possible effects of microbial ecto-nucleoside triphosphate ...the interaction of the pathogen with...

MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Dec. 2008, p. 765–781 Vol. 72, No. 41092-2172/08/$08.00�0 doi:10.1128/MMBR.00013-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Possible Effects of Microbial Ecto-Nucleoside TriphosphateDiphosphohydrolases on Host-Pathogen Interactions

Fiona M. Sansom,1 Simon C. Robson,2 and Elizabeth L. Hartland1*Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia,1 and Department of

Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 022152

INTRODUCTION .......................................................................................................................................................765MAMMALIAN NTPDases: REGULATION OF THROMBOSIS AND INFLAMMATION .............................766

Effects on Vascular Homeostasis ..........................................................................................................................768Modulation of the Host Inflammatory Response ...............................................................................................768

NTPDases OF THE APICOMPLEXAN PARASITES AND TRYPANOSOMATIDS ........................................768Purine Salvage and the Apicomplexan Parasites ...............................................................................................768

Toxoplasma gondii ................................................................................................................................................768Neosporum caninum.............................................................................................................................................769Sarcocytis neurona................................................................................................................................................769Plasmodium falciparum .......................................................................................................................................770

Trypanosomatids.....................................................................................................................................................770Trypanosoma species ...........................................................................................................................................770Leishmania species ..............................................................................................................................................770

NTPDase ACTIVITY IN OTHER PARASITES AND FUNGI..............................................................................771Schistosoma mansoni ...............................................................................................................................................771Trichomonas vaginalis..............................................................................................................................................771Other Protozoa........................................................................................................................................................773Fungi.........................................................................................................................................................................773

LEGIONELLA PNEUMOPHILA: THE PROKARYOTIC PUZZLE ....................................................................774CD39 EXPRESSION AND CAPTURE BY HIV .....................................................................................................775PURINERGIC SIGNALING AND THE HOST-PATHOGEN INTERACTION.................................................775

Interference with P2 Receptor Signaling by Parasites ......................................................................................775Possible effects on vascular homeostasis.........................................................................................................775Modulation of the host inflammatory response .............................................................................................775

Interference with P2 Receptor Signaling by Bacteria........................................................................................775Adenosine Generation by Pathogens....................................................................................................................776Nonadenine Nucleotide Signaling.........................................................................................................................776

CONCLUSIONS .........................................................................................................................................................777NTPDases as Possible Targets for Antimicrobial Therapy ..............................................................................777

ACKNOWLEDGMENTS ...........................................................................................................................................777REFERENCES ............................................................................................................................................................777

INTRODUCTION

In 1972, the term “purinergic” was first used by Burnstock todescribe signaling where ATP was the extracellular messengermolecule, which at the time was a rather radical idea (21).Since then the importance of purinergic signaling involving notjust ATP but other nucleoside triphosphates (NTPs) and nu-cleoside diphosphates (NDPs) has become increasingly evident(20). It is now understood that purinergic signaling involvesspecific purinergic type 1 (P1) and type 2 (P2) receptors and isimportant in both neuronal and nonneuronal processes, in-cluding the modulation of inflammation and specific immuneresponses. P1 receptors, of which there are four subtypes, areactivated by adenosine, which is generated by ecto-nucleo-tidases. In contrast, P2 receptors exist as two subtypes: P2X

receptors, which are specific for ATP, and P2Y receptors,which are activated by ATP, ADP, UTP, UDP, ITP, and nu-cleotide sugars. P2X receptors are ligand-gated ion channelreceptors, and seven subtypes have been identified, whereasP2Y receptors are G protein-coupled receptors that compriseeight known subtypes (20, 121).

Ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases) (gene family ENTPD) of the CD39 family areimportant ecto-nucleotidases that are characterized by thepresence of five “apyrase conserved regions” (ACR1 to ACR5)and by the ability to hydrolyze NTPs and NDPs to the mono-phosphate form. Nucleoside monophosphates may then becatalyzed to nucleosides such as adenosine by the action ofecto-5�-nucleotidases (for example, mammalian CD73). Bothof these ecto-enzymes are found on multiple cell types in avariety of eukaryotic organisms. The major function of theseenzymes so far appears to involve purine salvage and the reg-ulation of blood clotting, inflammatory processes, and immunereactions (69, 121, 151, 152). We and another group have

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, University of Melbourne, Grattan St.,Parkville, Victoria 3010, Australia. Phone: 61-3-8344-8041. Fax: 61-3-9347-1540. E-mail: [email protected].

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recently shown that CD39 is expressed on regulatory T (Treg)cells and mediates immune suppression (16, 39).

In mammals, NTPDases comprise at least eight members,with CD39/NTPDase 1, CD39L1/NTPDase 2, CD39L3/NTPDase 3, and NTPDase 8 located on the cell surface. Oth-ers are located on organelles and intracellular membranes orare secreted (14, 88, 121; H. Zimmermann, S. C. Robson, et al.,presented at the Second International Workshop on Ecto-ATPases and Related Ecto-Nucleotidases, Maastricht, TheNetherlands, 2000) (Fig. 1 and 2). The majority of these en-zymes are membrane bound with the active site facing eitherthe extracellular medium or the lumen of the organelle wherethey are sited (essentially communicating with the ecto-mem-branes), and are referred to as ecto-enzymes or ecto-NTP-Dases. An amino acid sequence alignment and phylogeneticanalysis of NTPDases using the sequence analysis tool ClustalW (147) shows that the surface-located mammalian NTPDasesare more closely related to each other than to mammalianNTPDases found in organelles (Fig. 1 and 3). NTPDases 5 and6, which are intracellular but undergo secretion after heterol-ogous expression (121), are also more closely related thanNTPDases 4 and 7, which are entirely intracellular (Fig. 3).

Surface-located or secreted NTPDases in several importantmicrobial pathogens of humans have also been described. Thenomenclature surrounding these enzymes is complex, andsince apyrases are classified by their ability to hydrolyze bothATP and ADP, here we have defined an NTPDase as a type ofapyrase that contains the five ACRs and which is related to theCD39/NTPDase 1 family by amino acid sequence similarity.We recently characterized the first bacterial member of theNTPDase family, present in Legionella pneumophila, whichenhances intracellular replication of the bacteria (123). Here

we review our current knowledge of microbial NTPDases withan emphasis on those that have been directly implicated invirulence. We examine the potential role of these enzymes inthe interaction of the pathogen with the immune system of thehost and propose that microbial NTPDases, in addition tohaving potential roles in purine salvage, may subvert inflam-matory and immune processes to modulate the host responseto infection.

MAMMALIAN NTPDases: REGULATION OFTHROMBOSIS AND INFLAMMATION

The prototypic member of the NTPDase family, CD39, hy-drolyzes ATP and ADP in an equivalent manner, with activitydependent on the presence of divalent cations, as is the casefor all NTPDases. CD39 utilizes either Ca2� or Mg2� as acofactor (83, 153). Recently the crystal structure of the extra-cellular domain of rat NTPDase 2 was solved (166). The struc-ture identified several key amino acid residues in ACR1 to -5that were variously involved in substrate and cofactor binding,although only ACR1 and ACR4 share homology with knownATP binding domains, namely, the actin-HSP 70-hexokinase�- and �-phosphate binding motif (121, 166). In particular,E165 in ACR3 was important for the positioning of a watermolecule for nucleophilic attack on the terminal phosphate ofATP or ADP. Several mutagenesis studies have confirmed therole of additional residues in ACR1 to -5 in the activity ofmammalian NTPDases (49, 50, 68, 87, 126, 134, 135, 160).However, the level of catalytic activity and the specificity ofNTPDases relate also to the membrane positioning of theprotein, as demonstrated by the regulation of activity by the N-and C-terminal transmembrane domains of CD39 (67, 155).

FIG. 1. Alignment of the amino acid sequences from ACR1 to -5 of known and hypothetical NTPDases present in eukaryotes and bacteria.Amino acid sequences were aligned using the ClustalW2 sequence alignment tool (http://www.ebi.ac.uk/Tools/clustalw2/).

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Furthermore, NTPDases form homo-oligomers, and the stateof oligomerization affects catalytic activity (67, 142, 155). CD39contains a total of 11 cysteine residues (126), and the regionbetween ACR4 and ACR5 in both CD39 and NTPDase 2contains eight conserved cysteine residues. The cysteine resi-dues play an important role in disulfide bond formation andoligomerization and have been shown to modulate substratespecificity (70, 166). Additionally, the N terminus containing

the Cys13 residue of CD39 undergoes palmitoylation, whichappears to contribute to the association of CD39 with lipidrafts (121). Cholesterol depletion therefore results in inhibi-tion of CD39 activity (113). We recently showed that the Nterminus of CD39 associates with the membrane scaffold pro-tein RanBPM and that this association results in downregula-tion of catalytic activity (158).

CD39 is responsible for inhibiting platelet aggregation. The

FIG. 2. Schematic representation of known and predicted localization of the NTPDases found in humans and pathogens, based on experi-mental evidence and sequence motifs, as listed in Table 1. Cylinders represent transmembrane domains, and black rectangles represent ACR 1to -5. (Adapted from reference 168 with permission of the publisher. Copyright 2001 Wiley-Liss.)

FIG. 3. Phylogenetic tree constructed from the amino acid sequence alignment in Fig. 1, with the resulting tree viewed and edited using thePhylodraw software. The accession numbers for the amino acid sequences used in the alignment are listed in Table 1.

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rapid phosphohydrolysis of ADP released from activated plate-lets is thought to prevent the paracrine amplification of ADP-induced platelet recruitment and aggregation (65, 99). Fur-thermore, CD39 plays a role in modulating inflammation andthe immune response by affecting the extracellular concentra-tions of ATP; this nucleotide serves as an immunostimulant ofthe immune system operative via P2 receptor pathways (105,119). During acute inflammatory reactions, the bioactivity ofCD39 is transiently but acutely compromised by oxidativestress (121), presumably resulting in a rise in extracellular ATPand increased activation of P2 receptors to stimulate host de-fenses.

Effects on Vascular Homeostasis

Platelets possess three types of P2 receptors: the P2X1 re-ceptor, activated by ATP, and two P2Y receptors, P2Y1 andP2Y12, which are both activated by ADP. Activation of P2Y1 isresponsible for initiation of aggregation, whereas activation ofP2Y12 is essential for completion of the ADP-induced aggre-gation response and for potentiation of platelet aggregationinduced by other compounds such as thromboxaneA2. Activa-tion of the P2X1 receptor results in rapid calcium influx andcontributes to platelet activation induced by low concentra-tions of collagen. Signaling through the P2X1 receptor mayalso play a priming role in the subsequent activation of theP2Y1 receptor by ADP (61).

In humans, NTPDase 2, which is expressed on pericytes andadventitial cells, stimulates platelet aggregation by preferen-tially hydrolyzing ATP to produce ADP, resulting in activationof P2Y1 and P2Y12 receptors (8). However CD39, which isdominantly expressed on the endothelium, hydrolyzes ADP toAMP, thus limiting platelet activation (8). CD39 is a trueplatelet modulator, as deletion of this gene in mice results inpericellular nucleotide flux that causes desensitization of P2Y1platelet receptors and a subsequent bleeding phenotype (51).Given the intrinsic role of CD39 and NTPDase 2 in the regu-lation of vascular homeostasis, it is possible that NTPDasesproduced by parasites, particularly by those parasites with ex-tended bloodstream phases in their life cycles, also modulateplatelet defense mechanisms to promote optimal parasite in-fection and growth.

Modulation of the Host Inflammatory Response

ATP and other nucleotides such as UDP and UTP may bereleased from activated, stressed, or injured mammalian cells,resulting in a rise in the concentration of extracellular nucle-otides, which function as a “danger signal” for the immunesystem. Extracellular ATP and other nucleotides then signalthrough P2 receptors to modulate the immune and inflamma-tory response in a variety of cell types, including immune andnonimmune cells (17, 20). In particular, extracellular ATPtriggers the release of proinflammatory cytokines, includinginterleukin-1� (IL-1�) (55) and IL-2 and gamma interferon(93). NTPDase expression modulates this response by control-ling the level of extracellular nucleotides. In addition, CD39 ishighly expressed on activated Treg cells, which mediate im-mune suppression (16, 39). The hydrolysis of ATP by CD39expressed on the surface of activated Treg cells functions first to

control the cytolytic effects of high concentrations of ATP butalso to control purinergic signaling and ensuing effects on theinflammatory process (16).

Interestingly, CD39 activity has been further linked tothe inhibition of IL-1 release from endothelial cells (77), andthe expression of CD39 on Treg cells has also been linked todecreased activation of dendritic cells (16). Therefore, theeffect of NTPDases on the immune response is one of immunesuppression, a process that may be mimicked by microbialNTPDases.

NTPDases OF THE APICOMPLEXAN PARASITESAND TRYPANOSOMATIDS

The mechanisms by which pathogen-associated NTPDasesmay affect virulence have not been precisely elucidated. Sev-eral lines of experimental evidence suggest that hydrolysis ofATP and ADP by these enzymes has the potential to subvertand avoid host defense mechanisms. In addition, further hy-drolysis of AMP to adenosine by either host or parasitic en-zymes could have a number of effects, as adenosine plays animportant role in limiting the inflammatory response and somepathogens may scavenge adenosine for growth (60).

Purine Salvage and the Apicomplexan Parasites

The apicomplexan parasites, which comprise protozoa char-acterized by the presence of the unique organelle known asthe apical complex, include a number of important humanand animal pathogens, some of which are known to expressNTPDases. These include Toxoplasma gondii, the causativeagent of the zoonotic disease toxoplasmosis, which can haveserious sequelae in pregnant women and immunocompro-mised people, and Plasmodium falciparum, the major cause ofmalaria, currently one of the most devastating infectious dis-eases worldwide. Apicomplexan parasites such as T. gondii andP. falciparum lack the ability to synthesize the purine ring denovo and instead rely on capturing purines from the host cellas an essential nutrient. The host purines are then transportedinto the parasite by nucleoside transporters and converted topurine nucleotides (44). Thus, the purine salvage pathway isvital to the metabolism of the parasites, and as such the en-zymes and transporters involved in these pathways are attrac-tive drug targets, provided that inhibitors of these componentsdo not affect similar host enzymes. It has been suggested thatthe NTPDases known to be expressed by some parasites areinvolved in purine salvage, and therefore inhibitors specific forparasitic NTPDases have potential therapeutic uses (66). Herewe discuss the NTPDases associated with the apicomplexanparasites and the evidence for a potential role in purine sal-vage, in addition to the other potential roles in pathogenesissuch as the modulation of purinergic signaling.

Toxoplasma gondii. T. gondii is capable of infecting bothphagocytic and nonphagocytic cells (130), where it replicateswithin a parasitophorous vacuole that remains mostly seques-tered from the endosomal network of the host cell (2, 33). Over20 years ago, a highly active enzyme purified from T. gondii wasshown to hydrolyze both ATP and ADP, although ADPaseactivity was only 18% of ATPase activity. It was designated an



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NTP hydrolase (NTPase) and found to require activation bythiol compounds, presumably to induce essential disulfidebonds, similar to CD39 (5, 7).

Further work demonstrated that the purified NTPase wasactually a mix of two isoenzymes, NTPase I and NTPase II,with identical molecular weights that differed in bioactivity butarose through gene duplication (6). In fact, three open readingframes encoding proteins with predicted NTPDase similarityare present in the genome of the virulent RH strain of T.gondii, and two are translated into proteins designated NTPase1 and NTPase 3 (11), which correspond to the two isoenzymesNTPase II and NTPase I identified previously (6). The aminoacid sequences of these two proteins show all five ACRs, andso the enzymes will be referred to as NTPDases from here on(6). Although both hydrolyze ATP, GTP, CTP, and UTP,NTPDase 3 has less than 1% of the relative activity of hydro-lysis of ADP, GDP, CDP, and UDP but is 4.5 times moreefficient at hydrolyzing ATP than NTPDase 1 (6). Interestingly,a 12-residue block of amino acids in the C termini of theNTPDase isoforms appears to dictate the substrate specificity.Through the synthesis of protein chimeras, amino acids FITGREMLASID and IVTGGGMLAAIN near the C termini ofNTPDase 1 and NTPDase 3, respectively (residues 488 to 499),have been shown to affect specificity for NTPs and NDPs (107).This difference is also antigenically distinct, as sera from asmall fraction of T. gondii patients can discriminate betweenNTPDase 1 and NTPDase 3 on the basis of these 12 residues(81).

In T. gondii, the NTPDases are present in dense granules inextracellular tachyzoites. Both proteins possess typical signalpeptides for secretion, and following infection of the host cell,both NTPDases are secreted into the lumen of the parasito-phorous vacuole (Fig. 1) (11, 130). As T. gondii is a purineauxotroph, the NTPDase activity may be part of a pathway thatprocesses host cell nucleotides prior to uptake by the parasite(6, 130). Depletion of host cell ATP decreases the metabolismof the tachyzoites, leading to the suggestion that ATP is usedby the parasite, either as energy for parasite processes or forpurine salvage after processing by an NTPDase (137). How-ever, T. gondii lacks a 5�-nucleotidase, the enzyme required toconvert AMP to adenosine, suggesting that the NTPDases ofT. gondii play an additional, more complex role than simplypurine salvage, although it is also possible that T. gondii mayexploit some host enzymes in the process of purine salvage(110).

NTPDase 3 is expressed in the actively replicating tachyzoiteform of T. gondii but is downregulated in the dormant brady-zoite form of the parasite, which is involved in chronic hostinfection (108). The similarity of the genes encoding theNTPDases makes it difficult to create a specific mutant, andother evidence also suggests that the gene encoding NTPDase3 is essential for viability (109). Antisense RNA studies showedthat NTPDase 3 activity was necessary for intracellular repli-cation but not for parasite invasion of the host cell (109). Incontrast to that study, the pretreatment of parasites with amonoclonal antibody that recognized and inhibited bothNTPDases resulted in decreased invasion of Vero cells, sug-gesting that NTPDase activity did contribute to invasion ofhost cells (84). NTPDase 3 is also indirectly implicated in

virulence, as most virulent strains of T. gondii possess the genebut avirulent strains carry only the gene encoding NTPDase 1(6, 107).

Apart from purine salvage, NTPDase activity may be directlyrelated to virulence by influencing intracellular replication andexit from host cells (131). When NTPDase 3 is secreted by theparasite, it remains largely oxidized despite the reducing envi-ronment of the host cell. However, since parasitic infectionsgenerate host nitric oxide and other free radicals, which resultsin oxidative and nitrative stress (165), it may be that T. gondiiitself is responsible for the reduction and therefore activationof NTPDase 3. Upon activation of the enzyme by exogenousthiols, the level of ATP in the host cell is rapidly depleted, andthe parasites exit the host cell within a minute of thiol treat-ment (131). Exit of parasites in response to thiol compounds isCa2� dependent, suggesting that ATP depletion releases Ca2�

stores that are controlled by ATP (141). These data all suggestthat the activation of a secreted NTPDase must be tightlyregulated by the parasite (131). Recently, it has been shownthat the reducing agent glutaredoxin (GRX) is secreted by theparasite as replication increases. GRX can activate NTPDase 3in vitro, suggesting that GRX may be also secreted by theparasite to control the reduction and therefore activation ofNTPDase 3, thereby stimulating exit from host cells (140).Therefore, either NTPDase 1 or NTPDase 3 activity hasbeen implicated at each stage of parasite invasion, replica-tion, and exit.

Neosporum caninum. The genome of the closely related api-complexan parasite N. caninum contains a single gene encod-ing a protein with 69% identity to T. gondii NTPDase that ismost similar to NTPDase 3. Correspondingly, the N. caninumNTPDase can hydrolyze NTPs but not NDPs. The enzymepossesses a typical N-terminal signal peptide for secretion andis also localized to the dense granules of N. caninum, suggest-ing that it may be exported in a manner similar to that for thedense granule proteins of T. gondii, which are secreted viaspecific exocytosis (4, 32). While the role of this enzyme inpathogenesis is unclear, the inability of the enzyme to hydro-lyze NDPs suggests that it is unlikely to play an independentrole in purine salvage (110).

Sarcocytis neurona. Another apicomplexan parasite relatedto T. gondii is S. neurona, which is a cause of equine myeloen-cephalitis. However, unlike T. gondii, it resides free in thecytoplasm of host cells during intracellular replication. Theparasite also has demonstrable NTPDase activity, exhibitinghydrolysis of both ATP and ADP that is activated by thiolcompounds. A single gene encoding a putative NTPDase ispresent in the genome of S. neurona, and specific polyclonalantibodies demonstrate that the protein is secreted into culturesupernatant by parasites in vitro, which is consistent with thepresence of a signal peptide. Other localization studies showthat the NTPDase of S. neurona is absent during much ofintracellular replication, but when present, it is apically local-ized and is detectable on newly invaded merozoites. It thendisappears before reappearing on newly formed merozoitesonce they are completely mature but before exit from the hostcell. This pattern of expression suggests the NTPDase is in-volved either in exit from host cells or in infection of new hostcells, or possibly in both (167). Alternatively, it may be that theNTPDase is required when the parasite is extracellular, to



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modulate host levels of extracellular ATP and perhaps protectagainst platelet activation and the immune response of thehost.

Plasmodium falciparum. NTPDase activity has not been de-scribed for the apicomplexan parasite P. falciparum, but asearch of the genome of the virulent strain 3D7 shows thepresence of an open reading frame encoding a predicted pro-tein containing all five ACRs (64; B. Cooke, personal commu-nication.). Similar to the case for CD39, the putative proteincontains two predicted transmembrane domains, one locatedat the N terminus and the other at the C terminus, suggestingthat the protein could be anchored in the membrane of theparasite with the active site facing the external medium. It isintriguing to note that whereas the NTPDases present in theother apicomplexan parasites appear to be closely related, theputative P. falciparum NTPDase appears to be evolutionarilydistinct, suggesting that it may fulfill a different role in P.falciparum (Fig. 3).

Trypanosomatids

Trypanosoma species. Trypanosoma protozoa cause a rangeof diseases in both humans and animals, and NTPDase activityhas been demonstrated in a number of species. The presenceof Mg2�-dependent ecto-ATPase activity was first demon-strated for T. cruzi, the causative agent of American trypano-somiasis, also known as Chagas’ disease, a serious infectionaffecting the heart and gastrointestinal system which can befatal (15, 57, 86). After entry into host cells, the vacuole con-taining T. cruzi undergoes lysosomal fusion, and the parasitesubsequently escapes the vacuole and replicates inside the cy-tosol of the host cell (3).

The dependence of the ecto-ATPase activity on Mg2� sug-gested that the activity might be due to a member of theCD39/NTPDase family. Further evidence for the presence ofan NTPDase in T. cruzi came from a second study that dem-onstrated a range of NTPDase activities in intact parasites andthat anti-T. gondii NTPDase antibodies cross-reacted with aprotein on the surface of the parasite (57). An open readingframe encoding a predicted secreted protein with similarity toNTPDases is present in the genome of T. cruzi, although it hasnot been determined if this gene does indeed encode the sur-face-located NTPDase identified by immunofluorescence.NTPDase activity is present in all forms of the parasite, al-though the activity of intact parasites at different developmen-tal stages varies for infective trypomastigotes but not for non-infective epimastigotes (57). The infective trypomastigote alsodisplays up to 20 times higher ecto-ATPase activity than theepimastigote stage (15). Both parasite stages are able to hy-drolyze ATP and ADP, but the ratio of ATP to ADP hydrolysisfor trypomastigotes is 2:1 while for epimastigotes it is 1:1.Epimastigotes have also been shown to hydrolyze GTP, GDP,UTP, and UDP, and the highest activity was observed againstGTP (57).

Ecto-ATPase activity is inhibited by suramin and 4,4�-diiso-thiocyanostylbene 2�,2�-disulfonic acid, both of which areknown inhibitors of NTPDase activity (15), Furthermore thepresence of either inhibitor reduced the number of parasitesattaching to and infecting mouse peritoneal macrophages. Incontrast, the addition of 200 �M ATP resulted in approxi-

mately 30% more parasite-infected macrophages. The additionof suramin to epimastigotes also stimulated a fourfold increasein the ecto-ATPase activity of this parasite form, presumably inan effort to overcome the inhibition. These parasites were thenmuch more capable of adhering to mouse macrophages thanuntreated epimastigotes (15). An additional study also dem-onstrated Mg2�-dependent ecto-ATPase activity in T. cruziand showed that ATPase activity is higher in the infectivetrypomastigote and amastigote stages than in the noninfectiveepimastigotes, again linking NTPDase activity to virulence(104).

Like the apicomplexan parasites, trypanosomes are unableto synthesize the purine ring (28), and the ability to hydrolyzeNTPs and NDPs may be part of an essential nutrient salvagepathway (57). Additionally, it has been suggested that in-creased hydrolysis of ATP in the infective stage of T. cruzi mayreflect modulation of the immune system by the parasite (57).ADPase activity may also be important for avoidance of hostdefenses, particularly since platelet recruitment results in theremoval of opsonized T. cruzi from the circulation (149).

Trypanosoma brucei causes both African sleeping sickness inhumans and nagana in livestock. Unlike T. cruzi, T. brucei is anextracellular pathogen that proliferates in the mammalianbloodstream (100). The genome of T. brucei encodes a putativesecreted NTPDase, and assays using intact parasites demon-strate that T. brucei exhibits Mg2�-dependent surface-locatedNTPDase activity that hydrolyzes ATP, GTP, CTP, UTP, andADP. Interestingly, catalytic activity may be stimulated byother divalent cations, including zinc (47).

Another species, Trypanosoma rangeli, is capable of infectinghumans and animals. Little is known about the biology of thisparasite in vertebrate hosts, other than that it may cause dis-ease following infection (136). T. rangeli also exhibits Mg2�-dependent ecto-NTPDase activity that is highest against ATP,although ADP and other NTPs may also be hydrolyzed.NTPDase activity is stimulated by a number of carbohydrates,which has led to the suggestion that NTPDase activity mayhave a role in adhesion to the intermediate insect host, ascarbohydrates on insect salivary glands play a part in adhesionby Trypanosoma species (59).

Leishmania species. Leishmania parasites are responsible fora number of clinical syndromes known as visceral, cutaneous,and mucosal leishmaniasis, which are a consequence of para-site replication inside macrophages of the mononuclear phago-cyte system, dermis, and naso-oropharyngeal mucosa, respec-tively. All of these syndromes have serious sequelae, butvisceral leishmaniasis is particularly life-threatening (71).Leishmania replicates inside macrophages within a parasito-phorous vacuole, the morphology of which varies between par-asite species (23).

Mg2�-dependent surface NTPDase activity has been de-tected in both Leishmania tropica and Leishmania amazonensis(13, 103), two of the species responsible for cutaneous leish-maniasis (71). However, as the genes encoding the proteinsresponsible for this activity have not been identified, it is notknown if the proteins are actually members of the CD39/NTPDase 1 family. A BLAST search of the Leishmania ge-nomes revealed that putative NTPDases containing the fiveACRs are present in Leishmania major, Leishmania infantum,and Leishmania braziliensis, suggesting that it is likely that



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other species of Leishmania also possess putative NTPDases.All the hypothetical proteins encoded by these Leishmaniaspecies possess either a predicted N-terminal transmembranedomains or an N-terminal signal peptide (Fig. 2; Table 1),suggesting that they may be anchored on the membrane sur-face of the parasite or secreted. Unlike many NTPDases, theenzymes identified in L. tropica and L. amazonensis cannotutilize Ca2� instead of Mg2� (13, 103). The enzymes also showdifferent substrate specificities. The L. tropica enzyme can hy-drolyze both ATP and ADP, although ADPase activity is only21% of ATPase activity, and the enzyme also hydrolyzes otherNTPs (103). Like that of a number of other parasiteNTPDases, L. tropica NTPDase activity is stimulated by de-fined carbohydrates, namely, dextran sulfate, although it isunclear how the carbohydrates stimulate catalytic activity(116). In contrast to that of L. tropica, the L. amazonensisenzyme appears to utilize only NTPs as substrates (13). Thelocation of the enzyme on the surface of L. amazonensis hasbeen confirmed by immunogold electron microscopy using an-ti-CD39 antibodies, and this cross-reaction with anti-CD39suggests that the protein is indeed a member of the CD39/NTPDase 1 family (118). Enzyme activity is highest toward theend of logarithmic replication and is higher in virulent strainsthan in avirulent strains. In addition, activity is increased morethan 10-fold in the obligate intracellular amastigote stage com-pared to the promastigote stage (13, 118). Further evidence fora role in virulence comes from the observation that treatmentwith the anti-CD39 antibodies reduces the interaction of theparasites with mouse peritoneal macrophages (118). Finally, itwas recently shown that ecto-ATPase activity of L. amazonen-sis is increased when the parasites undergo heat shock (115),which is of particular interest because it has been suggestedthat the heat shock response may play a role in invasion byparasites (120).

The high activity of the enzyme toward the end of parasitereplication may indicate a role in exit from the infected cell,while the effect of the anti-NTPDase antibodies also suggests arole in host cell entry (118). In this context, the similarity ofACR1 and ACR4 to the actin-HSP 70-hexokinase �- and�-phosphate binding motif is particularly curious (134). Phago-cytosis of pathogens involves rearrangement of the actin cy-toskeleton of the cell (101), and it may be that NTPDases areone of the pathogenic factors involved in disrupting normalphagocytic events and enabling intracellular pathogen suchas Leishmania to establish their replicative niche. Addition-ally, it may reflect a need for the parasite to express theprotein when extracellular in order to regulate host levels ofextracellular ATP.

NTPDase ACTIVITY IN OTHER PARASITES AND FUNGI

Schistosoma mansoni

NTPDases have also been identified in the worm Schisto-soma mansoni, the cause of intestinal schistosomiasis. S. man-soni is capable of surviving for several years in the humanmesenteric vasculature by evading the host defenses, resultingin a chronic debilitating disease (151). S. mansoni possessestwo isoenzymes containing the five ACRs that have NTPDaseactivity and which are encoded by two genes, SmATPDase 1

and SmATPDase 2 (45, 151). While SmATPDase 1 is locatedon the surface, SmATPDase 2 is secreted by the parasite (Fig.2), suggesting distinct roles for the two enzymes (45, 96). Aswith many other NTPDases, activity is stimulated by Ca2� andMg2� ions (148), and both ATP and ADP as well as otherNTPs and NDPs are hydrolyzed (53). Since S. mansoni lives inthe portal venous bloodstream, it has been postulated thatADPase activity prevents recruitment and aggregation ofplatelets, enabling the worm to avoid host hemostatic defenses(152). Recently, a new class of antischistosomal drugs, N-alkyl-aminoalkanethiosulfuric acids, were shown to partially inhibittegumental S. mansoni NTPDase activity, suggesting that in-hibiting the NTPDase activity of the parasite may compromiseits survival (97).

Trichomonas vaginalis

Trichomonas vaginalis is a flagellate protozoan that lives inthe human urogenital tract. T. vaginalis is an extracellularpathogen that is cytotoxic for mammalian cells and the cause oftrichomoniasis, a disease usually characterized by vaginitis(128). T. vaginalis-induced vaginitis is likely the most prevalentnonviral sexually transmitted disease worldwide (150). Com-parison of the NTPDase activities of intact and disrupted T.vaginalis cells has demonstrated the presence of Ca2�- orMg2�-dependent surface NTPDase activity that catalyzes thehydrolysis of ATP, ADP, and other nucleotides (40, 42). Theenzyme(s) responsible for this observed activity has not beenpositively identified, but a search of the T. vaginalis G3 genomesequence revealed the presence of four genes encoding hypo-thetical proteins (locus tags TVAG_063220, TVAG_167570,TVAG_351590, and TVAG_397320) that all contain regionssimilar to the five ACRs found in NTPDases. This suggeststhat one or more of this class of enzyme is present in T.vaginalis and responsible for the NTPDase activity. Further-more, the proteins encoded by these genes all possess pre-dicted C-terminal transmembrane domains, implying that theyare anchored in the membrane of the cell and function asecto-enzymes (Fig. 2). D-Galactose, which is involved in adhe-sion of T. vaginalis to host cells, increases NTPDase activity by90%, suggesting that the enzyme may play a role in adhesion,although to date no further evidence for this is available (42).Nevertheless, fresh isolates of T. vaginalis have increased sur-face NTPDase activity compared to a laboratory-adaptedstrain, suggesting a possible role in virulence (42, 146).

The concentration of free purine nucleotides in the vaginaduring disease can reach 10 mM as they are released fromepithelial cells lysed during infection, and 90% of the nucleo-tides released are ATP (106). ATP itself is cytolytic towardmammalian cells not expressing NTPDase activity on theirsurface (58), but ATP does not lyse T. vaginalis, which may bea result of hydrolysis of ATP by the parasite. T. vaginalis is alsoa purine auxotroph, and so NTPDase activity may be part of apurine salvage pathway for the parasite (145). Aside frompossessing higher ATPase and ADPase activities than lab-oratory-adapted strains, fresh isolates also exhibit variationin the ratio of ATP to ADP hydrolysis, suggesting they mayhave two or more NTPDases (146), as predicted by the T.vaginalis genome sequence.

The related parasite Tritrichomonas foetus, which inhabits



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the urogenital tract of cattle and may lead to abortion, alsopossesses Mg2�-dependent NTPDase activity. The enzyme isunable to hydrolyze ADP but hydrolyzes ATP and other NTPs,and activity is stimulated by D-mannose and D-galactose, sug-gesting it may have a similar role in adhesion as postulated forT. vaginalis (80). For the NTPDases of both T. foetus and T.vaginalis, iron-depleted medium reduces the activity of theNTPDase, suggesting that iron may exert a positive regulatoryrole, although the significance of this is not yet known (43).

Other Protozoa

Entamoeba histolytica, an enteric protozoan that causes in-vasive colitis and liver abscesses, also exhibits Mg2�-dependentNTPDase activity that catalyzes the hydrolysis of ATP and to alesser extent ADP, with still lower activity rates for other NTPs(10). Pathogenic E. histolytica has a higher rate of activity thannonpathogenic E. histolytica or free-living Entamoeba moshk-ovskii, suggesting a role in virulence. Additionally, D-galactosestimulates NTPDase activity, as for a number of other parasiticNTPDases (10). A second amoebic pathogen, Acanthamoeba,can cause fatal encephalitis and keratitis in humans, and theamoebae show ecto-ATPase activity, which is dependent ondivalent cations and inhibited by the NTPDase inhibitorsuramin (133). Increased enzyme activity is associated withvirulent isolates rather than environmental isolates, again sug-gesting a role in host-pathogen interactions. Acanthamoebabinds to endothelial cells using a mannose binding protein, andit has been shown that alpha-mannose stimulates enzyme ac-tivity. Furthermore, suramin inhibition of enzyme activity re-sults in decreased cytotoxicity and binding to host cells, allsupporting a role in virulence (133).

While the enzyme activities detected for Acanthamoebaand Entamoeba species appears to indicate the presence ofNTPDases, a BLAST (tblastn) search of the genomes of bothAcanthamoeba castellanii and E. histolytica using the CD39amino acid sequence did not reveal any significant homologuescontaining five ACRs. Although these studies support the hy-pothesis that ecto-ATPase activity is related to the virulence ofmany parasites, it seems probable that the observed catalyticactivities are due to enzymes unrelated to the CD39/NTPDase1 family.

Most recently, Mg2�-stimulated ecto-ATPase activity wasdemonstrated in Giardia lamblia, a flagellated protozoan thatcan cause small intestinal diarrhea (46). Hydrolysis of ADPand AMP was also observed, but Mg2� did not stimulate thisactivity. BLAST searching of the G. lamblia genome using theCD39 amino acid sequence also failed to reveal any NTPDasehomologues, suggesting that this ecto-enzyme activity is notdue to a member of the NTPDase family. Furthermore, therole of this enzymatic activity in the virulence of the parasite iscurrently unknown.

Fungi

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closely related to the mammalian NTPDases 4, 5, 6, and 7,which are localized in the cytoplasm of the cell (Fig. 3). SurfaceNTPDase activity was recently described for the pathogenicyeast Cryptococcus neoformans, an important cause of pneu-monia and meningoencephalitis, particularly in immunocom-promised individuals. The NTPDase activity was stimulated byMg2� and showed high rates of ATP, ITP, GTP, CTP, andUTP, but not ADP, hydrolysis (82). The genome of C. neofor-mans encodes a hypothetical protein containing all five ACRs,but this is predicted by pTARGET to be Golgi localized, so itis unclear if the observed enzyme activity is due to a memberof the CD39/NTPDase 1 family or an unrelated enzyme. It isworth noting however, that the predicted localization of theseenzymes often does not correspond with the known localiza-tion (Table 1). Ecto-ATPase activity was also recently demon-strated for the pathogen Fonsecaea pedrosoi, the cause of chro-moblastomycosis, a chronic, subcutaneous fungal infection(29). Enzyme activity was stimulated by Mg2� and was postu-lated to play a role in fungal physiology and/or pathogenesis(29). The protein(s) responsible for this activity is also yet tobe identified.

LEGIONELLA PNEUMOPHILA: THEPROKARYOTIC PUZZLE

Expression of the CD39/NTPDase 1 family of enzymes isextremely rare in prokaryotes. These ecto-enzymes appear tohave been acquired only upon association with eukaryotes.While CD39/NTPDase 1 family members are present in allhigher eukaryotes, the presence of the enzymes in lower eu-karyotes is variable and not necessarily consistent with theputative phylogenetic relationships and evolutionary develop-ment of eukaryotic organisms. Therefore, it remains unclearwhen and why the CD39/NTPDase 1 family of enzymesevolved.

Recently, we characterized the first prokaryotic NTPDasein Legionella pneumophila, the major causative agent ofLegionnaires’ disease. Legionnaires’ disease is a systemicdisease characterized by a severe pneumonia and possiblerenal impairment which may be fatal even with appropriateantibiotic therapy (38, 123). Key to the pathogenesis ofLegionnaires’ disease is the ability of L. pneumophila toreplicate inside cells, in particular alveolar macrophages.The innate capacity of L. pneumophila and other species ofLegionella to replicate inside eukaryotic cells is widely at-tributed to their long-standing association with environmen-tal protozoa (143). L. pneumophila is a parasite of manyprotozoan species, particularly free-living amoebae such asAcanthamoeba, Hartmanella, and Naegleria species, whichalso serve as useful infection models to study L. pneumo-phila intracellular replication (56). In both mammalian cellsand amoebae, the L. pneumophila vacuole avoids the endocyticpathway and interacts instead with the exocytic pathway of the cellso that the replicative vacuole ultimately develops characteristicsof the endoplasmic reticulum (75, 76).

Possible DNA transfer events between the bacteria andtheir environmental eukaryotic hosts may have facilitatedthe acquisition of genes encoding proteins that mimic eu-karyotic signaling and protein interaction motifs, therebyallowing Legionella to interfere with eukaryotic signaling

and trafficking pathways. Indeed the L. pneumophila ge-nome encodes a surprising abundance of proteins that har-bor eukaryotic motifs (18, 25, 41).

Two genes, annotated as lpg1905 and lpg0971 in the L.pneumophila Philadelphia genome, each encode predictedproteins that contain all five ACRs typical of eukaryoticNTPDases (26). The presence of NTPDase genes in L. pneu-mophila is surprising since the lower environmental eukaryotesthat Legionella typically associates with, such as amoebae, donot possess CD39/NTPDase 1 enzymes. Therefore, although itis widely assumed that L. pneumophila coevolved with free-living protozoa and acquired many eukaryotic-like determi-nants from this relationship (41), in this case it is not clearwhich eukaryote was the source of the L. pneumophilaNTPDases.

Lpg1905 is a functional NTPDase, able to hydrolyze bothATP/ADP and GTP/GDP with similar efficiencies (123, 124).Lpg1905 is secreted by the bacterium in vitro, although its siteof action during infection is unclear. The enzyme has maximalactivity at neutral pH (124), and given that the purpose ofLegionella virulence determinants is to enhance bacterialreplication in amoebae, the enzyme presumably has an in-tracellular function. Indeed, inactivation of lpg1905 resultsin defective replication of L. pneumophila within amoebae,epithelial cells, and macrophages (123). Substantial levels ofenzyme activity are also required for full virulence of L.pneumophila in an A/J mouse model of Legionnaires’ dis-ease, although it is not clear if it is ATP/ADPase or GTP/GDPase activity or both that contribute to L. pneumophilainfection (124). The second putative secreted NTPDase,Lpg0971, is dispensable for replication within amoebae andmacrophages (123) but contributes to virulence in the A/Jmouse lung infection model, similar to Lpg1905 (F. M. San-som et al., unpublished data). Although it is not yet proventhat Lpg0971 possesses NTPDase activity, the additionalrequirement of lpg0971 for lung infection suggests that theenzymes may mediate effects on the host response over andabove any role in intracellular replication of L. pneumo-phila. The contribution of NTPDases to L. pneumophilavirulence and replication is unlikely to relate to purine scav-enging, as L. pneumophila is not purine auxotroph (117).Rather, it may relate to effects on host nucleotide levels andsubsequent effects on P2 receptor signaling.

A putative NTPDase is also annotated in the genome of theplant pathogen Pseudomonas syringae pv. tomato strainDC3000 (locus tag PSPTO_3560) (19). The predicted proteinpossesses a signal peptide for secretion but to date remainsuncharacterized. Finally, a BLAST search of all known bacte-rial sequences revealed only one other putative NTPDase, anuncharacterized hypothetical protein containing all five ACRs(locus tag Patl_3457) encoded in the genome of the marinebacterium Pseudoalteromonas atlantica, which is a pathogen ofcrabs (34; A. Copeland, S. Lucas, A. Lapidus, K. Barry, J. C.Detter, T. Glavina del Rio, N. Hammon, S. Israni, E. Dalin, H.Tice, S. Pitluck, E. Saunders, T. Brettin, D. Bruce, C. Han, R.Tapia, P. Gilna, J. Schmutz, F. Larimer, M. Land, L. Hauser,N. Kyrpides, E. Kim, A. C. Karls, D. Bartlett, B. P. Higgins,and P. Richardson, unpublished data).



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CD39 EXPRESSION AND CAPTURE BY HIV

Increased expression of CD39, with a concomitant increasein observed NTPDase activity, occurs in lymphocytes isolatedfrom patients infected with human immunodeficiency virus(HIV) (94). In addition, it now appears that CD39 is incorpo-rated into HIV virions, where it remains enzymatically active(9). The CD39-bearing virions can inhibit platelet aggregation,although the importance of this in pathogenesis is unclear, assevere bleeding is not common in HIV/AIDS patients (9).Enhanced CD39 expression may alternatively have effects onextracellular ATP concentrations and purinergic signaling, in amanner similar to that for other pathogens bearing surface-located NTPDases, although this remains to be proven.

PURINERGIC SIGNALING AND THEHOST-PATHOGEN INTERACTION

Interference with P2 Receptor Signaling by Parasites

Possible effects on vascular homeostasis. The expression ofNTPDases on the surface of pathogens that invade the blood-stream has the potential to alter platelet activity. Hydrolysis ofATP and ADP by NTPDases may influence the activation ofP2X1, P2Y1, and P2Y12 receptors, resulting in interferencewith normal thrombotic defense mechanisms. This is particu-larly relevant to the metazoan parasite S. mansoni as it entersthe bloodstream. Experimental percutaneous infection of micewith S. mansoni results in a transient thrombocytopenia 2 daysafter infection, at about the stage that the organism reaches thebloodstream, suggesting that platelet activation and attach-ment of platelets to larvae is a host defense mechanism againstinfection (139). This is supported by the observation that plate-lets can attach to larvae in vitro. However, a few days afterinfection, platelet levels are similar in infected and controlanimals, suggesting that the larvae become resistant to plateletdefense mechanisms (139). Since the larval forms of S. man-soni express both SmATPDase 1 and SmATPDase 2 (45, 96),it is possible that upon entry of the parasite into the blood-stream, exposure to platelets leads to increased expression ofthe enzymes. The subsequent hydrolysis of ADP and limitationof platelet activation could then result in resistance of theparasite to platelet defense mechanisms.

Another parasite with a significant bloodstream phase is P.falciparum, and while suppression of platelet aggregation inhumans infected with P. falciparum has been reported (138),other work has suggested that P. falciparum infection enhancesplatelet aggregation (112). Even though the precise effect ofthe parasite on platelets is unclear, the presence of a putativeNTPDase gene in the genome of P. falciparum warrants fur-ther investigation. In particular, determining if the protein islocated on the parasite surface and what, if any, preference theenzyme displays for ATP and/or ADP would assist in clarifyingthe possible effect of P. falciparum on platelet activation duringinfection.

The life cycles of both African and American trypanosomesinvolve blood-borne stages, and it has been suggested that theNTPDases on the surface of the parasites are involved ininhibiting platelet recruitment (57, 99). In the case of T. brucei,where the parasite remains extracellular and replicates withinthe bloodstream, the ability to inhibit platelet defense mecha-

nisms would be highly advantageous. However, it is difficult toreconcile this with the fact that both T. brucei and T. cruzi areknown to cause platelet aggregation and resultant thrombocy-topenia (111, 144). One explanation may be that in all threetrypanosome species in which NTPDases have been studied,the enzymes hydrolyze ATP preferentially, resulting in libera-tion of ADP and transient platelet activation. Although pref-erential ATPase activity would provide ADP for activation ofP2Y1 and P2Y12 receptors, ADP hydrolysis by these enzymes isnevertheless still relatively efficient (47, 57, 59). Again, morework is needed to clarify the contribution of NTPDases toplatelet responses during infection.

Modulation of the host inflammatory response. Extracellu-lar ATP and other nucleotides constitute potent “danger sig-nals” for the host immune and inflammatory responses (17).ATP in particular can trigger the release of proinflammatorycytokines such as IL-1� through P2 receptor signaling (93).Extracellular ATP also activates dendritic cells and inducessecretion of IL-12 (125). CD39 has been shown to inhibitATP-stimulated cytokine release from mammalian cells (77),and the expression of CD39 on Treg cells has also been linkedto decreased activation of dendritic cells (16). Therefore thehydrolysis of extracellular nucleotides by microbial NTPDasescould potentially mimic the action of mammalian NTPDasesby influencing the immune response during infection, includingthe induction of inflammatory responses, Treg cell activation,and/or dendritic cell maturation. Interestingly, Leishmania, T.cruzi, and T. gondii have all been shown to inhibit IL-12 pro-duction by dendritic cells (122), and the invasion of dendriticcells by live T. gondii does not result in dendritic cell matura-tion (102). Similarly, dendritic cells pulsed with live L. pneu-mophila do not undergo phenotypic maturation and secretelower levels of cytokines (85). Nevertheless, despite this link, itremains to be seen if microbial NTPDases can function in amanner similar to that for CD39 in vivo.

Interference with P2 Receptor Signaling by Bacteria

Extracellular ATP has already been shown to be involved inhost defense mechanisms during certain bacterial infections(52, 92). Although in general bacterial pathogens do not pos-sess CD39/NTPDase 1 enzymes, several organisms have none-theless been shown to interfere with ATP-mediated signalingby other means (162, 163). For example, extracellular ATPappears to stimulate killing of intracellular Mycobacterium bo-vis and Mycobacterium tuberculosis by infected human macro-phages in a manner dependent on P2X7 receptors (52, 92).Mycobacterium spp. are intracellular pathogens of macro-phages that inhibit phagosome maturation as a path to intra-cellular replication. Stimulation with extracellular ATP over-comes the inhibition of phagolysosome fusion and drivesmaturation of the Mycobacterium vacuole to a mature phagoly-sosome where the bacteria are killed. Interestingly, a recentstudy showed that extracellular ATP does not induce the kill-ing of intracellular Mycobacterium avium subsp. paratuberculo-sis in bovine mononuclear phagocytes, demonstrating that spe-cies differences do exist (157). Although the exact mechanismbehind ATP-induced killing of Mycobacterium is unknown,polymorphisms in the P2X7 receptor are associated with in-creased susceptibility to the dissemination of mycobacterial



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growth and clinical disease (54). M. bovis is known to secrete atleast two enzymes that alter ATP concentration, an ATPaseand a NDP kinase (Ndk). Filtered culture supernatants pre-vent ATP-induced macrophage death, although the initialstudy describing this result did not demonstrate which en-zyme(s) secreted by the bacteria was responsible for this pro-tective effect (163). More recently, purified Ndk secreted by M.tuberculosis was shown to have ATPase and GTPase activities.Surprisingly, the same study also demonstrated that Ndk actu-ally enhanced ATP-induced cytotoxicity in macrophages,which was inhibited in the presence of a P2 receptor antagonist(27). As Ndk catalyzes the transfer of the terminal phosphategroup of NTPs to NDPs, the authors postulated that this effecton cytotoxicity resulted from Ndk-mediated conversion of ATPinto other nucleotides that act as better agonists toward P2receptors, although there is no direct evidence for this hypoth-esis.

Recently, the intracellular pathogenic bacterium Porphy-romonas gingivalis was shown to interfere with P2X7 receptor-mediated apoptosis in epithelial cells (162). While P. gingivalisdoes not possess an NTPDase, it also secretes an Ndk intoculture supernatants. In contrast to the case for M. tuberculosis,the secretion of this enzyme is associated with the inhibition ofP2X7-mediated apoptosis, suggesting that Ndk secreted fromdifferent pathogens results in varied effects on the host. Thereasons for this are not understood, but it may result fromdifferences in other nucleotide concentrations or other, as-yet-unknown factors. Pseudomonas aeruginosa also secretes severalATP-utilizing enzymes (including Ndk and an ATPase). Thepresence of purified ATPase or Ndk reduces the cytotoxicityassociated with ATP-induced P2X7 receptor activation and thesubsequent loss of macrophage viability. This effect presum-ably results from hydrolysis of ATP by either enzyme and/orsequestration of ATP from the P2X7 receptors (164).

Extracellular ATP (but not other nucleotides) when incu-bated with macrophages infected with Chlamydia trachomatisreduces the number of infectious bacteria within the macro-phages. However, the bacteria themselves appear to also in-terfere with apoptosis by decreasing the activity of the P2X7receptor (35), although the mechanism by which this occurs isunclear. As is the case for M. tuberculosis, treatment of infectedmacrophages with extracellular ATP results in the death ofbacteria from activation of phospholipase D in a manner de-pendent on activation of P2X7 receptors (36).

Mycoplasma hominis, an extracellular organism that colo-nizes the human urogenital tract, also possess an ecto-ATPase,although this enzyme is not a member of the NTPDase family(74). Surprisingly, a recent study demonstrated that this en-zyme was able to induce apoptosis, and although the mecha-nism by which this occurred was not elucidated, it was postu-lated that it may be due to the generation of ADP and/or otherbreakdown products of ATP, again suggesting that ATP-uti-lizing enzymes of pathogens may influence purinergic signaling(73).

While none of these bacteria possess a typical NTPDase ofthe CD39/NTPDase 1 family, the degradation of extracellularATP by pathogens has a proven effect on host cell viability andthe capacity of the host to clear an infection. Therefore, mi-crobial NTPDases are likely to interfere with similar processesin pathogen-infected macrophages. In particular, the observa-

tion in a number of studies that secreted ATP-utilizing en-zymes from intracellular pathogens interfere with purinergicsignaling strongly suggests a similar role for the secreted andsurface-located NTPDases expressed by the microbial patho-gens reviewed here.

Adenosine Generation by Pathogens

The immune suppression elicited by Treg cells also relies onthe concurrent expression of CD73 by these cells (39). CD73 isan ecto-5�-nucleotidase that hydrolyzes AMP to produce aden-osine, a molecule that signals through P1 receptors, specificallyA2A receptors, to induce a number of immunosuppressiveeffects such as the inhibition of effector T-cell activation andsuppression of proinflammatory cytokine expression (39).Therefore, the immunosuppressive effect of CD39 results notonly from the inhibition of P2 receptor signaling by the deg-radation of ATP but also from the activation of P1 receptors byadenosine.

Ecto-5�-nucleotidase activity has also been detected on thesurface of T. vaginalis (145). While the parasite is a purineauxotroph and may utilize some of the adenosine produced forgrowth, free adenosine may also play a secondary role as amediator of immune suppression. The trypanosomes Leishma-nia and S. mansoni have all been shown experimentally tohydrolyze AMP to generate adenosine at the surface of theparasite (13, 47, 118, 132), suggesting a similar role for theenzyme in these organisms. Of particular interest is the obser-vation that C57BL/6 mice are able to control infection by L.braziliensis but develop chronic lesions when infected with L.amazonensis (98). The L. amazonensis parasites exhibit higherlevels of AMP hydrolysis, thus producing adenosine in largeramounts, and mice infected with L. amazonensis show de-creased production of proinflammatory cytokines, thereby rais-ing the possibility that adenosine production by Leishmaniaparasites contributes to immune suppression.

In contrast, apicomplexan parasites are not known to pos-sess ecto-5�-nucleotidases (110), so it is unclear if adenosine isgenerated by these parasites. Similarly, L. pneumophila doesnot appear to possess an ecto-5�-nucleotidase (24, 26) and doesnot reportedly hydrolyze AMP. Although to date there is noevidence of adenosine generation by the apicomplexan para-sites and Legionella, it is possible that host enzymes such asCD73 could theoretically mediate the hydrolysis of excessAMP resulting from the action of microbial NTPDases.

Nonadenine Nucleotide Signaling

It is interesting that of the microbial NTPDases described sofar, most hydrolyze purine and pyrimidine bases such as GTP,UTP, and CTP with efficiencies similar to those for ATP andADP. Unusually, Lpg1905 from L. pneumophila hydrolyzesonly ATP/ADP and GTP/GDP and shows very limited activityagainst CTP/CDP and UTP/UDP (124). The NTPDases thatdo efficiently hydrolyze nonadenine nucleotides include thosefound in the apicomplexan parasites T. gondii (6) and N. cani-num (4) and those found in T. vaginalis (40), T. foetus (80), thetrypanosomes (47, 57, 59), and S. mansoni (53). In addition totheir activity against NTPs, the NTPDases from T. gondii (6),T. vaginalis (40), T. cruzi (57), and S. mansoni (53) hydrolyze



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other NDPs. Nonadenine NTPs and NDPs such as UTP andUDP are agonists for a number of P2Y receptors and in somecases have higher affinity for these receptors than ATP (20).UTP has also been shown to stimulate expression and releaseof the proinflammatory cytokine IL-6 (48, 89). Furthermore,UDP, a selective P2Y6 agonist, stimulates production and re-lease of IL-8 and tumor necrosis factor alpha in human mono-cytic cell lines (37, 156). Extracellular nucleotides are alsoinvolved in lipopolysaccharide-induced neutrophil migration(90). Therefore, the microbial NTPDases may potentially uti-lize several substrates to interfere with P2 receptor signaling.

CONCLUSIONS

In recent years the identification of an increasing number ofhuman pathogens with secreted and plasma membrane-asso-ciated NTPDase activity has raised a number of intriguingquestions regarding the role in of these enzymes in interactionswith the mammalian host. The similarities between microbialand mammalian NTPDases suggest that pathogens havethe capacity to interfere with pathways mediated by hostNTPDases, such as modulation of vascular homeostasis andinflammatory and immune responses. While further work isrequired to elucidate the mechanism(s) by which microbialNTPDases influence virulence, it is evident from the workreviewed here that investigating the effects of these enzymes onpurinergic signaling may yield valuable information.

NTPDases as Possible Targets for Antimicrobial Therapy

In addition, the characterization of crucial differences be-tween the microbial NTPDases and mammalian NTPDasesmay lead to the development of selective inhibitors that targetmicrobial survival and enhance antimicrobial immune re-sponses. The fact that the enzymes have an external location,are implicated in parasite survival, and are widely distributedamong different eukaryotic human pathogens make them ap-pealing targets for the development of antimicrobial agents.However, the success of microbial NTPDases as pharmacolog-ical targets will depend on the identification of inhibitors thatare selectively toxic for microbial pathogens but have littleeffect on the mammalian enzymes. Some promise has beenshown already with the new N-alkylaminoalkanethiosulfuricacid class of antischistosomal drugs that partially inhibit para-site NTPDase activity (54).

We recently observed that Lpg1905 from L. pneumophilaexhibited different sensitivities to inhibition by novel polyoxo-metalate NTPDase inhibitors than mammalian NTPDases(124). Although these differences were not pharmacologicallyuseful, this demonstrated that there are likely to be structuraldifferences between the enzymes that could be exploited forinhibitor development.

The importance of protein structures for understanding en-zyme function and for the development of NTPDases as tar-gets for antimicrobial therapy is paramount. The rational de-sign of inhibitors that have greater potency against themicrobial enzymes than against the host will depend on iden-tifying structural differences between the microbial and mam-malian enzymes. The recent elucidation of the crystal structureof rat NTPDase 2 will form an important basis for studying

structural differences within this family of enzymes (166). Thiswork needs to take place in concert with detailed pathogenesisstudies of the role of microbial NTPDases in infection so thatwe can approach a clear understanding of the contribution ofthese enzymes to parasite survival and host immunity.

ACKNOWLEDGMENTS

This work was supported by Australian National Health and Med-ical Research Council (NHMRC) grants awarded to E.L.H. and byNIH grants (HL57307, HL63972, and HL076540) to S.C.R.

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