Identification and Characterization of a Novel Calcium-
Activated Apyrase from Cryptosporidium Parasites and
Its Potential Role in Pathogenesis
Patricio A. Manque1,2.¤a, Ute Woehlbier1,2.¤b, Ana M. Lara1,2, Fernando Tenjo1,2,3, Joa ˜o M. Alves1,2,
Gregory A. Buck1,2*
1Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia, United States of America, 2Center for the Study of Biological
Complexity, Virginia Commonwealth University, Richmond, Virginia, United States of America, 3Department of Biology, Virginia Commonwealth University, Richmond,
Virginia, United States of America
Herein, we report the biochemical and functional characterization of a novel Ca2+-activated nucleoside diphosphatase
(apyrase), CApy, of the intracellular gut pathogen Cryptosporidium. The purified recombinant CApy protein displayed
activity, substrate specificity and calcium dependency strikingly similar to the previously described human apyrase, SCAN-1
(soluble calcium-activated nucleotidase 1). CApy was found to be expressed in both Cryptosporidium parvum oocysts and
sporozoites, and displayed a polar localization in the latter, suggesting a possible co-localization with the apical complex of
the parasite. In vitro binding experiments revealed that CApy interacts with the host cell in a dose-dependent fashion,
implying the presence of an interacting partner on the surface of the host cell. Antibodies directed against CApy block
Cryptosporidium parvum sporozoite invasion of HCT-8 cells, suggesting that CApy may play an active role during the early
stages of parasite invasion. Sequence analyses revealed that the capy gene shares a high degree of homology with apyrases
identified in other organisms, including parasites, insects and humans. Phylogenetic analysis argues that the capy gene is
most likely an ancestral feature that has been lost from most apicomplexan genomes except Cryptosporidium, Neospora and
Citation: Manque PA, Woehlbier U, Lara AM, Tenjo F, Alves JM, et al. (2012) Identification and Characterization of a Novel Calcium-Activated Apyrase from
Cryptosporidium Parasites and Its Potential Role in Pathogenesis. PLoS ONE 7(2): e31030. doi:10.1371/journal.pone.0031030
Editor: Gordon Langsley, Institut National de la Sante ´ et de la Recherche Me ´dicale - Institut Cochin, France
Received October 3, 2011; Accepted December 30, 2011; Published February 7, 2012
Copyright: ? 2012 Manque et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grant U54 AI057168 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
¤a Current address: Center for Genomics and Bioinformatics, Universidad Mayor, Santiago, Chile
¤b Current address: Institute of Biomedical Sciences, University of Chile, Santiago, Chile
Cryptosporidiosis, caused by the obligate intracellular protozo-
an Cryptosporidium, is a ubiquitous infectious disease that can cause
a persistent and chronic diarrhea . Most Cryptosporidium
infections in humans are self-limiting, but severe disease may
occur in immunodeficient hosts, in particular in AIDS patients .
Although Cryptosporidium hominis (C. hominis) is the most prevalent
cause of endemic disease in humans, Cryptosporidium parvum (C.
parvum) also can cause serious outbreaks when humans are exposed
to contaminated water supplies or are otherwise in close contact
with the non-human carriers of this parasite. Effective vaccines
and safe non-toxic anti-cryptosporidial drugs are not yet available.
The cellular and molecular mechanisms of infection are poorly
understood, mostly due to difficulties in propagation of the parasite
A Cryptosporidium infection is initiated when the host ingests
oocysts from which invasive sporozoites emerge and infect
enterocytes . Clearly, interactions between macromolecules of
the parasites and host cells are of critical importance for the
establishment of the infection and consequent survival of the
parasite. Thus, pathogenic factors such as parasite proteins or
macromolecules responsible for attachment or invasion, or factors
that block host cell responses, are ideal targets for drug and
Nucleotide mediated signaling plays a central role in maintain-
ing homeostasis in many tissues. Thus, ecto-nucleotidases are
major players in the regulation of purinergic signaling, modulate
inflammation and immune responses in Langerhans cells , and
lead to cardioprotection and protective responses to hypoxia/
ischemia in mice [5,6]. As signaling molecules, extracellular
nucleotides also serve as danger signals induced by pathogen
infection as well as cell or tissue injury, triggering various cellular
events such as proliferation, differentiation and chemotaxis .
Recently, high ecto-nucleotidase activity of several protozoan
parasites - including Toxoplasma gondii, Entamoeba histolytica,
Leishmania tropica, Leishmania amazonesis, Trypanosoma cruzi, Trypano-
soma brucei, and Tritrichomonas foetus - has been shown to interfere
with the extracellular signaling of the host and affect the virulence
and pathogenesis of these organisms [8,9,10,11,12,13,14,15,
PLoS ONE | www.plosone.org1February 2012 | Volume 7 | Issue 2 | e31030
16,17]. Thus, it has been suggested that these enzymes play a role
in the pathogenicity of these parasites by controlling the host cell
response to infection, specifically by: (i) protecting the parasite
from the cytolytic effects of extracellular ATP, (ii) regulating
ectokinase substrate concentrations, (iii) preventing activation of
signal transduction cascades associated with cellular injury, and (iv)
facilitating cellular adhesion [18,19,20,21,22,23,24,25,26,27,28],
reviewed in .
Among ecto-nucleosidases, Ecto-ATPases, or E-ATPases, are
cell-surface enzymes that hydrolyze a range of extracellular
nucleoside triphosphates (NTPs) and nucleoside diphosphates
(NDPs). Most of the E-ATPases are apyrases (ATP dipho-
sphohydrolases, EC 184.108.40.206), enzymes that were originally defined
as those that catalyze the hydrolysis of both adenosine triphos-
phate (ATP) and adenosine diphosphate (ADP) to adenosine
monophosphate (AMP) and inorganic phosphate (Pi) . The
majority of known apyrases belong, on basis of sequence
homology, to the CD39 family. CD39, also known as ENTPD1
(ectonucleoside triphosphate diphosphohydrolase 1), is an integral
plasma membrane protein with two transmembrane domains and
a large heavily glycosylated extracellular region with nucleoside
triphosphate diphosphohydrolase activity [18,29,30]. However, a
novel and evolutionarily distinct apyrase, that differs from the
CD39 family in amino acid sequence as well as its exclusive
calcium-dependent functionality, has been identified in the
salivary glands of blood-sucking bed bug Cimex lectularius . A
series of homologs to the Cimex gene were recently found in other
blood-sucking insects, as well as in vertebrates, including humans,
indicating that these enzymes represent an evolutionarily wide-
spread family of proteins [31,32,33,34,35,36,37,38].
Herein we describe for the first time the biochemical and
functional characterization of an apyrase from C. hominis,
designated CApy, its potential role during the infection, and the
resulting implications for pathogenesis during cryptosporidiosis.
Materials and Methods
All mice were housed at the vivarium at Virginia Common-
wealth University (VCU) in an AAALAC-accredited facility and
experimentation followed VCU Institutional Animal Care and
Use Committee approved protocols (VCU IACUC Approved
Protocol AM10329, Cryptosporidium Reverse Vaccinology). This
study was carried out in strict accordance with the recommenda-
tions in the Guide for the Care and Use of Laboratory Animals of
the National Institutes of Health (NIH).
Mammalian cell culture and C. parvum
Intestinal epithelial HCT-8 cells were obtained from ATCC
(CCL-244) and grown in 75-cm2flasks in Dulbecco’s modified
Eagle’s medium (DMEM, Invitrogen) containing 10% fetal calf
serum, 25 mM HEPES, 100 units penicillin, and 100 mg of
streptomycin per ml at 37uC in 5% CO2as described . C.
parvum oocysts were purchased from the University of Arizona.
Oocysts were stored at 4uC until use.
The sequence encoding the C. hominis apyrase gene (CApy)
(Chro. 60194) lacking the N-terminal signal sequence was
obtained by PCR amplification from C. hominis genomic DNA
and cloned into the pTriEx-4 Ek/LIC vector (Novagen) using the
following primers: apyLICF 59GACGACGACAAGATGATAGA-
CGAAAGGAGGGTTTG39, and apyLICR 59GAGGAGAAGC-
59 end of the primers incorporated the LIC (ligation independent
cloning) sequences (underlined). The amplified CApy product was
ligated into pTriEx-4 after treatment with LIC-qualified T4 DNA
polymerase as described by the manufacturer (Novagen). E. coli
strain NovaBlue (Novagen) and E. coli strain BL21(DE3) (Novagen)
were used for plasmid maintenance and protein expression,
respectively. The resulting protein is fused to an N-terminal His6-
and S-tag with a predicted molecular mass of 41,014 Da, and is
referred to as recombinant CApy, designated rCApy.
For production of an unrelated control protein (composed of an
N-terminal His6-tag, Nus-protein, and C-terminal His6- and S-
tag), the pET44 Ek/LIC vector (Novagen) transformed into E. coli
strain BL21(DE3) was used. The resulting protein with a
molecular mass of 61,523 Da is herein referred to as Nus.
Expression and purification of rCApy protein
The E. coli strain BL21(D3) transformed with pTriEx-4/CApy
was cultured aerobically in TB medium (Overnight ExpressTM
Autoinduction System, Novagen) supplemented with ampicillin
(100 mg/ml) at 37uC under constant agitation. The rCApy protein
C-terminally fused to a His6/S-Tag was expressed in inclusion
bodies (not shown). Cell pellets were resuspended in BugBuster
protein extraction reagent (Novagen) with LysonaseTMsolution
(Novagen), and incubated for 30 min at room temperature to
induce lysis. After centrifugation at 390006g (Sorvall SS-34 rotor)
for 30 min at 4uC, the supernatant was discarded and the pellet
was resuspended in BugBuster protein extraction reagent with
rLysozymeTMsolution (Novagen). Following incubation for 5 min
at room temperature, 6 volumes of 1:10 diluted BugBuster protein
extraction buffer was added and mixed by vortexing for 1 min.
The suspension was centrifuged for 15 min at 390006g (Sorvall
SS-34 rotor) at 4uC and the supernatant was discarded. rCApy
inclusion bodies were resuspended in 20 mM sodium phosphate,
pH 7.4, 500 mM NaCl, 6 M guanidine hydrochloride (buffer 1),
and vortexed for 5 min. The buffer was adjusted to 20 mM
sodium phosphate pH 7.4, 500 mM NaCl, 4 M guanidine
hydrochloride, 10 mM imidazole (buffer 2) and applied to Ni2+
chelate affinity chromatography using a column with a 1 ml bed
volume (GE Healthcare). The solubilized rCApy inclusion bodies
were loaded onto a Ni2+- column in buffer 2 (GE Healthcare).
Bound proteins were washed with buffer 3 (buffer 1 with 25 mM
imidazole) and eluted with buffer 4 (buffer 1 with 500 mM
imidazole). The eluted material was renatured by dialysis
overnight against a minimum of 50 volumes of 100 mM Tris,
pH 8.0, 1 M arginine, 2 mM EDTA, 1 mM GSH (glutathione
reduced), 0.1 mM GSSG (glutathione oxidized), 5% glycerol at
4uC. Finally, after additional dialysis overnight at 4uC against a
minimum of 200 volumes of PBS (phosphate buffered saline),
pH 7.4, or 20 mM MOPS, pH 7.4, the purity of the CApy protein
preparation was examined by SDS-PAGE analysis. Protein
concentrations were determined according to Bradford (Biorad).
To purify Nus, the supernatant after bacterial cell lysis as
described above was diluted 1:1 with 20 mM sodium phosphate
pH 7.4, 500 mM NaCl, 10 mM imidazole (buffer 5) and loaded
onto a Ni2+- column (GE Healthcare) equilibrated with buffer 5.
Column bound proteins were washed with buffer 6 (buffer 5 with
50 mM imidazole) and eluted with buffer 7 (buffer 5 with 500 mM
imidazole). The eluted material was dialyzed overnight 4uC
against a minimum of 200 volumes of PBS, pH 7.4.
Production of antibodies against rCApy
Groups of five female 6 to 8 week old C57BL/6 mice (Jackson
Laboratory, MA) were immunized i.p. on day 0 with 20 mg
purified rCApy or Nus formulated in Freund’s complete adjuvant.
Cryptosporidium Calcium-Activated Apyrase (CApy)
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Mice were boosted i.p. twice on day 21 and 42 with 20 mg purified
rCApy or Nus formulated in incomplete Freund’s adjuvant. Blood
samples were collected on day 63 and sera were prepared, pooled,
and stored at 220uC. The serum against Nus protein was used as
an unrelated control serum.
SDS-PAGE and Western blot analysis
To obtain sporozoites, 16108C. parvum oocysts were washed
three times (5,0006g for 4 minutes at 4uC) with Hanks’ Balanced
Salt Solution, transferred to excystation medium (0.75% Sodium
Taurocholate and 0.25% Trypsin in Hanks’ medium) and
incubated 37uC for one hour.
For preparation of soluble and insoluble fractions of oocysts and
sporozoites, parasites were lysed in a Nonidet P-40 based lysis buffer
(1%NonidetP-40,50 mMTris/HCl,pH 7.4,150 mMNaCl,20%
glycerol, proteinase inhibitor cocktail (Roche)) for 20 min at room
temperature. After centrifugation at 160006g and 4uC the
supernatant (soluble fraction) was collected and the pellet (insoluble
fraction) was dissolved in Nonidet P-40 based lysis buffer.
To remove N and O-linked oligosaccharides from native CApy,
sporozoites obtained as described above were treated with the
Enzymatic Carbo Release KitTM(QAbio, San Mateo, CA) as
recommended by the manufacturer. In brief, 26108sporozoites in
385 ml of water were mixed with 55 ml of Carbo Release buffer.
After addition of 27.5 ml denaturation buffer, the sample was boiled
for 5 min, chilled on ice, and supplemented with 27.5 ml Triton-X.
For incubation with various combinations of carbohydrate
removing enzymes, the sporozoite extract was distributed into
1.5 ml microfuge tubes (45 ml each), and 1 ml each of the following
enzymes was added: PNGase, sialidase, ß-galactosidase, glucosami-
nidase and/or O-glycosidase, as recommended by manufacturer.
After incubation at 37uC for 16 hr, protein loading buffer was
added, the samples were incubated at 100uC for 5 min, and an
equivalent of 2.56106sporozoites was subjected SDS-PAGE (12%
polyacrylamide). Separated proteins were transferred to PVDF
nitrocellulose membrane (Millipore) for 90 min at 350 mA and
4uC. Following blocking for 1 h at room temperature with 10 mM
Tris, pH 8.0, 150 mM NaCl, 0.05% Tween20 (TBST) containing
2% milk powder (blocking buffer); the nitrocellulose membrane was
incubated for 1 hour at room temperature with mouse anti-rCApy
serum diluted 1:500 in blocking buffer. After 3 washes with TBST,
the membrane was further incubated with anti-mouse IgG HRP-
conjugate (Sigma) diluted 1:5000 in blocking buffer. The final
reaction was revealed by chemiluminescence using ECL Western
blotting detection reagent (Pierce) and BioMax light film (Kodak) as
recommended by the manufacturers.
Apyrase activity assays
Apyrase activity assays were conducted in 96-well microtiter
plates at 23uC in a final volume of 80 ml of 20 mM MOPS,
pH 7.4, 100 mM NaCl containing 0.5 mM rCApy and 2.5 mM
NTPs, NDPs, or NMPs. Additionally, CaCl2, MgCl2 and/or
EDTA the solutions to an end concentration of 5 mM. After
10 min, 20 ml of Chan’s reagent (SensoLyteTMMG Phosphate
Assay Kit, AnaSpec) was added to determine the amount of
inorganic phosphate (Pi) formed. After 5 min of gentle shaking,
absorbance was measured at 630 nm. The units used for enzyme
activity are mmol of Pigenerated per mg of protein per hour.
HCT-8 cell binding assay
Cell binding assays were performed similar as previously
described . Briefly, HCT-8 cells (16105/well) were seeded in
96-well microtiter plates (Costar) and grown overnight at 37uC.
After fixation with 4% paraformaldehyde in PBS, pH 7.4, for 1 h
at RT, the cells were washed three times with PBS, pH 7.4,
blocked for 1 hour at room temperature with PBS, pH 7.4,
containing 10% FCS (PBS-FCS), washed three times with PBS,
pH 7.4, and incubated with varying concentrations of rCApy or
Nus protein as an unrelated protein control, in PBS-FCS. After
1 h at 37uC, the cells were washed four times with PBS, pH 7.4,
containing 0.05% Tween20 (PBS-Tween), and incubated for 1 h
at 37uC with mouse anti-His6-HRP conjugated antibody 1:10,000
(SantaCruz) diluted in PBS-FCS. After four washes, PBS-Tween
substrate buffer containing o-phenylenediamine (SIGMAFAST
OPDTM) was added, the cells were incubated for 30 min and the
OD was measured at 450 nm.
Cell invasion assay
Sporozoites (16106) were incubated with 1:10 diluted mouse
anti-rCApy serum or non-related control serum (anti-Nus) for
30 min at 37uC, washed three times with PBS, pH 7.4, and added
to 24-well plates containing a monolayer of HCT-8 cells in a ratio
of 4 parasite:1 cell. Following incubation under culture conditions
for 3 hours, extracellular parasites were removed by washing three
times with PBS, pH 7.4, and the plates were returned to the
incubater. Cells were collected 24 hours post-infection RNA was
extracted from infected cells using the RNAqueous system
(Ambion) following the manufacturer’s recommendations. RNA
samples were stored at 280uC until quantification of C. parvum
rRNA by RT-PCR.
Real-time quantitative RT-PCR
RT-PCR was used to quantify the in vitro infection rate of HCT-
8 cells by C. parvum. Thus, RNA samples from HCT-8 cells
infected with C. parvum sporozoites pretreated with mouse anti-
rCApy serum and the corresponding control (mouse anti-Nus
serum) were incubated with TURBO DNA-free DNase (Ambion)
following the manufacture’s instructions and used for Real-Time
RT-PCR analysis using TaqManTMtechnology. Primers and
probes specific for Cryptosporidium rRNA and human rRNA were
designed using Primer ExpressH version 2.0 (ABI). For each target,
forward and reverse primers and an internal probe were
synthesized. Probes were synthesized with 59 end linked FAM
(6-carboxyfluoresceine) and 39 end fluorescent TAMRA (6-
carboxytetramethylrhodamine) dyes. Amplification and analysis
were performed in our ABI7900HT instrument. The infection rate
was obtained by calculating the ratio of human rRNA versus
Cryptosporidium rRNA, with 100% infection represented by the
negative control (treated with anti-Nus serum).
Putative signal peptide cleavage sites and asparagine-linked
glycosylation sites of CApy were determined using SignalP 3.0
(http://www.cbs.dtu.dk/services/SignalP/) and NetNGlyc 1.0
molecular weights and isoelectric points were determined with
Phylogenetic analysis of CApy
Putative orthologs of CApy were identified by BLAST  search
of the CApy protein sequence against NCBI GenBank non-
redundant protein database (nr), with a threshold of 1E-6 for the
expectancy value E. All the identified protein sequences were aligned
with ClustalX  and manually checked for adequate aligned
length (.75% of the protein aligning). To expedite subsequent
bootstrap analyses and simplify the final tree, phylogenetic analysis
was performed by maximum likelihood using PHYML  in two
Cryptosporidium Calcium-Activated Apyrase (CApy)
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steps: first, a preliminary analysis (without bootstrap and using less
thorough tree space search strategies) identified possible in-paralogs
as well as out-paralogs that reflected the same phylogenetic pattern.
These sequences were then removed from the alignment, and the
sequences were realigned and reanalyzed to check whether the
phylogeny remained stable after removal of the paralogs. The second
analysis, involving only sequences remaining after the first step, was
performed with one hundred bootstrap replicates and the SPR
strategy  for the search of the optimal trees. The amino acid
substitution model used was LG with 4 categories of gamma-
distributed substitution rates, as selected by ModelGenerator .
Table S1 lists the GenBank accession numbers for the 109 sequences
utilized in phylogenetic reconstruction.
Identification and sequence analysis of a C. hominis
Analysis of the recently sequenced C. hominis and C. parvum
genomes identified a single ecto-ATP diphoshohydrolase (apyrase,
EC 220.127.116.11) in each sequence that displays high similarity to the
human soluble calcium-activated nucleotidase 1 (SCAN-1, Fig. 1A)
(GenBank accession numbers EAL36710, and EAK89829). The
Cryptosporidium apyrase (CApy) is a single copy gene with a predicted
openreadingframeof1035 bpcodingfora 345aminoacid protein.
Further characterization of CApy amino acid protein sequence
predicted a signal peptide with a cleavage site between residues 22
and 23. The cleavage results in a protein of 323 amino acids, with a
predicted molecular mass of 36,932 Da and an isoelectric point of
5.23 (Fig. 1B). No GPI anchor or other known cell targeting signals
were found, suggesting that the protein is either attached to the
membrane by a hydrophobic stretch or secreted. In addition, N-
linked glycosylation sites were not detected.
Expression, Purification and Refolding of recombinant
The C. parvum and C. hominis apyrase amino acid sequences are
98% identical. Herein, we describe the cloning, expression and
purification of recombinant CApy derived from C. hominis,
designated rCApy. Thus, the DNA sequence encoding amino
acid residues 34–345 excluding the signal peptide of the gene was
amplified from C. hominis genomic DNA. The resulting 936-bp
fragment was inserted into the pTriEx-4 vector (Novagen) C-
terminally fused to a His6-S-tag. As shown in Fig. 2, rCApy was
expressed in E. coli in inclusion bodies (IBs) and was therefore
solubilized as described in the Materials and Methods. Subsequent
purification by Ni2+- chelate chromatography under denaturing
conditions led to a highly purified and homogenous product (Fig. 2,
lane 7). During refolding of rCApy, little or no protein was lost due
to precipitation (Fig. 2, lane 8). Nevertheless, in subsequent dialysis
against PBS, pH 7.4, or MOPS pH 7.4 (Fig. 2, lanes 9 and 10), a
significant fraction of the refolded protein precipitated when
protein concentrations exceeded approximately 0.35 mg/ml. A
similar dependency on protein concentration for successful
refolding was reported for human SCAN-1 .
Expression and localization of CApy in infective forms of
To determine the expression and localization of the CApy
protein in Cryptosporidium parasites, we performed Western blot
experiments. Since C. hominis is very difficult to obtain, we used C.
parvum in this and the following experiments where parasites are
required. Since the genomes of these two parasites exhibit ,95%
sequence identity and the amino acid sequences of the CApy
proteins are nearly 98% identical [46,47], we do not expect major
differences in results obtained using C. parvum parasites. Western
blot analysis using polyclonal antibodies against rCApy showed
that the native protein is in the detergent-soluble fraction of
oocysts and sporozoites with an apparent molecular mass of
,50 kDa (Fig. 3). The apparent ,50 kDa molecular mass
suggests that the protein may be subject to post-translational
modifications. Higher molecular mass bands were also observed,
particularly in the oocyst fraction (Fig. 3), suggesting that CApy
could form multimer complexes. Previously we showed using
polyclonal mouse antibodies raised against rCApy that this
parasite protein is membrane associated with polar localization
in the infected sporozoites . The intriguing possibility that
CApy is associated with and secreted by the apical complex in
sporozoites remains to be further explored.
To investigate the possible presence of N- and O- linked
experiments on the proteins in sporozoite extracts and analyzed the
resulting proteins by Western blot analysis using CApy-specific
antibodies (Fig. 4). Treatment with glucosamidase, O-glucosidase,
or sialidase showed little effect on the migration of CApy. In
contrast, treatment with PNGase or ß-galactosidase revealed a
major shift in the molecular mass of CApy, with ß-galactosidase
having the most pronounced effect. Nevertheless, even after
deglycosylation with both PNGase and ß-galactosidase the most
prominent band detected in sporozoites was significantly higher,
,50 kDa,than the expected molecular weightforCApy,,39 kDa.
Moreover, reductionofsamples inthe presence100 mMDTT(1,4-
dithio-DL-threitol) before separation in SDS-PAGE did not change
the outcome of the Western blot (data not shown). These results
suggest that CApy in its mature form is posttranslationally
glycosylated in an N-linked fashion. Additional posttranslational
modifications may also be present since a larger than the expected
protein species was obtained after deglycosylation.
Activity of CApy requires Ca2+and is highest against UDP
substrates and divalent cations. ATPase and ADPase activities were
measured at 2.5 mM total nucleotide concentrations in the absence
of divalent cations, as well as in presence of 5 mM CaCl2, or MgCl2
(Fig. 5A). Nucleotidase activity was only detected in presence of
calcium ions. To determine enzyme substrate specificity of CApy, a
variety of nucleoside mono-, di-, and triphosphates were used as
substrates (2.5 mM final nucleotide concentrations) in the presence
of 5 mM CaCl2(Fig. 5B). The activity for the preferred substrates
UDP and GDP is high, whereas activities towards ATP and GTP
are lowest. No measurable hydrolysis of any investigated mono-
phosphate was detected. These results reflect those expected for
human apyrase SCAN-1.
Recombinant CApy binds to the HCT-8 cells in a dose
To assess the possibility that CApy may play a role in the
attachment of Cryptosporidium sporozoites to enterocytes, we
measured the ability of recombinant CApy to bind HCT-8 cells.
Binding assays were performed by adding various concentrations
of rCApy to paraformaldehyde fixed HCT-8 cells as described in
the Materials and Methods. As shown in Fig. 6, rCApy binds to
HCT-8 cells in a dose-dependent and saturable manner. No
binding was observed when an unrelated protein (bacterial Nus)
was used in identical assays.
Cryptosporidium Calcium-Activated Apyrase (CApy)
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Antibodies directed against rCApy block invasion of HCT-
8 cells by C. parvum
To further gain insight into the role of CApy during parasite
invasion, we performed in vitro invasion assays using HCT-8 cells.
Thus, sporozoites were pre-incubated with mouse anti-rCApy
serum or unrelated control serum and the infection rate was
measured using quantitative RT-PCR (Fig. 7). In these experi-
ments, antibodies against rCApy were able to reduce the invasion
of enterocytes by sporozoites by over 60%, suggesting that CApy
plays a role during the early stages of infection.
Sequence and phylogenetic analysis of apyrase
To perform a reconstruction of the evolutionary history of
CApy, we identified putative orthologs in as many genomes as
possible (Table S1) and used these sequences to perform a
Figure 1. Schematic outline of CApy and multiple sequence alignment of the apyrase domain of Cryptosporidium sp. with apyrase
homologs of their respective hosts. A. Alignment of selected apyrase sequences. Sequence alignment was performed using the CLUSTAL 2W
algorithm. The GenBank accession numbers of sequences are given in parentheses. CH, Cryptosporidium hominis (XP_666945); CP, Cryptosporidium
parvum (XP_627524); CM, Cryptosporidium muris (XP_002140694); HS, Homo sapiens (NP_620148); MM, Mus musculus (EDL34666), BT, Bos Taurus
(XP_596269). The enzymatic activity of the human apyrase has been established by biochemical analysis , therefore residues important for
nucleotide and Ca2+binding in the human apyrase and the predicted counterparts in corresponding apyrases of other species are indicated by gray
shadows and pluses. Residues that differ from the human sequence at positions with potential impact on enzymatic activity are colored red. Cysteine
residues are shadowed light blue. B. CApy is a protein comprising 345 aa, including a 22-aa signal peptide (SP), and an apyrase domain spanning
Cryptosporidium Calcium-Activated Apyrase (CApy)
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phylogenetic analysis (Fig. 8, and see Materials and Methods). The
analysis showed that among the Unikonta [49,50], a CApy
ortholog is present in Choanozoa, the Amoebozoa, in one
Opisthokonta of uncertain phylogenetic affiliation (Capsaspora),
and in several Metazoa, but not in any fungi. Among the Bikonta,
CApy orthologs only seem to be present in the Chromalveolata,
comprised of Alveolata (which includes apicomplexan parasites)
and Stramenopiles, and in one Excavata, Naegleria gruberi, which
grouped at a deep divergence, and with low bootstrap support,
with the Unikonta. The grouping of Apicomplexa and Strameno-
piles has strong bootstrap support, as have the internal groupings
of cryptosporidia, diatoms and oomycetes. The branch connecting
Toxoplasma and Neospora with Cryptosporidium is relatively well
supported, with a bootstrap value of 75 (only values above 50 are
shown on Fig. 8). Other known Bikonta (Excavata, Plantae and
Rhizaria) genomes seem to lack an ortholog of this protein, with
the exception of the excavate Naegleria, as noted above.
Multiple sequence alignment of these orthologs from protozoan,
invertebrate, and vertebrate organisms shows that most variations
occur in the N-terminus, possibly reflecting differences in the
Figure 2. Expression, Purification and Refolding of recombi-
nant CApy. The electrophoretic analysis (SDS-PAGE, 12% PAA under
reducing conditions) shows extracts of non-induced and induced E. coli
cultures (lane 1 and 2) bearing the CApy gene in the pTriEx-4 expression
vector from samples taken at different steps of protein purification
(lanes 3–10). Following bacterial cell lysis the soluble (supernatant, lane
3) and insoluble (pellet, lane 4) fractions show that CApy was mostly
found in the insoluble fraction in form of inclusion bodies (IBs). The
solubilized IBs (see Materials and Methods) were loaded onto a Ni2+
chelate column and purified under denaturing conditions, flow-through
(lane 5), wash (lane 6), and elution (lane 7) fractions were collected. The
eluate containing the purified CApy was refolded by dialysis against
folding buffer (lane 8), which was subsequently dialysed against PBS,
pH 7.4 (lane 9) or 20 mM MOPS, pH 7.4 (lane10).
Figure 3. Expression of CApy in oocysts and sporozoites. CApy
is expressed in oocysts and sporozoites. Aliquots of NP-40 soluble (S) or
insoluble (I) C. parvum oocyst and sporozoite fractions were resolved by
12% SDS-PAGE, transferred to nitrocellulose and probed with mouse
anti-rCApy serum as described in the Materials and Methods.
Figure 4. Glycosylation of CApy in sporozoites. C. parvum
sporozoites were treated as recommended in the Enzymatic Carbo
Release KitTM(QAbio, San Mateo, CA) for identification of glycosylation.
In brief, sporozoites were suspended in Carbo Release buffer, and
denaturation buffer was added. After incubation at 100uC for 5 min,
samples were chilled on ice and Triton-X was added. The enzymes
PNGase, Sialidase, ß-Galactosidase, Glucosaminidase, O-Glycosidase
were added alone or in combinations. Following incubation at 37uC
for 16 hours, protein loading buffer was added and samples were
incubated again at 100uC for 5 min. Proteins were separated by 12%
SDS-PAGE, transferred to nitrocellulose and probed with mouse anti-
Cryptosporidium Calcium-Activated Apyrase (CApy)
PLoS ONE | www.plosone.org6 February 2012 | Volume 7 | Issue 2 | e31030
secretory systems, as well as possible different localizations of the
protein, depending on the specific function in the respective
organism. Nevertheless the apyrase domain, and in particular the
structurally important regions, seem to be highly conserved (data
not shown). Surprisingly, orthologs occur in various infectious
disease causing eukaryotic parasites, as well as their respective
hosts, yet they are apparently absent in yeast and bacteria.
Overall, our analysis argues that CApy was present in the
progenitors of Cryptosporidium, but was lost in other apicomplexans
except Toxoplasma and Neospora, or, less likely, that the gene was
picked up by the parasites from more distantly related organisms
by lateral gene transfer.
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
Figure 5. Enzymatic characterization of recombinant CApy. Apyrase activity was measured in 20 mM MOPS buffer, pH 7.4, and a final
nucleotide concentration of 2.5 mM. Activity units are micromoles of Piliberated per milligram of protein per hour at 23uC. The graphs present the
mean and standard deviations of triplicate experiments. A. Cofactor specificity of rCApy. ATPase and ADPase activity of rCApy was measured in
presence of either 5 mM CaCl2or 5 mM MgCl2with or without addition of 5 mM EDTA. B. Substrate preference of rCApy. Assays were done in
presence of 5 mM CaCl2.
Figure 6. Binding of recombinant CApy to intestinal epithelial
cells. Increasing concentrations of protein in the range of 5–60 mg/ml,
rCApy or control protein, were added to 96-well plates containing
formaldehyde-fixed adherent HCT-8 cells (as described in Material and
Methods. Interactions of proteins, both carrying a His6-tag, with the
HCT-8 cell surface were detected by ELISA using anti-His6 HRP-
conjugated antibody. The bound antibody was revealed by using o-
phenylenediamine as a substrate. Respective results of one of three
experiments are shown, and are expressed as absorbance at 450 nm.
The values are the means of one experiment performed in triplicates,
error bars represent standard deviations. An unrelated bacterial protein
(Nus) was used as a control (C).
Figure 7. CApy-specific serum blocks C. parvum infection of
HCT-8 cells. C. parvum sporozoites were preincubated with mouse
anti-rCApy serum or unrelated control serum (dilution 1:10), followed
by incubation with HCT-8 cells for 24 hours and infection was
quantified by RT-PCR as described in the Materials and Methods. The
values are the means three independently performed experiments
performed, error bars represent standard deviations.
Cryptosporidium Calcium-Activated Apyrase (CApy)
PLoS ONE | www.plosone.org7 February 2012 | Volume 7 | Issue 2 | e31030
Cryptosporidium Calcium-Activated Apyrase (CApy)
PLoS ONE | www.plosone.org8 February 2012 | Volume 7 | Issue 2 | e31030
Unikonta, including arthropods and vertebrates [31,32,33,34,
36,37], but much less prevalent in the Bikonta, which includes the
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 [34,38], 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
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 [10,52].
Furthermore chemical inhibition of the ecto-NTPDase led to a
reduction of the virulence of T. cruzi in in vivo experiments .
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 [7,55] 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 inflamma-
tion 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 success-
ful parasite propagation. Further studies are necessary to test this
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, Neospora,
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, Theileria) and the Ciliata (e.g. Tetrahymena and
Paramecium). While Toxoplasma, Neospora and Cryptosporidium branch
together, they do so with moderate bootstrap support of 75. This is
likely due to the greater sequence divergence of the Toxoplasma and
Neospora 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 participa-
tion 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.
Database accession number of sequences used in
DNA synthesis and sequencing, and RT-PCR were performed in the
Nucleic Acids Research Facilities at Virginia Commonwealth University.
Figure 8. Maximum likelihood phylogenetic tree of all identified CApy orthologs. Numbers at the nodes represent bootstrap support
(only numbers above 50 are shown).
Cryptosporidium Calcium-Activated Apyrase (CApy)
PLoS ONE | www.plosone.org9 February 2012 | Volume 7 | Issue 2 | e31030
Conceived and designed the experiments: PAM UW. Performed the
experiments: PAM UW AML FT. Analyzed the data: PAM UW JMA
GAB. Contributed reagents/materials/analysis tools: GAB. Wrote the
paper: PAM UW JMA GAB.
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