Secreted Variant of Nucleoside Diphosphate Kinase from the Intracellular Parasitic Nematode Trichinella spiralis

Abstract

The molecular components involved in the survival of the parasitic nematode Trichinella spiralis in an intracellular environment are poorly characterized. Here we demonstrate that infective larvae secrete a nucleoside diphosphate kinase when maintained in vitro. The secreted enzyme forms a phosphohistidine intermediate and shows broad specificity in that it readily accepts ␥-phosphate from both ATP and GTP and donates it to all nucleoside and deoxynucleoside diphosphate acceptors tested. The enzyme was partially purified from culture medium by ATP affinity chromatography and identified as a 17-kDa protein by autophosphorylation and reactivity with an antibody to a plant-derived homologue. Secreted nucleoside diphosphate kinases have previously been identified only in prokaryotic organisms, all of them bacterial pathogens. The identification of a secreted variant of this enzyme from a multicellular eukaryote is very unusual and is suggestive of a role in modulating host cell function.Nucleoside diphosphate kinases (NDPKs) play a key role in the maintenance of intracellular pools of deoxynucleoside triphosphates (dNTPs) and NTPs via the transfer of phosphate from an NTP donor to an NDP acceptor. In addition, certain variants of these enzymes are involved in a variety of cellular processes unrelated to their catalytic activity, such as differentiation, proliferation, and suppression of tumor metastasis (8).In particular, nm23-H2/NDPK B has been identified as a DNA-binding protein and transcriptional activator of the human c-myc gene, previously known as PuF (29,31). NDPKs are typically intracellular enzymes, although recently an ectoenzyme has been detected on the surface of mammalian cells (19,20), and NDPKs have been reported to be secreted by the prokaryotic pathogens Mycobacterium bovis, Pseudomonas aeruginosa, and Vibrio cholerae (32,38,39).Trichinella spiralis is a ubiquitous nematode parasite of a wide variety of mammalian species, including humans, and is remarkable among multicellular parasites in adopting an intracellular habitat, both in the systemic phase of infection in skeletal muscle cells and in the enteral phase, in which it invades and migrates through mucosal epithelial cells (9). It is likely that secreted products are involved in survival and development of parasites in both environments. Consistent with this assumption, infective larvae possess a large organelle termed the stichosome, which is the major source of secreted proteins which can be recovered from in vitro culture of parasites (9).We have previously demonstrated that T. spiralis infective larvae secrete serine/threonine protein kinases (3). During the course of these studies, it became apparent that phosphorylation of a protein with an estimated mass of 17 kDa was regulated independently of both exogenous substrates and the major endogenous parasite substrate for protein kinase activity, a doublet of 50 and 55 kDa. We hypothesized that this may result from the activity of an additional enzyme and demonstrate here that T. spiralis se...

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

“…Reported here for the first time is that Leishmania secrete NdK, as reported previously for pathogenic bacteria [4] and parasitic nematodes [50,51]. NdK activity in the Leishmania culture supernatants is far too substantial (~40% of the total NdK activities and proteins) to be accounted for by inadvertent cytolysis.…”

Purinergic regulation of angiogenesis by human breast carcinoma-secreted nucleoside diphosphate kinase

“…The secretion of NDPK orthologues by intracellular parasites as a possible survival tactic (Gounaris et al, 2001;Chopra et al, 2003) and the secretion of NDPK by various solid and haematological malignancies OkabeKado and Kasukabe, 2003) lead us to propose the pathological role of secreted NDPK in promoting cancer and tumour angiogenesis. Extracellular NDPK in the tumour vasculature would elevate nucleotide signalling and subsequent release of vasoactive factors with angiogenic effects (Ziche and Morbidelli, 2000).…”

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).…”