diernhoferi. Each mycobacterial species exhibited distinct isoenzyme pattern for LDH.

Effect of purified protein derivative and sonicates of Mycobacterium leprae and Mycobacterium bovis BCG on thromboplastin response in human monocytes in vitro.

Human monocytes isolated from peripheral blood responded with increased thromboplastin expression upon stimulation in vitro with three mycobacterial antigens: tuberculin purified protein derivative and sonicates of Mycobacterium boviS BCG and Mycobacterium leprae. The stimulating principle of mycobacteria is probably a cell wall constituent since crude extracts of cell walls were 2.5 to 25 times more potent in stimulating thromboplastin synthesis than were whole sonicates. This thromboplastin response was inhibited by inhibitors of RNA and protein synthesis, dexamethasone, and agents that caused elevation of intracellular cyclic AMP. The presence of lymphocytes did not enhance the monocyte thromboplastin response significantly during the first 24 h of incubation. For M. bovis BCG and M. leprae sonicates, the thromboplastin response correlated with general activating effects measured by determining the release of lysozyme and beta-glucuronidase. The role of thromboplastin in chronic inflammatory reactions is discussed.

[Photometric studies of the activity of dehydrogenases and nonspecific esterases in leprosy]. / Fotometricheskie issledovaniia aktivnosti degidrogenaz i nespetsificheskikh ésteraz pri lepre.

The enzymatic activity of dehydrogenases and nonspecific esterases in skin affections in leprosy and their dynamics under the effect of treatment were investigated photometrically. It has been found that pentosophosphate pathway oxidation was mostly pronounced in microbe-containing macrophages of the infiltrate in untreated patients with the lepromatous type of leprosy and less according to the Krebs cycle and glycolysis. The intensity of oxidative processes is reduced under the influence of treatment, mainly, due to considerable inhibition of glycose-6-phosphatdehydrogenase. The activity of nonspecific esterases before the treatment is high in leprosy macrophages and low in the epidermis (as compared with the norm). Under the effect of antileprosy therapy the esterase content in the epidermis is restored and in the macrophages is decreased significantly.