Leishmania-released nucleoside diphosphate kinase prevents ATP-mediated cytolysis of macrophages

Abstract

Leishmania amazonensis was found to release nucleoside diphosphate kinase (NdK) -a stable enzyme capable of decreasing extracellular ATP. The release of this enzyme from Leishmania results in its progressive accumulation extracellularly as they replicate, peaking at the stationary phase in vitro. The released NdK is immunoprecipitable and constitutes ~40% of its total activities and proteins. The retention of a known cytosolic protein by wild type cells and a fluorescent protein by DsRed transfectants at stationary phase, which release NdK, indicates that this is a spontaneous event, independent of inadvertent cytolysis. Recombinant products of Leishmania NdK prepared were enzymatically and immunologically active. Both recombinant and native Leishmania NdK utilized ATP to produce expected nucleoside triphosphates in the presence of nucleoside diphosphates in excess. Both native and recombinant Leishmania NdK were also found to prevent ATP-induced cytolysis of J774 macrophages in vitro, as determined by assays for lactate dehydrogenase release from these cells and for their mitochondrial membrane potential changes. The results obtained thus suggest that Leishmania NdK not only serves its normal house-keeping and other important functions true to all cells, but also prevents ATP-mediated lysis of macrophages, thereby preserving the integrity of the host cells to the benefit of the parasite.

Secretory nucleoside diphosphate kinases from both intra- and extracellular pathogenic bacteria are functionally indistinguishable

“…Based on this hypothesis, we utilized an assay system in which the cytotoxic effect or behaviour of ATP was studied by monitoring the release of the cytoplasmic marker enzyme LDH from J774 cells (Kolli et al, 2008;Melnikov et al, 2000;Punj et al, 2000). The system displayed ATP-dependent release of LDH from J774 cells (Fig.…”

“…P]ATP, 1 mM NDP or a mixture of NDPs and 1 mM MgCl 2 (Melnikov et al, 2000;Kolli et al, 2008). The reaction was performed at room temperature for 10 min and then stopped by incubating at 4 uC (over ice).…”

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.…”

A novel stage-specific glycosomal nucleoside diphosphate kinase from Trypanosoma cruzi

“…In addition, the Leishmania spp. gene orthologous was associated to microsomal fractions and it was found to be secreted regulating macrophage infection (de Oliveira et al 2006, Kolli et al 2008.…”