INFECTION AND IMMUNITY,0019-9567/01/$04.0010 DOI: 10.1128/IAI.69.6.3658–3662.2001

June 2001, p. 3658–3662 Vol. 69, No. 6

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Secreted Variant of Nucleoside Diphosphate Kinase from theIntracellular Parasitic Nematode Trichinella spiralis

KLEONIKI GOUNARIS,* SIMON THOMAS, PILAR NAJARRO, AND MURRAY E. SELKIRK

Department of Biochemistry, Imperial College of Science, Technology and Medicine,London SW7 2AY, United Kingdom

Received 22 December 2000/Returned for modification 11 February 2001/Accepted 7 March 2001

The molecular components involved in the survival of the parasitic nematode Trichinella spiralis in anintracellular environment are poorly characterized. Here we demonstrate that infective larvae secrete anucleoside diphosphate kinase when maintained in vitro. The secreted enzyme forms a phosphohistidineintermediate and shows broad specificity in that it readily accepts g-phosphate from both ATP and GTP anddonates it to all nucleoside and deoxynucleoside diphosphate acceptors tested. The enzyme was partiallypurified from culture medium by ATP affinity chromatography and identified as a 17-kDa protein by auto-phosphorylation and reactivity with an antibody to a plant-derived homologue. Secreted nucleoside diphos-phate 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 issuggestive of a role in modulating host cell function.

Nucleoside diphosphate kinases (NDPKs) play a key role inthe maintenance of intracellular pools of deoxynucleosidetriphosphates (dNTPs) and NTPs via the transfer of phosphatefrom an NTP donor to an NDP acceptor. In addition, certainvariants of these enzymes are involved in a variety of cellularprocesses unrelated to their catalytic activity, such as differen-tiation, proliferation, and suppression of tumor metastasis (8).In particular, nm23-H2/NDPK B has been identified as aDNA-binding protein and transcriptional activator of the hu-man c-myc gene, previously known as PuF (29, 31). NDPKs aretypically intracellular enzymes, although recently an ectoen-zyme has been detected on the surface of mammalian cells (19,20), and NDPKs have been reported to be secreted by theprokaryotic pathogens Mycobacterium bovis, Pseudomonasaeruginosa, and Vibrio cholerae (32, 38, 39).

Trichinella spiralis is a ubiquitous nematode parasite of awide variety of mammalian species, including humans, and isremarkable among multicellular parasites in adopting an in-tracellular habitat, both in the systemic phase of infection inskeletal muscle cells and in the enteral phase, in which itinvades and migrates through mucosal epithelial cells (9). It islikely that secreted products are involved in survival and de-velopment of parasites in both environments. Consistent withthis assumption, infective larvae possess a large organelletermed the stichosome, which is the major source of secretedproteins which can be recovered from in vitro culture of par-asites (9).

We have previously demonstrated that T. spiralis infectivelarvae secrete serine/threonine protein kinases (3). During thecourse of these studies, it became apparent that phosphoryla-tion of a protein with an estimated mass of 17 kDa was regu-lated independently of both exogenous substrates and the ma-

jor endogenous parasite substrate for protein kinase activity, adoublet of 50 and 55 kDa. We hypothesized that this may resultfrom the activity of an additional enzyme and demonstratehere that T. spiralis secretes an NDPK which is autophospho-rylated as part of its catalytic mechanism.

MATERIALS AND METHODS

Parasites. Infective larvae of T. spiralis were recovered from outbred rats 2months after oral infection as previously described (3). Parasites were cultured inserum-free RPMI 1640 containing 0.25% glucose, 2 mM L-glutamine, penicillin(100 U ml21), streptomycin (100 mg ml21) and gentamicin (20 mg ml21) at 37°Cin 5% CO2 for up to 72 h with a daily change of medium. Pooled secretedproducts were cleared through 0.2-mm-pore-size filters, dialyzed against 25 mMHEPES (pH 7.5), concentrated by passage through Centricon 10 microconcen-trators (Amicon), and assayed for protein content using the bicinchoninic acidmicroplate assay (Pierce).

NDPK phosphotransferase assay. Phosphotransferase assays were conductedby incubating 1 mg of secreted proteins or 100 ng of a partially purified enzymefraction at 37°C for 30 min in 25 mM HEPES (pH 7.5)–50 mM NaCl–10 mMMgCl2–10 mM dithiothreitol (DTT) in a final volume of 10 ml with 1 mCi or[g-32P] ATP or [g-32P] GTP and a range of NDP acceptors at 10 mM. Controlreactions were set up in the absence of parasite proteins or acceptor. Reactionswere terminated with 1 ml of 500 mM EDTA, and 1 ml was resolved by thin-layerchromatography (TLC) on cellulose MN 300 polyethyleneimine-impregnatedplates (Macherey-Nagel) developed with 0.75 M KH2PO4 (pH 3.65). Plates weredried and exposed to autoradiography.

NDPK autophosphorylation and protein phosphorylation assays. For NDPKautophosphorylation assays, parasite secreted proteins (3 to 4 mg) were incu-bated in 25 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM MgCl2, 0.8 mM CaCl2,5 mM KCl, and 5 mM EDTA in the presence of 10 mCi of [g-32P] ATP at 37°Cfor 30 min in a final volume of 10 ml. For protein phosphorylation assays, 2 mgof proteins was incubated in 25 mM HEPES (pH 7.5)–50 mM NaCl–10 mMMgCl2–10 mM DTT in the presence of 10 mCi of [g-32P] ATP for 30 min at 37°C.Reactions were terminated by addition of Laemmli sample buffer, resolved bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 15or 20% gel, and exposed to autoradiography.

ATP-agarose chromatography. Cyanogen bromide-activated ATP agarose(linked through C-8; nine-atom spacer; Sigma) was washed and equilibrated with25 mM HEPES-KOH (pH 7.5)–2 mM MgCl2–25 mM KCl–0.1 mM EDTA–0.05mM DTT. Secreted parasite proteins were loaded onto the column, and thecolumn was washed with the above buffer. Bound proteins were eluted with 0.5M KCl in the above buffer, followed by elution with 2 mM ATP (pH adjusted to7.5). Fractions thus obtained were concentrated by passage through Centricon 10

* Corresponding author. Mailing address: Department of Biochem-istry, Imperial College of Science, Technology and Medicine, LondonSW7 2AY, United Kingdom. Phone: 44 20 7594 5209. Fax: 44 20 75945207. E-mail: k.gounaris@ic.ac.uk.

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microconcentrators (Amicon) and extensively washed with 25 mM HEPES (pH7.5).

Western blotting. Protein samples were resolved by SDS-PAGE on a 15% gel,transferred to nitrocellulose membranes, and overlaid with a 1:400 dilution of arabbit polyclonal antibody to NDK-P1 from Pisum sativum, a kind gift of Paul A.Millner (12). Binding was determined by standard procedures utilizing horse-radish peroxidase-conjugated secondary antibodies and enhanced chemilumines-cence (Amersham RPN 2209).

Acid/alkali stability of phosphorylated NDPK residues. Samples in which theNDPK was autophosphorylated as described above were resolved by SDS-PAGEand transferred to polyvinylidene difluoride (PVDF) membranes. Radioactivitywas localized by autoradiography, and the areas on the membrane correspondingto NDPK were isolated and treated as described elsewhere (5). Briefly, pieces ofPVDF membrane were incubated for 2 h at 45°C in 200 ml of the appropriatebuffer containing 5% methanol, and radioactivity released was measured byscintillation counting. The following buffers were used: 50 mM KCl-HCl (pH 1),50 mM glycine-HCl (pH 3), 0.1 M Tris-HCl (pH 7), 50 mM KCl-NaOH (pH 12),and 1 M KOH (pH 14). The membranes were then subjected to a further 2-hincubation at either pH 1 or pH 14 as indicated.

RESULTS

In previous studies, we had observed a phosphoprotein withan estimated mass of 17 kDa in the products secreted byinfective larvae of T. Spiralis. We hypothesized that this mightbe the result of an autocatalytic phosphorylation event cata-lyzed by an NDPK enzyme, although this would be unusual inthat NDPKs are typically intracellular enzymes. We thereforescreened for an activity in secreted products which transferredthe terminal g-phosphate from radiolabeled NTP donors toNDP acceptors; as shown in Fig. 1, the requisite phosphotrans-ferase activity was indeed present in parasite secreted prod-ucts. The enzyme was capable of utilizing both ATP (Fig. 1A)and GTP (Fig. 1B) as donors, and all NDPs as acceptors.Furthermore, the enzyme could efficiently transfer phosphateto dNDP acceptors (data not shown).

In all NDPKs investigated, autophosphorylation of an ac-tive-site histidine is an intermediate in the catalytic mecha-nism. The parasite secreted NDPK was therefore identified viareaction with [g-32P] ATP under conditions which have beenpreviously defined to optimize autophosphorylation (4). Theresults are presented in Fig. 2, which illustrate the phosphor-ylation of a 17-kDa protein (lane 1), which was completelyabolished by the inclusion of 10 mM NDP acceptor in thereaction buffer (lane 2). Although, as stated above, NDPKs areknown to phosphorylate on an active-site histidine residue,there have been numerous reports of additional serine (1, 7,15, 22, 26) as well as aspartate and glutamate phosphorylation(36). In particular, phosphoserine formation has been linked toa variety of cellular processes involving NDPK activity otherthan the transfer of terminal phosphate between nucleosides.As shown in Fig. 3, we observed that 80 to 90% of the radio-activity was acid labile and alkali stable, indicative of histidinephosphorylation. Furthermore, alkali-stable radioactivity wasreleased by subsequent acid treatment. It has been reportedthat addition of cyclic AMP in the reaction medium inhibitsserine phosphorylation of human NDPK/nm23-H1 (22). Addi-tion of excess cyclic AMP in the reaction medium resulted in

FIG. 1. NDPK activity in secreted products of infective larvae. (A)Transfer of g-32P from ATP to UDP (lane 1), CDP (lane 2), and GDP(lane 3). (B) Transfer of g-32P from GTP to UDP (lane 1), CDP (lane2), and ADP (lane 3). Positions of migration of NTPs in TLC areindicated, as are the positions of liberated radiolabeled phosphate (Pi)and the origin (O).

FIG. 2. Identification of the secreted NDPK as a 17-kDa protein byautophosphorylation. Lanes: 1, reaction performed in the absence ofan NDP acceptor; 2, reaction performed in the presence of 10 mMGDP. Reaction products were resolved by SDS-PAGE (20% gel) andexposed to autoradiography. Autophosphorylated NDPK is indicatedby an asterisk.

FIG. 3. pH stability of autophosphorylated secreted NDPK. Treat-ment of NDPK immobilized on PVDF membranes was carried out asdescribed in Materials and Methods. Values show percentage of totalradioactivity released after incubation for 2 h in solutions of differentpH (white bars) or after a subsequent incubation at pH 1 (hatchedbars) or pH 14 (black bars). Values are averages of three independentexperiments (standard deviations are indicated).

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no significant changes in the profile that we observed (data notshown). We did, however, reproducibly observe 5 to 10% acid-resistant phosphorylation and therefore carried out phospho-amino acid analysis after acid hydrolysis of the phosphorylatedNDPK, which revealed no phosphorylated serine residues(data not shown).

We proceeded to purify the NDPK by ATP-agarose chro-matography. Figure 4 shows the profiles of total secreted prod-ucts (lane 1), unbound proteins (lane 2), and those eluted by0.5 M KCl (lane 3) and subsequently by 2 mM ATP (lane 4).Proteins with apparent masses of 70, 58, 30, and 17 kDa wereeluted in the KCl fraction, and a single protein of 70 kDa waseluted by ATP. Western blotting and reaction with a polyclonalantibody to NDPK from P. sativum demonstrated reactivity tothe 17-kDa protein in total secreted products and the KCl-eluted fraction, conclusively identifying this protein as a para-site-secreted NDPK. Antibody binding was occasionally ob-served to a protein with apparent molecular mass of 70 kDa inthe latter fraction, possibly indicative of a multimeric associa-tion of the enzyme, although there was no reactivity to the70-kDa protein eluted with ATP (lane 4).

Given that we had previously identified serine/threonineprotein kinase activity in T. spiralis secreted products (3) andthat NDPKs have been demonstrated to phosphorylate otherproteins with which they form close association (10, 11), wesought to discriminate between these activities to determinewhether they indeed were catalyzed by two distinct enzymessecreted by these organisms. Figure 5A demonstrates thatNDPK activity (assayed by transfer of phosphate to CDP) waspresent in both total secreted products and the KCl-elutedfraction of the ATP column but absent from in the flowthrough from the same column. These fractions were thenassayed under conditions optimal for protein phosphorylation.Figure 5B shows that the total secreted products phosphory-lated a triplet of proteins between 50 and 60 kDa and a proteinof 17 kDa (lane 1). Phosphorylation of the 17-kDa proteinalone was observed in the KCl-eluted fraction, whereas phos-phorylation of the 50- to 60-kDa triplet alone was obtainedwith the flowthrough. These data therefore demonstrate that

both NDPK and protein kinase activities are present in para-site secreted products and that they may be effectively sepa-rated by ATP-agarose chromatography under the conditionsdescribed.

A number of secreted T. spiralis proteins have been shown topossess a novel family of tri- and tetra-antennary N-glycanscapped by unusual tyvelose residues, and it has been demon-strated that tyvelose-specific monoclonal antibodies block in-vasion of epithelial cells by infective larvae in vitro (2). Wetherefore examined whether any of the proteins which boundto the ATP column were modified in this manner via Westernblotting with a tyvelose-specific monoclonal antibody desig-nated 18H, provided by Judith A. Appleton (23). We observedno binding to any of these proteins (data not shown), andfound the expected profile of reactivity against other secretedproducts, and therefore conclude that the ATP-binding pro-teins described here are not modified by N-glycans incorporat-ing tyvelose residues. In addition, we carried out deglycosyla-tion reactions on the autophosphorylated form of the NDPKwith N-glycanase, the results of which suggested that this en-zyme is not glycosylated (data not shown).

DISCUSSION

The data presented here show that infective larvae of T.spiralis secrete NDPK. This was demonstrated by phospho-transferase activity, autophosphorylation, and reactivity withan antibody to NDPK from a plant source (P. sativum). Both ofthe latter procedures identified a protein of 17 kDa in secretedproducts, and inhibition of autophosphorylation by GDP iden-tified this as an NDPK rather than a substrate for a proteinkinase.

It was further observed that NDPK and protein kinase ac-tivities could be separated by ATP affinity chromatography.The identities of the other nucleotide-binding proteins at 30,58, and 70 kDa are not known. NDPK and protein kinaseactivities could also be effectively distinguished by manipula-

FIG. 4. Partial purification of the enzyme by ATP affinity chroma-tography. Samples from the various fractions were stained with Coo-massie blue (A) or transferred onto nitrocellulose and reacted with anantibody to NDPK from P. sativum (B). Lanes 1, total secreted pro-teins; 2, unbound proteins; 3, proteins eluted with 0.5 M KCl; 4,proteins eluted with 2 mM ATP. Proteins were resolved by SDS-PAGE(15% gel). The molecular masses of marker proteins are shown inkilodaltons.

FIG. 5. ATP affinity chromatography separates NDPK and proteinkinase activities. (A) Phosphotransferase assay with [g-32P] ATP donorand CDP acceptor resolved by TLC. The migration of NTPs is indi-cated. (B) Phosphorylation assays performed under conditions de-scribed in Materials and Methods. Proteins were resolved by SDS-PAGE (15% gel), and the molecular masses of marker proteins areshown on the right. Lanes: 1, total secreted proteins; 2, unboundproteins; 3, proteins eluted with 0.5 M KCl.

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tion of the Mg21 concentration. Thus, when EDTA was usedto generate low levels of Mg21, NDPK was the only proteinphosphorylated in total secreted products (Fig. 2). It has beenpreviously shown that in Candida albicans, NDPK autophos-phorylation occurs with Mg21 in the nanomolar range, andanalogous to our findings, under optimal conditions it was theonly protein phosphorylated in crude extracts (4).

We tested for the formation of the high-energy phosphoen-zyme intermediate and showed that the autophosphorylatedenzyme donates all of the phosphate to GDP (Fig. 2) and thatalmost all of the radioactivity is acid labile (Fig. 3). We there-fore conclude that in this secreted variant of NDPK only thehigh-energy phosphoenzyme is formed. The low levels of acid-resistant radioactivity could be due to nonenzymatic transphos-phorylation (5), and release of radioactivity at pH 7 can beaccounted for by the low thermal stability of histidine-associ-ated phosphate at neutral pH as previously reported (5, 6, 21).Secreted NDPK from T. spiralis copurified, under our condi-tions, with other proteins (Fig. 4). The identities of these pro-teins are unknown, but NDPKs from other sources have alsobeen shown to copurify with a number of other proteins (10,28).

NDPKs are ubiquitous enzymes which, in keeping with theirrole in the maintenance of intracellular nucleotide pools, ineukaryotes are found in the nucleus, the mitochondria andchloroplasts, and the cytosol (8). Different isoforms of NDPKshow discrete patterns of expression in different tissues andduring differentiation, although generally they are consideredintracellular enzymes. An ectoenzyme was recently found to beassociated with the surface of a human astrocytoma cell line(20), and ecto-NDPKs were subsequently described for a va-riety of other cell lines (19). It was suggested that this extra-cellular transphosphorylating activity might play a role in mod-ulating adenine and uridine nucleotides in order to influencecellular functions via the P2 receptor class of signaling proteins(19, 20).

Only three examples of NDPK secretion, all from bacterialpathogens, appear to have been reported in the literature. M.bovis (and M. smegmatis) secrete both NDPK and ATPase(38). Extrinsic ATP acting through P2Z receptors on macro-phages has been shown to induce both cell death by apoptosisand killing of resident mycobacteria (18). As secreted productsfrom mycobacteria prevent ATP-induced macrophage apopto-sis, it was suggested that depletion of extracellular ATP bythese enzymes may act to promote survival of mycobacterium-infected cells (38). This postulate assumes that nucleotide-utilizing enzymes secreted by mycobacteria resident in phago-somes gain egress to the external environment. Moreover, thepotential role of an NDPK in this process is less clear than thatprovided by an ATPase, as in addition to ATP depletion, thephosphotransferase activity of NDPK would generate ATPfrom other extracellular NDPs.

The other organisms for which NDPK secretion has beenreported are P. aeruginosa (39) and V. cholerae (32). In bothcases, NDPK was one of multiple nucleotide-utilizing enzymessecreted, including ATPase, 59-nucleotidase, and adenylate ki-nase. Rather than protecting cells from ATP-induced apopto-sis, the secreted products from these organisms are cytotoxicfor macrophages and mast cells. It was suggested that thedichotomy in postulated functions for these enzymes lay on the

one hand in the intracellular habitat of mycobacteria and onthe other in the extracellular environment of P. aeruginosa andV. cholera, in which leukocytes present a potential threat ratherthan a requirement for survival (32, 39). Although it is unclearhow these contrasting functions might be regulated, it is ofinterest that in the case of P. aeruginosa, NDPK secretion wasobserved only from virulent mucoid strains isolated from cysticfibrosis patients, not from avirulent strains (39). A specific rolehad previously been proposed for intracellular NDPK in theprovision of GTP for synthesis of the exopolysaccharide algi-nate, associated with the transition to mucoidy (33).

To the best of our knowledge, the current data provide thefirst documented example of NDPK secretion by a eukaryoticorganism, although significantly this is another infectiousagent. A possible function for a T. spiralis secreted NDPKmight lie in regulation of host cell proliferation and differen-tiation. Six isoforms of NDPK in humans, termed nm23-H1 to-H6, have been described (24, 25, 30, 34). One of these (nm23-H2) has been shown to act as a positive regulator of c-myctranscription (31), whereas nm23-H1 is a potential negativeregulator of growth factor genes (8), and nm23-H1, -H2, and-H3 have all been implicated in arrest of differentiation indifferent cell types (17, 27, 35). Infective larvae of T. spiralis areisolated from nurse cells, a modified compartment of skeletalmuscle with hypertrophic nuclei and endoplasmic reticulum(9). Cell cycle reentry and arrest in apparent G2/M phase is afeature of these cells, as are extensive alterations in gene ex-pression which result in the loss of characteristics associatedwith differentiated muscle cells (16). A number of parasitesecreted proteins have been localized in host cell nuclei, al-though their identities and functions are unknown, and therelative contributions from host and parasite in cell cycle re-entry and altered gene expression are unclear (37). One couldpotentially envisage a role for the T. spiralis secreted NDPK inparticipating in the alterations in gene expression associatedwith intramuscular development of the parasite.

Alternatively, a secreted NDPK could have a role in thesubsequent intestinal phase of the life cycle, as it is possiblethat under the culture conditions used to maintain the para-sites in vitro, they acquire certain characteristics of more ad-vanced parasitic stages. Given the crucial involvement of mastcells in expulsion of T. spiralis from the gut (13, 14), it isinteresting that secreted products of P. aeruginosa and V. chol-erae show ATP-dependent cytotoxicity toward mast cells (32,39), although this has not been specifically linked to NDPK perse. We therefore intend to investigate the potential involve-ment of the T. spiralis secreted NDPK in directed cytotoxicityagainst a variety of cell types. We are also in the process ofcloning genes, localizing the enzymes, and determining theirdynamics of expression throughout the life cycle in order toelucidate the roles of this multifunctional protein.

ACKNOWLEDGMENTS

This work was supported by the BBSRC and the Wellcome Trust,the latter via a research leave award to K.G.

We are grateful to Paul A. Millner for providing the antibody toNDK-P1 and to Judith A. Appleton for monoclonal antibody 18H.

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