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

demonstrate whether these enzymes may be used by mycobacteria to sequester ATP from the macrophage P2Z receptors, thereby preventing phagosome-lysosom e fusion or macrophage apoptotic death. Mycobacterium bovis BCG secretes tw o ATP-scavenging enzymes, nucleoside diphosphate kinase (Ndk) and ATPase, d uring growth in Middlebrook 7H9 medium. In synthetic Sauton medium without any protein supplements, there is less secretion of these two enzymes unles s proteins such as bovine serum albumin (BSA), ovalbumin or extracts of mac rophages are added to the medium. There is a gradient of activity among var ious 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. Purificat ion of the enzymes from the culture filtrate of 7H9-grown M, bovis BCG cell s 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 AT Pase use ATP as a substrate, the physiological significance of the secretio n of these two ATP-utilizing enzymes was explored. External ATP is importan t in the activation of macrophage surface-associated P2Z receptors, whose a ctivation has been postulated to allow phagosome-lysosome fusion and macrop hage cell death. We demonstrate that the presence of the filtrate containin g these enzymes prevents ATP-induced macrophage cell death, as measured by the release of an intracellular enzyme, lactate dehydrogenase, In vitro com plexation 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 phag osome-lysosome fusion or macrophage apoptotic death.