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

Abstract

SummaryMycobacterium 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. Puri®cation of the enzymes from the culture ®ltrate 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 signi®cant identity with the Ndk of Myxococcus xanthus. As both Ndk and ATPase use ATP as a substrate, the physiological signi®cance 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 ®ltrate 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 puri®ed 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±lyso-some fusion or macrophage apoptotic death.

Comprehensive Analysis of the Secreted Proteins of the Parasite Haemonchus contortus Reveals Extensive Sequence Variation and Differential Immune Recognition

“…Nucleoside diphosphate kinase (NDK) (spot 157) is responsible for maintaining the levels of intracellular nucleotide pools but is also involved in cell growth, differentiation, and tumor metastasis (66). A secreted form has been found only in some bacteria (67)(68)(69) and in the nematode Trichinella spiralis (70). A function is suggested by observed ATP-dependent cytotoxicity for macrophages and mast cells and prevention of cells from apoptosis (67,71).…”

Schistosome apyrase SmATPDase1, but not SmATPDase2, hydrolyses exogenous ATP and ADP

“…An ability to cleave ATP and/or ADP in the extracellular environment has been described in several pathogens including bacteria, protozoans, and nematodes [34][35][36][37][38][39][40]. This conserved feature of several pathogens is also found in schistosomes, here mediated by SmATPDase1.…”

The P2X7 receptor and intracellular pathogens: a continuing struggle

“…Infection by several pathogens has now been shown to inhibit ATP-induced apoptosis of the host cell, including mycobacteria, chlamydiae, Porphyromonas gingivalis, and Leishmania [45,[49][50][51]. However, the mechanism by which these pathogens protect the host cell has been characterized mainly for mycobacteria and P. gingivalis [49,50], both of which secrete an enzyme, nucleoside diphosphate kinase (NDK), that consumes extracellular ATP, thus depriving P2X 7 of its physiological ligand.…”

“…However, the mechanism by which these pathogens protect the host cell has been characterized mainly for mycobacteria and P. gingivalis [49,50], both of which secrete an enzyme, nucleoside diphosphate kinase (NDK), that consumes extracellular ATP, thus depriving P2X 7 of its physiological ligand.…”

“…Approximately 70% homology was reported between the N terminus of M. smegmatis NDK and those of P. aeruginosa, E. coli, and Myxococcus xanthus. Mycobacterium bovis BCG is capable of secreting NDK upon stimulation with eukaryotic proteins (bovine serum albumin or ovalbumin) [50]. Later, recombinant M. tuberculosis NDK was crystallized by Chen et al, who showed that it has 45% sequence identity to human NDK with similar secondary and tertiary structures and exists as a hexamer, unlike most other bacteria [54].…”