In this study, we have identified and characterized a C. hominis
gene, CApy, which encodes an ecto-apyrase homolog of a group of calcium-dependent apyrases that catalyze hydrolysis of both nucleoside triphosphate (NTP) and nucleosine diphosphate (NDP) to nucleoside monophosphate (NMP) and inorganic phosphate (Pi
). These enzymes are highly conserved among the Unikonta, including arthropods and vertebrates 
, but much less prevalent in the Bikonta, which includes the apicomplexans.
Initial analysis of the CApy sequence revealed that the protein displays a classical signal peptide sequence, suggesting that the protein is either located on the surface of the parasite or secreted. No GPI anchor or other transmembrane signal was identified. Interestingly, immunofluorescence experiments suggested that the protein is mainly located in the apical region of the parasite, a highly specialized structure containing several organelles known to secret a series of proteins necessary for invasion of the host 
. Although bioinformatic analysis did not strongly suggest the presence of glycosylation sites, our observations suggest that CApy is heavily glycosylated. It remains to be investigated to what extent glycosylation influences the function of the molecule.
Our recombinant CApy functions as a soluble apyrase with exclusive calcium-dependent activity, consistent with its human homolog ortholog SCAN-1 
, as well as various insect apyrases from Cimex lectularius
, Phlebotomus papatasi
, and Lutzomyia longipalpis
. As observed for SCAN-1, UDP and GDP, rather than ADP or ATP, are the preferred substrates of CApy 
Several indications suggest that CApy could be involved in the invasion of human enterocytes by Cryptosporidium
. Thus, CApy seems to be found on the apical surface of sporozoites, binds to the host cell in a dose-dependent manner suggesting the presence of a potential receptor on the host cell surface, and elicits antibodies that significantly block invasion. Our observations support previous reports that indicate that ecto-apyrases play various roles during host pathogen interaction 
. Recently it was reported, that antibodies directed against an ecto-NTPDase from Trypanosoma cruzi
and Leishmania amazonesis
are able to significantly reduce the rate of infection of these parasites 
. Furthermore chemical inhibition of the ecto-NTPDase led to a reduction of the virulence of T. cruzi
in in vivo
. Moreover, Bissagio et al. 2003 
suggested that Mg2+
-dependent ecto-ATPase activity on the surface of T. cruzi
could stimulate the adherence of the parasite to the host cell and protect from neutrophil attack.
The ability of CApy to inactivate extracellular nucleotides, which generally serve as danger signals that are rapidly produced during the onset of immune responses, suggests other possible roles of CApy during parasite pathogenesis. Moderate to severe infection with Cryptosporidium
is characterized by mucosal inflammation with neutrophils and macrophages in the lamina propria underlying intestinal epithelial cells, as well as the presence of intraepithelial neutrophils (
, reviewed in 
). Cell and tissue damage, hypoxia, leukocyte activation, decreased pH and other stress factors previously described to be caused by this pathogen 
may lead to the release of large amounts of extracellular nucleotides into the intestinal lumen at the site of inflammation. Our in vitro
data indicate that CApy may interfere with the interaction of ADP, ATP, UDP, and UTP with different subsets of purinergic receptors, thereby possibly impacting various signaling pathways normally activated during inflammation resulting in modulation of cellular immune responses. In addition to its potential role in attachment, an apyrase expressed on the surface or secreted by the parasite may be an effective counter measure to the release of extracellular nucleotides rapidly secreted at local sites of infection which would otherwise display potent innate immune-enhancing activities undermining successful parasite propagation. Further studies are necessary to test this hypothesis.
Apyrases are broadly distributed among the tree of life and are present in many pathogenic parasites 
. To gain insight into the evolution of this pathogenic factor we performed phylogenetic analysis of CApy and all orthologs that we could identify from available sequences. Our analysis, using recent multi-gene phylogenies of eukaryotes as a benchmark 
, suggests that CApy was present in the apicomplexan progenitor, but the gene was lost from apicomplexans other than Cryptosporidium
, and Toxoplasma
. Moreover, the phylogenetic analysis indicates that all Chromalveolata sequences form a monophyletic group, suggesting that CApy might have been an ancestral feature of the group that was later lost in other Apicomplexa (e.g., Plasmodium
) and the Ciliata (e.g. Tetrahymena
). While Toxoplasma
branch together, they do so with moderate bootstrap support of 75. This is likely due to the greater sequence divergence of the Toxoplasma
apyrases, which can lead to lower phylogenetic signal and more homoplasy with other sequences. Assuming that the phylogenetic groups Bikonta and Unikonta are natural, the absence of this apyrase in other bikonts (all Excavata but Naegleria
, all Plantae, and possibly Rhizaria, for which no complete genomes were available at analysis time) suggests that either the CApy gene is ancient but was lost in these bikont lineages, or that this gene is novel and was transferred from unikonts to Chromalveolata (or vice-versa) early in evolution of these groups. Our current taxonomic sampling of this gene does not allow a definitive conclusion, emphasizing the need for further genomic studies of more diverse taxa among the unikonts, Chromalveolata, Rhizaria, as well as basal Plantae, e.g. rhodophytes and glaucophytes.
In summary, herein we described a new potential pathogenic factor, the CApy apyrase, in Cryptosporidium. CApy may play multiple roles during the infection, including an active participation in the attachment and invasion of Cryptosporidium to the host cells. In addition, we provide indirect evidence suggesting that CApy could potentially interfere with extracellular nucleotide signaling responsible for triggering an inflammatory response in injured tissues, possibly delaying it and providing an opportunity for the parasite to successfully establish the infection. Phylogenetic evidence suggests that either this gene was acquired very early in evolution but lost in many of the nearest relatives of Cryptosporidium, explaining its otherwise broad distribution among the tree of life and among pathogenic parasites, or, less likely, that Cryptosporidium, Toxoplasma, and Neospora may have acquired the gene by lateral gene transfer. Finally the evidence provided by this study indicates that CApy might be a potential drug target and/or vaccine candidate against cryptosporidiosis.