Evidence for Mucin-Like Glycoproteins That Tether Sporozoites of … · Immucell Corp., Portland, MA. Rabbit antibodies were diluted 1:100 or 1:200 and incubated with C. parvum for1hatRT,andwashed

EUKARYOTIC CELL, Jan. 2010, p. 84–96 Vol. 9, No. 11535-9778/10/$12.00 doi:10.1128/EC.00288-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Evidence for Mucin-Like Glycoproteins That Tether Sporozoites ofCryptosporidium parvum to the Inner Surface of the Oocyst Wall�†

Anirban Chatterjee,1 Sulagna Banerjee,1 Martin Steffen,2 Roberta M. O’Connor,3Honorine D. Ward,3 Phillips W. Robbins,1 and John Samuelson1*

Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts 021181;Department of Genetics and Genomics, Boston University School of Medicine, Boston, Massachusetts 021182; and Division of

Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, Massachusetts 021113

Received 6 October 2009/Accepted 12 November 2009

Cryptosporidium parvum oocysts, which are spread by the fecal-oral route, have a single, multilayered wallthat surrounds four sporozoites, the invasive form. The C. parvum oocyst wall is labeled by the Maclura pomiferaagglutinin (MPA), which binds GalNAc, and the C. parvum wall contains at least two unique proteins(Cryptosporidium oocyst wall protein 1 [COWP1] and COWP8) identified by monoclonal antibodies. C. parvumsporozoites have on their surface multiple mucin-like glycoproteins with Ser- and Thr-rich repeats (e.g., gp40and gp900). Here we used ruthenium red staining and electron microscopy to demonstrate fibrils, which appearto attach or tether sporozoites to the inner surface of the C. parvum oocyst wall. When disconnected from thesporozoites, some of these fibrillar tethers appear to collapse into globules on the inner surface of oocyst walls.The most abundant proteins of purified oocyst walls, which are missing the tethers and outer veil, wereCOWP1, COWP6, and COWP8, while COWP2, COWP3, and COWP4 were present in trace amounts. Incontrast, MPA affinity-purified glycoproteins from C. parvum oocysts, which are composed of walls andsporozoites, included previously identified mucin-like glycoproteins, a GalNAc-binding lectin, a Ser proteaseinhibitor, and several novel glycoproteins (C. parvum MPA affinity-purified glycoprotein 1 [CpMPA1] toCpMPA4). By immunoelectron microscopy (immuno-EM), we localized mucin-like glycoproteins (gp40 andgp900) to the ruthenium red-stained fibrils on the inner surface wall of oocysts, while antibodies to the O-linkedGalNAc on glycoproteins were localized to the globules. These results suggest that mucin-like glycoproteins,which are associated with the sporozoite surface, may contribute to fibrils and/or globules that tether sporo-zoites to the inner surface of oocyst walls.

Cryptosporidium parvum and the related species Cryptospo-ridium hominis are apicomplexan parasites, which are spreadby the fecal-oral route in contaminated water and cause diar-rhea, particularly in immunocompromised hosts (1, 12, 39, 47).The infectious and diagnostic form of C. parvum is the oocyst,which has a single, multilayered, spherical wall that surroundsfour sporozoites, the invasive forms (14, 27, 31). The outermostlayer of the C. parvum oocyst wall is most often absent fromelectron micrographs, as it is labile to bleach used to removecontaminating bacteria from C. parvum oocysts (27). We willrefer to this layer as the outer veil, which is the term used fora structure with an identical appearance on the surface of theoocyst wall of another apicomplexan parasite, Toxoplasma gon-dii (10). At the center of the C. parvum oocyst wall is a pro-tease-resistant and rigid bilayer that contains GalNAc (5, 23,43). When excysting sporozoites break through the oocyst wall,the broken edges of this bilayer curl in, while the overall shapeof the oocyst wall remains spherical.

The inner, moderately electron-dense layer of the C. parvumoocyst wall is where the Cryptosporidium oocyst wall proteins

(Cryptosporidium oocyst wall protein 1 [COWP1] and COWP8)have been localized with monoclonal antibodies (4, 20, 28, 32).COWPs, which have homologues in Toxoplasma, are a familyof nine proteins that contain polymorphic Cys-rich and His-rich repeats (37, 46). Finally, on the inner surface of C. parvumoocyst walls are knob-like structures, which cross-react with ananti-oocyst monoclonal antibody (11).

Like other apicomplexa (e.g., Toxoplasma and Plasmodium),sporozoites of C. parvum are slender, move by gliding motility,and release adhesins from apical organelles when they invadehost epithelial cells (1, 8, 12, 39). Unlike other apicomplexa, C.parvum parasites are missing a chloroplast-derived organellecalled the apicoplast (1, 47, 49). C. parvum sporozoites have ontheir surface unique mucin-like glycoproteins, which contain Ser-and Thr-rich repeats that are polymorphic and may be modifiedby O-linked GalNAc (4–7, 21, 25, 26, 30, 32, 34, 35, 43, 45). TheseC. parvum mucins, which are highly immunogenic and are poten-tially important vaccine candidates, include gp900 and gp40/gp15(4, 6, 7, 25, 26). gp40/gp15 is cleaved by furin-like proteases intotwo peptides (gp40 and gp15), each of which is antigenic (42).gp900, gp40, and gp15 are shed from the surface of the C. parvumsporozoites during gliding motility (4, 7, 35).

The studies presented here began with electron micro-scopic observations of C. parvum oocysts stained with ru-thenium red (23), which revealed novel fibrils or tethers thatextend radially from the inner surface of the oocyst wall tothe outer surface of sporozoites. We hypothesized that atleast some of these fibrillar tethers might be the antigenic

* Corresponding author. Mailing address: Department of Molecularand Cell Biology, Boston University Goldman School of Dental Med-icine, 715 Albany Street, Evans 425, Boston, MA 02118. Phone: (617)414-1054. Fax: (617) 414-1041. E-mail: [email protected].

† Supplemental material for this article may be found at http://ec.asm.org/.

� Published ahead of print on 30 November 2009.

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mucins, which are abundant on the surface of C. parvumsporozoites. To test this hypothesis, we used mass spectros-copy to identify oocyst wall proteins and sporozoite glyco-proteins and used deconvolving and immunoelectron mi-croscopy (immuno-EM) with lectins and anti-C. parvumantibodies to directly label the tethers.

MATERIALS AND METHODS

Preparation of C. parvum oocysts for fluorescence microscopy. C. parvumoocysts (Iowa strain), which were passaged through newborn calves, were pur-chased from Bunch Grass Farm, Dury, ID. Contaminating fecal bacteria wereremoved by two different methods. (i) C. parvum oocysts were enriched bycentrifugation three times at 2,000 � g in cold phosphate-buffered saline (PBS),pH 7.5. The pellet containing the oocysts was resuspended in PBS, applied to a1.2 M cesium chloride cushion, and centrifuged at 16,000 � g for 2 h. By thismethod, the outer veil on the surface of C. parvum oocysts was removed. (ii)Alternatively, contaminating bacteria were lysed by 5 to 10 pulses of sonicationin PBS, and C. parvum oocysts were recovered in the pellet of a 2,000 � g spinfor 10 min. By this method, which was used for fluorescence microscopy andelectron microscopy in Fig. 3 and 6 to 9, the outer veil was left intact. The washwas repeated three times, and oocysts were fixed in 2% paraformaldehyde in PBSfor 10 min at 4°C.

Sporozoites were induced to excyst by incubating oocysts in RPMI 1640 me-dium with 0.75% Na taurocholate (pH 7.5) at 37°C for 30 to 60 min. Sporozoitesand broken oocyst walls were fixed in 2% paraformaldehyde in the presence orabsence of 0.05% Triton X-100 to permeabilize membranes. For labeling the C.parvum surface, unfixed sporozoites were incubated for 60 min on ice in 200 mMcarbonate-bicarbonate buffer (pH 9.3) with 20 �g/ml of the amine-reactive probeAlexa Fluor 610 5-tetrafluorophenyl (5-TFP) (Molecular Probes) (13). AlexaFluor-labeled sporozoites were washed three times with PBS to remove excessfluorochrome, and parasites were fixed in paraformaldehyde before incubationwith lectins or antibodies.

Plant lectins, including Maclura pomifera agglutinin (MPA), Ulex europaeusagglutinin I (UEA1), and Lens culinarus hemagglutinin (LCH) were purchasedfrom EY Labs, Inc. The anti-retroviral, mannose-binding lectin cyanovirin-N(CVN) was a generous gift of Barry O’Keefe of the NCBI, Frederick, MD (2).Five hundred micrograms of each lectin was labeled with Alexa Fluors that haveabsorbance peaks at 488 nm or 610 nm, and unreacted fluorochromes wereremoved by dialysis versus PBS. Fixed oocysts, oocyst walls, and sporozoites wereincubated with 4 �g/ml of each lectin for 1 h at room temperature (RT) in PBSand washed three times in PBS.

Mouse monoclonal antibodies to an N-linked epitope on gp900 (4G12) and toO-linked GalNAc on gp900 and gp40 (4E9), which have been described previ-ously (7, 42), were diluted 1:250 and incubated with C. parvum oocysts, walls,and/or sporozoites for 1 h at RT in PBS. Bovine serum albumin (BSA) (1%) inPBS was used as a blocking reagent. Preparations were washed three times inPBS and then incubated with a secondary goat anti-mouse antibody conjugatedwith Alexa Fluor for 1 h at RT. Control incubation mixtures contained preim-mune mouse sera or no primary mouse antibody. C. parvum parasites werewashed three times with PBS.

Polyclonal mono-specific rabbit antibodies made against recombinant C. par-vum gp40 and against recombinant C. parvum gp15 have been described previ-ously (6, 26). A polyclonal rabbit antibody made against recombinant COWP1,which has been described previously (4), was a generous gift of Joe Crabb,Immucell Corp., Portland, MA. Rabbit antibodies were diluted 1:100 or 1:200and incubated with C. parvum for 1 h at RT, and washed parasites were incu-bated with a secondary goat anti-rabbit antibody conjugated with Alexa Fluordyes. BSA (1%) in PBS was used as a blocking reagent. Control incubationmixtures contained preimmune rabbit sera or no primary rabbit antibody.

Alexa Fluor-stained C. parvum parasites were incubated with 0.1 �g/ml 4�,6�-diamidino-2-phenylindole (DAPI) to stain nuclei (blue), and organisms werevisualized with a DeltaVision deconvolving microscope (Applied Precision, Is-saquah WA) with a channel for each fluorochrome. Images were taken at aprimary magnification of �100 and deconvolved using Applied Precision’s soft-WoRx software.

Transmission electron microscopy (TEM) and immuno-EM of oocysts. ForTEM or immuno-EM, intact oocysts and oocyst walls after excystation wereprepared as described above for fluorescence microscopy. In addition, “soni-cated” oocyst walls were prepared as follows: C. parvum oocysts, which wereseparated from bacteria by cesium chloride centrifugation, were treated with 200pulses (each for 10 s at 500 W) from a probe sonicator (model W250R; Ultra-

sonic, Inc.) in PBS on ice. Oocyst walls were separated from debris by centrifu-gation one or two times through a 60% sucrose cushion in PBS at 14,000 � g for1 h. In addition, some oocyst walls were treated with pronase E (protease XIV;Sigma, St Louis, MO) in the presence of 50 mM sodium bicarbonate (pH 8.5) at37°C for 20 h.

Intact oocysts, excysted walls, and sonicated walls were fixed in 2% paraform-aldehyde and 1% glutaraldehyde buffered by 0.1 M sodium cacodylate (pH 7.3)for �12 hours at 4°C for conventional TEM. Intact oocysts were frozen andthawed three times after fixation to induce an ice artifact that allows fixatives topenetrate through the C. parvum wall (17). C. parvum oocysts were postfixed for�12 h at 4°C in 1% osmium tetroxide in the presence or absence of 0.5 mg/mlruthenium red in cacodylate buffer (23). Water was removed from pellets in agraded series of solutions of ethanols and propylene oxide, and organisms wereembedded in Epon at the Harvard Medical School Electron Microscopy Facility.Ultrathin sections (�60 to 80 nm) were cut on a Reichert Ultracut-S microtome,picked up and placed on copper grids, stained with 1% uranyl acetate and 0.2%lead citrate, and examined in a Tecnai G2 Spirit BioTWIN transmission electronmicroscope. Images were taken with a 2,000 Advanced Microscopy Techniques(AMT) charge-coupled-device (CCD) camera.

For immuno-EM, C. parvum parasites were fixed in 2% paraformaldehyde and0.1% glutaraldehyde, postfixed in ruthenium red without osmium, dehydrated,and embedded in LR White resin. Ultrathin sections were picked up on coppergrids, which were incubated with primary anti-C. parvum rabbit or mouse anti-bodies used for fluorescence microscopy. Primary rabbit antibodies were local-ized using protein A-gold, while primary mouse antibodies were incubated withrabbit anti-mouse antibodies prior to localization with protein A-gold. Controlincubation mixtures omitted the primary mouse antibody.

Mass spectroscopy of purified oocyst walls and of total oocyst proteins puri-fied on MPA affinity column. For mass spectroscopy, oocysts were purified bycesium chloride centrifugation. Oocyst walls were prepared from excysted oo-cysts or sonicated oocysts by being centrifuged twice through a sucrose cushion.A portion of the C. parvum oocyst walls was examined by TEM for purity, and therest of the excysted or sonicated C. parvum oocyst walls were treated with 10 �gof mass spectroscopy-grade trypsin (Promega), which was dissolved in 50 mMacetic acid and then diluted in 100 mM ammonium acetate (final pH 8) for 1 hat 37°C and then 16 h at 4°C. Undigested oocyst wall material was removed bycentrifugation. Tryptic peptides from C. parvum oocyst walls were purified byreverse-phase chromatography on C18 Zip tip (Millipore) or PepClean (Pierce).

To affinity purify C. parvum glycoproteins, we sonicated oocysts, which containwalls and sporozoites, with 150 5-second pulses on ice and extracted proteinswith 0.1% Triton X-100 and EDTA-free Complete protease inhibitor cocktail(Roche). Insoluble material was removed by centrifugation (�12,000 � g), andsoluble proteins were applied to an MPA Sepharose column (EY Laboratories,Inc.) (41). Because there was nonspecific binding to this column, C. parvumglycoproteins were selectively eluted with 100 mM galactose rather than withsodium dodecyl sulfate (SDS). C. parvum glycoproteins were digested in solutionwith sequencing-grade trypsin.

Mass spectroscopy of tryptic peptides from oocyst walls or from MPA affinity-purified oocyst glycoproteins was performed using a nano-high-performanceliquid chromatography (nano-HPLC) pump and autosampler (Surveyor andMicroAS, respectively; Thermo Finnigan, San Jose, CA) on a 10-cm by 100-�minner diameter (ID) Magic C18 reverse-phase capillary column (Michrom, Au-burn, CA) at the Boston University Proteomics Core Facility or at the Massa-chusetts Institute of Technology (MIT) Center for Cancer Research (CCR)Biopolymers Laboratory. Peptides, which were separated using gradients of 2%to 98% acetonitrile over 30 to 200 min in the presence of 0.1% formic acid (48),were analyzed using a LTQ ProteomeX ion trap mass spectrometer (ThermoFinnigan, San Jose, CA).

Mass spectra were compared to tryptic digestions of predicted C. parvumproteins from whole-genome sequencing (1, 47) using SEQUEST. Tryptic pep-tides with a SEQUEST XCorr score of �1.75, 2.5, or 3.5 for charge states �1,�2, and �3, respectively, a peptide probability of �0.01, and a protein proba-bility of �0.001, and proteins with two or more high-scoring tryptic peptides wereconsidered to be present in the sample. An estimate of the relative abundance ofeach protein in a sample was calculated from the area under the peak for allpeptides belonging to that particular protein.

C. parvum proteins, which were identified by mass spectroscopy, were classi-fied as secreted or membrane proteins if they contained either an N-terminalendoplasmic reticulum (ER)-targeting sequence and/or one or more predictedtransmembrane helices (19, 24). Conversely, C. parvum proteins were classifiedas nucleocytosolic if they lacked both N-terminal sequences and transmembranehelices. C. parvum proteins were compared to the conserved domain databaseand to proteins in the NR database at the NCBI (22).

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FIG. 1. Ruthenium red-stained fibrils appear to tether C. parvum sporozoites to the inner surface of oocyst walls. (A and B) Low- magnification(A) and higher-magnification (B) images of intact C. parvum oocysts reveal an intact outer veil (OV), which is separated from the oocyst wall (OW)by an electron-translucent layer. On the inner surface of the oocyst wall, there are dozens of ruthenium red-stained globules (glob). In addition,there are ruthenium red-stained fibrils that appear to act as tethers (Teth) between the surface of the sporozoites (Spz) and the inner aspect ofthe oocyst wall. (C) Low-magnification image of a partially excysted oocyst reveals loss of the outer veil due to purification of oocysts (prior toexcystation) by centrifugation through a cesium chloride cushion. Radially arranged, ruthenium red-stained fibrils or tethers extend between theouter surface of a vacuolated sporozoite and the inner surface of the oocyst wall. (D) At higher magnification, the wall of an excysted oocyst, whichis also missing the outer veil, includes an outer glycocalyx (Glx), an electron-dense bilayer (Bil) surrounding an electron-translucent layer, an innermoderately electron-dense layer (Inn), numerous tethers, and large ruthenium red-stained globules. The glycocalyx on the outer surface of theoocyst wall is visualized only when the outer veil has been removed, suggesting the possibility that the outer veil is preventing staining of theelectron-translucent layer. Bars, 500 nm (A and C) and 100 nm (B and D).

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Analysis of C. parvum oocyst glycoproteins by lectin blots and nucleotide sugartransport assays. Two orthogonal methods were used to characterize the car-bohydrates on the glycoproteins of C. parvum oocysts. First, total oocyst proteinswere obtained by sonicating (200 pulses) C. parvum oocysts in a solution of 0.1%Triton X-100, PBS (pH 7.5), and anti-fungal protease inhibitor cocktail (Roche).Walls, nuclei, and cell debris were removed by centrifugation, and half thesample was digested exhaustively with 2.5 �g of peptide–N-glycanase F (PNGaseF from New England Biolabs) for 6 h at 37°C. C. parvum oocyst proteins, beforeor after PNGase F treatment, were run in parallel on SDS-polyacrylamide gelscontaining a 4 to 20% gradient of acrylamide, blotted onto polyvinylidene diflu-oride (PVDF) membranes, and probed with horseradish peroxidase (HRP) con-jugated to MPA, UEA1, and cyanovirin-N.

Second, sporozoites from excysted oocysts were sonicated briefly in hypotonicbuffer (10 mM HEPES-KOH [pH 7.2], 10 mM MgCl2, 25 mM KCl, and anti-fungal protease inhibitor cocktail [Roche]). C. parvum membranes were isolatedby centrifugation at 100,000 � g for 45 min, and nucleotide sugar transport andtransfer assays were performed with radiolabeled nucleotide sugars as describedpreviously (3). In these assays, nucleotide sugar transport and transfer to glyco-proteins are measured at the same time. The negative control is treatment of themembranes with 0.1% Triton X-100 to make open vesicles, which fail to con-centrate nucleotide sugars (15). The amount of sugar transferred to N-glycanswas determined by treating radiolabeled pellets with PNGase F, while theamount of sugar transferred to O-glycans was determined by treating pellets with0.1 N NaOH (�-elimination).

FIG. 2. TEMs of excysted and sonicated C. parvum oocyst walls. (A) The oocyst wall (OW) of a sonicated oocyst, which has not been purifiedon cesium chloride gradient, contains the outer veil (OV). Some dense globules (Glob) are present on the inner surface of the oocyst wall, but theyare difficult to visualize in the absence of ruthenium red staining. (B and C) The outer veil is absent from the walls of an excysted C. parvum (B) anda sonicated C. parvum (C), which were purified using centrifugation through a cesium chloride cushion. The fibrillar tethers (Teth) on the innersurface of the oocyst wall are long and irregular in their shape (B). (D) These fibrils and the moderately electron-dense layer on the inside of theoocyst wall (Inn) are removed by protease treatment of the oocyst walls. After protease treatment, the rigid double bilayer (Bil) is all that remainsof oocyst walls. Bars, 100 nm (A and B) and 250 nm (C and D).

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RESULTS AND DISCUSSION

Fibrillar tethers extend in a radial manner from the innersurface of the oocyst wall to the surface of C. parvum sporo-zoites. Ruthenium red, which is a polycationic dye, has previ-ously been used to demonstrate a glycocalyx on the surfaces of

C. parvum oocysts and sporozoites (23). Here we used ruthe-nium red to reveal an abundant set of fibrils, which extendradially from the inner surface of the oocyst wall to the outersurface of sporozoites (Fig. 1). These ruthenium red-stainedfibrils, which are unbranched, were best seen when sporozoitesin hypertonic fixative pull away from the intact oocyst wall orwhen sporozoites are in the process of excystation, suggestingthe possibility that the fibrils are exaggerated by the method ofpreparing C. parvum oocysts for TEM. However, we do notbelieve that the ruthenium red-stained fibrils are a simpleartifact of fixation. These ruthenium red-stained fibrils areattached to a moderately electron-dense inner layer of theoocyst wall, where COWP has been localized by immuno-EM(33). When the ruthenium red-stained fibrils are no longer

FIG. 3. (Set A) COWP1, which is present on the inner surface of the oocyst wall (stained red with MPA), is accessible to anti-COWP1antibodies (green) only after oocyst walls are broken. In contrast, C. parvum walls of intact oocysts (labeled intact) are not stained withanti-COWP1 antibodies. (B) Excysted, fixed, and permeabilized sporozoites have a surprisingly large number of vesicles that stain greenwith anti-COWP1 antibodies. Nuclei in panels B and D are stained blue with DAPI. Fucose-containing material, which is stained green with theplant lectin UEA1 in the Set C panels, is also present on the inner surface of C. parvum oocyst walls stained red with MPA. (D) MPA (green) andUEA1 (red) label nonoverlapping vesicles in excysted C. parvum sporozoites. Bars, 2.5 �m (Sets A and C), 1 �m (B), and 0.5 �m (D).

FIG. 4. (A) Blotting shows that the GalNAc-binding lectin MPAbinds to numerous C. parvum glycoproteins, and this binding is notaffected by PNGase F treatment. (B) The fucose-binding lectin UEA1also binds to numerous C. parvum glycoproteins, some of which areaffected by PNGase F treatment. (C) In contrast, the mannose-bind-ing, anti-retroviral lectin cyanovirin-N no longer binds to C. parvumglycoproteins treated with PNGase F. The structure of the predictedN-glycan of C. parvum, which binds cyanovirin-N, is Man5GlcNAc2(after removal of a terminal Glc) (29). The positions of molecular sizemarkers (in kilodaltons) are shown to the left of the blot in panel A.

TABLE 1. Cryptosporidium oocyst wall proteins identified bymass spectroscopy

Proteina CryptoDB geneidentification

%Abundanceb

No. ofpeptides/expt

Likely oocyst wall proteinsCOWP1 cgd6_2090 55 21–43COWP8 cgd6_200 13 5–9COWP6 cgd4_3090 8 2–13COWP3 cgd4_6070 tr 6COWP4 cgd8_3350 tr 3COWP2 cgd7_1800 tr 2gp40/gp15 cgd6_1080 0.8 2

Possible oocyst wall proteinsthat are unique toC. parvum

CpPOWP1 cgd2_490 4 2–4CpPOWP2 cgd2_850 2 2–3CpPOWP3 cgd3_1540 1 2–3CpPOWP4 cgd2_2510 2 3–12CpPOWP5 cgd8_2670 1 2–4

Proteins also found inapicomplexa withoutoocyst walls

FNPA cgd7_4810 2 2–6CCP2 cgd7_1730 3 2–11CCP3 cgd7_300 3 2–9CCP1 cgd2_790 tr 1–4

Chaperones present in mosteukaryotes

HSP70 cgd2_20 2 6HSP90 cgd3_3770 1 1–5PDI cgd1_800 1 2–3

a COWP1 to COWP8 are Cryptosporidium oocyst wall proteins. CpPOWP1to CpPOWP5 are possible oocyst wall proteins in Cryptosporidium parvum. Thesequences of the CpPOWPs are shown in Fig. S1 in the supplemental material.

b The average values for six experiments (three excysted walls and three son-icated walls, which were essentially the same) are shown. The relative abundanceof each protein was determined by areas of the peptide peaks and is crudelyestimated. There were 5% to 10% nucleocystolic contaminants. tr, trace.

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attached to sporozoites, the fibrils appear to collapse into elec-tron-dense globules, which resemble “knobs” stained by anti-C.parvum antibodies (11). Because these ruthenium red-stainedfibrils appear to attach the sporozoites to the inner surface ofthe oocyst wall, we will refer to them as “tethers.”

Ruthenium red also stained a narrow band, which is part ofthe outer veil on the outermost aspect of the C. parvum oocystwall (Fig. 1 and 2). The C. parvum outer veil, which has thesame appearance as that present on the surface of Toxoplasmaoocysts, also contains an electron-translucent layer (10). TheC. parvum outer veil is present on intact oocysts and on oocystwalls after excystation, but the outer veil is removed if oocystsare treated with bleach (27) or if the oocysts or walls arepurified on CsCl2 gradients (observed here).

COWP1 is by far the most abundant protein of purified C.parvum oocyst walls. In order to characterize the proteins in theC. parvum oocyst wall by mass spectroscopy, we allowed sporo-zoites to excyst and purified excysted walls, which have character-istic curled ends at the site of the fracture (Fig. 2). Alternatively,we sonicated oocysts and purified walls, which curl into scrolls.

Regardless of the method, the outer veil and numerous fibrillartethers were lost from oocyst walls during the purification onCsCl2 gradients. Treatment of the purified oocyst walls with tryp-sin, which removed all layers of the wall except for the rigiddouble bilayer, did not change their curled ends after excystationor their scroll-like appearance after sonication.

By far the most abundant protein identified by mass spec-troscopy from either excysted or sonicated C. parvum oocystwalls is COWP1, which makes up greater than 50% of theidentified peptides (Table 1). This result is consistent withCOWP1 being the most antigenic and the first C. parvum wallprotein that was molecularly characterized (20, 28, 33, 37, 44).COWP6 and COWP8 are also relatively abundant in oocystwalls. COWP2, COWP3, and COWP4 are present in traceamounts, while COWP5, COWP7, and COWP9 were not de-tected. Together, COWPs, which are also present in the pre-dicted proteome of Toxoplasma (37), account for �75% of theC. parvum oocyst wall proteins identified by mass spectroscopy.The relative abundance of C. parvum oocyst wall proteins is inagreement with recent mass spectroscopic analysis of totaloocyst proteins, which include proteins from oocyst walls andsporozoites (30).

The mucin-like glycopeptide gp40/gp15 (7, 26), which local-izes to the C. parvum sporozoite surface and to the innersurface of oocyst walls (see below), was a minor component ofthe oocyst walls by mass spectroscopy. Five other unique C.parvum proteins, which were present in much smalleramounts in purified C. parvum oocyst walls, are referred toas possible oocyst wall proteins (POWPs) (Table 1) (see Fig.S1 in the supplemental material). Possible oocyst wall pro-tein 1 (POWP1), POWP2, and POWP3 lack any conserveddomains and do not have homologues in other organisms.POWP3 is mucin-like in that it has a serine- and threonine-richrepeat near its C terminus. POWP4 and POWP5, which showa 20% amino acid identity with each other, each contain aglucose-methanol-choline (GMC) oxidoreductase domain thatis also present in plant, fungal, and bacterial enzymes (e.g.,cellobiose dehydrogenase) (22).

For two reasons, CCP1, CCP2, and CCP3, as well as FNPA,which has a carbohydrate-binding domain (38), (gene names inCryptoDB) were likely contaminants in C. parvum oocyst wall

FIG. 5. (A) Membranes isolated from C. parvum sporozoitestransport UDP-GalNAc, UDP-Gal, GDP-Fuc, and GDP-Man butfail to transport UDP-Glc, UDP-glucuronic acid (UDP-GlcA), andUDP-GlcNAc. Negative controls are membranes permeabilizedwith detergent, which fail to concentrate nucleotide sugars. (B) Thesugars (UDP-GalNAc, UDP-Gal, GDP-Fuc, and GDP-Man), whichare transported by C. parvum membranes, are transferred to O-linked glycans, which are released with strong base. In contrast, onlyGDP-Fuc and GDP-Man are transferred to N-glycans, which arereleased with PNGase F.

TABLE 2. MPA-enriched oocyst proteins identified bymass spectroscopy

Proteina CryptoDB geneidentication

%Abundanceb

No. ofpeptides/expt

gp40/gp15 cgd6_1080 28 5–25gp900 cgd7_4020 14 7–55CpMPA1 cgd1_660 15 6–22CpMPA2 cgd3_3370 14 2–53CpMPA3 cdg7_400 8 3–54CpMPA4 cgd4_3550 3 1–36CpMPA5 cdg4_3530 0.6 1–28CpMPA6 cdg3_1540 tr 2–10Gal lectin cdg2_430 2 2–12CCP2 cgd7_1730 3 1–2CCP3 cgd7_300 tr 1–2

a The sequences of the CpMPAs are shown in Fig. S4 in the supplementalmaterial.

b The average values for three independent experiments are shown. Therewere 10% to 20% nucleocystolic contaminants. tr, trace.

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samples. First and most important, these C. parvum proteinsare also present in all apicomplexa, including Plasmodium andTheileria, which are transmitted by insect vectors and so do notmake an oocyst wall. Second, these glycoproteins were abun-

dant in MPA affinity-purified proteins of C. parvum (below)and/or in mass spectroscopic analyses of sporozoite proteins(30, 32).

Proteins associated with N-glycan-independent quality con-

FIG. 6. (Set A and panels B and C) A monoclonal antibody (Ab) to O-linked GalNAc on gp40 and gp900 (4E9 labeled green) binds to the innersurface of broken oocyst walls (Set A), the surface of intact sporozoites (B), and to vesicles within permeabilized sporozoites (C). The brokenoocyst wall in Set A panels is stained red with the lectin WGA. Nuclei in panel C are stained blue with DAPI. (D) Immuno-EM shows proteinA-gold binding to 4E9 antibody, which reacts with globules (Glob) on the inner surface of the oocyst walls (OW) and to small electron-densevesicles (arrows) in sporozoites. Bars, 2 �m (Set A), 1 �m (B and C), and 200 nm (D).

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trol of protein folding in the ER, which include two chaperones(Hsp70 and Hsp90) and a protein disulfide isomerase, werealso likely contaminants of the C. parvum oocyst wall prepa-rations (Table 1). Similarly, nucleocytosolic proteins identifiedby mass spectroscopy were likely contaminants of the wallpreparations.

The GalNAc-binding lectin (MPA) binds to oocyst walls andto peptide–N-glycanase-resistant carbohydrates on oocyst gly-coproteins. The goal here was to determine whether lectinaffinity might be used to identify other oocyst wall glycopro-teins, which were not identified by mass spectroscopy of cesiumchloride-purified C. parvum walls. Both intact and fracturedoocyst walls of C. parvum densely stain with the GalNAc-binding lectin Maclura pomifera agglutinin (MPA) (Fig. 3).MPA also binds to the surface of excysted sporozoites andagglutinates them (7, 35, 43). MPA binds to vesicles inpermeabilized sporozoites, which are distinct from thoselabeled with the fucose-binding lectin Ulex europaeus agglu-tinin I (UEA1) (Fig. 3). MPA-labeled vesicles are also dis-tinct from those labeled with the anti-retroviral lectin cya-

novirin-N, which binds to high-mannose N-glycans of C.parvum (see Fig. S2 in the supplemental material) (2, 29).Cyanovirin-N binds weakly or not at all to C. parvum oocystwalls (data not shown).

Antibodies to COWP1 do not bind to the outer surface ofintact oocysts but bind to the inner surface of broken oocyst walls(Fig. 3). This result is consistent with the localization of COWP1to the inner, moderately electron-dense layer of the oocyst wall(33). COWP1 is for the most part absent from the surface ofsporozoites but is abundant within vesicles of permeabilizedsporozoites (Fig. 3). This result suggests that sporozoites retainsome COWP1 within secretory vesicles (the most likely explana-tion) or have phagocytosed COWP1 (the less likely explanation).

The fucose-binding lectin UEA1 binds to broken oocysts,but not intact oocysts (data not shown), suggesting its localiza-tion on the inside oocyst walls (Fig. 3). A second fucose-binding lectin, Lens culinarus hemagglutinin (LCH), binds tovesicles in permeabilized sporozoites, which are distinct fromthose labeled with cyanovirin-N (see Fig. S3 in the supplemen-tal material).

FIG. 7. (Set A and panel B) A monoclonal antibody (Ab) to gp900 (4G12 labeled green) binds to the inner surface of broken oocyst walls (SetA) and to the outer surface of a sporozoite (B). Oocyst walls are stained red with WGA, and the 4G12 antibody does not bind to intact oocysts.(C) Immuno-EM shows protein A-gold binding to anti-gp900 antibodies, which react with tethers (arrows) on the inner surface of the oocyst walls(OW) and to large, electron-translucent vesicles (ETV) in sporozoites (Spz). Bars, 2.5 �m (Set A), 1 �m (B), and 250 nm (C).

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The binding of MPA to blots of total C. parvum oocystproteins is not affected by treatment with peptide–N-glycanase(PNGase F) (Fig. 4). In contrast, the binding of UEA1 to blotsof C. parvum oocyst proteins is affected by PNGase F, althoughit is not an all-or-nothing effect, as was the case with cyanovi-rin-N. The blotting results and the nucleotide sugar transportassays (next section) strongly suggest that the GalNac-bindinglectin MPA is binding to O-linked glycans, while the fucose-binding lectin UEA1 is likely binding to both N- and O-linkedglycans.

C. parvum membranes have nucleotide sugar transportersfor GDP-fucose, GDP-mannose, UDP-galactose, and UDP-GalNAc. O-linked glycans, as well as complex N-linked glycans,are made in the Golgi apparatus using nucleotide sugars thatare transported from the cytosol (15). Membranes of C. par-vum sporozoites have nucleotide sugar transporter activitiesfor UDP-GalNAc, UDP-galactose, GDP-fucose, and GDP-mannose (Fig. 5). Treatment of the radiolabeled glycoproteinswith PNGase F and strong base to release N-glycans and O-glycans, respectively, suggest that GalNAc and galactose aretransferred for the most part to O-linked glycans, while fucose

and mannose may be transferred to both N- and O-linkedglycans (Fig. 5).

These results are consistent with the binding of the mono-clonal antibody 4E9 and other GalNAc-binding lectins, such asMPA to O-linked GalNAc on the surface of C. parvum sporo-zoites (7). These results suggest the use of MPA affinity chro-matography and mass spectroscopy to identify candidate gly-coproteins, which might be part of the tethers that connect theouter surface of sporozoites to the inner surface of the oocystwalls (next section).

MPA affinity-purified oocyst glycoproteins include numer-ous mucin-like glycoproteins. In the absence of any purifica-tion step, many of the proteins identified by mass spectroscopyfrom lysed C. parvum oocysts, which include oocyst walls andsporozoites, are nucleocytosolic, while secreted proteins areless abundant (our data not shown) (30, 32). In contrast, thevast majority of MPA affinity-purified proteins from C. parvumoocysts are secreted glycoproteins, which contain Ser- or Thr-rich repeats (gp900, gp40/15, and C. parvum MPA affinity-purified glycoprotein 1 [CpMPA1], CpMPA4, and CpMPA6)or are relatively rich in Ser and Thr (CpMPA2, CpMPA3, and

FIG. 8. (Set A and panels B and C) Polyclonal, mono-specific antibodies to recombinant gp40 (green) bind to the inner surface of oocyst walls(Set A), the surface of intact sporozoites (B), and vesicles within permeabilized sporozoites (C). The walls of C. parvum oocysts are labeled redwith MPA (Set A), while the nucleus in panel C is stained blue with DAPI. (D) Immuno-EM shows protein A-gold binding to anti-GP40 antibodies,which react with tethers (arrows labeled Teth) on the inner surface of the oocyst walls (OW). The outer veil (OV) is not labeled. Panels B andC are shown at the same magnification. Bars, 2 �m (Set A), 1 �m (C), and 200 nm (D).

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CpMPA6) (Table 2) (see Fig. S4 in the supplemental material)(4, 7). Again this result is consistent with the presence ofO-linked GalNAc on C. parvum glycoproteins (7).

MPA affinity-purified glycoproteins (CpMPA1 to CpMPA6),as well as gp40/gp15 and gp900, are unique C. parvum proteinsthat do not have conserved domains with the exception ofCpMPA2, which has 12 KAZAL domains. KAZAL domainsare small Cys-rich domains, which are present within manyextracellular matrix proteins and trypsin inhibitors of metazoabut are absent from fungi, plants, and other protists (22).

Other glycoproteins identified by MPA affinity chromatogra-phy and mass spectroscopy include a Gal/GalNAc-binding lec-tin (5), which binds to gp40 and gp900, and CpCCP2 andCpCCP3 that are likely contaminants in oocyst wall prepara-tions (see above).

COWPs, which are abundant in oocyst wall preparations(above), are absent from MPA affinity preparations, consistentwith the absence of mucin-like domains in COWPs (28). ERchaperones and protein disulfide isomerases, which are alsopresent in oocyst wall preparations, are absent from MPA

FIG. 9. (Set A) Polyclonal, mono-specific antibodies to recombinant gp15 (green) bind to the exterior of intact oocysts, where oocyst walls arelabeled red with WGA. (Set B) Antibodies to gp15 (green) agglutinate intact C. parvum sporozoites, which are labeled red on their surface withAlexa Fluor dye. (C) Immuno-EM shows protein A-gold binding to anti-gp15 antibodies, which react with the outer veil (OV) on the exterior ofoocyst walls but do not react with the outer wall (OW) or sporozoites (Spz). Bars, 2.5 �m (Set A), 1 �m (Set B), and 250 nm (C).

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affinity-purified proteins, presumably because O-linked Gal-NAc is added in the Golgi apparatus rather than the ER.

The MPA affinity-purified glycoproteins from C. parvumoocysts are much less complex than glycoproteins purifiedby lectin affinity methods from Giardia, Entamoeba, andTrichomonas (our unpublished data). Three explanations mayexplain this result. First, it is possible that a terminal fucose onC. parvum glycoproteins blocks binding of MPA to O-linkedGalNAc, as suggested by the distinct binding patterns of MPAand UEA1 in Fig. 3. Second, the MPA affinity methods usedhere may detect only C. parvum glycoproteins, which containSer- and Thr-rich repeats (so-called mucins) or are rich in Serand Thr (supported by the results in Table 2). Third, we mayonly be detecting the most abundant C. parvum glycoproteins,which include gp900 and gp40/gp15 (30, 32). Indeed otherrecently characterized C. parvum mucins (C. parvum mucin 4[CpMuc4] and CpMuc5), which are important for infectivity,are absent from the MPA affinity-purified proteins (25).

Antibodies to some C. parvum sporozoite mucins bind to theinner oocyst wall. The hypothesis tested here is that some C.parvum mucins, which are present on the surface of C. parvumsporozoites by indirect immunofluorescence (4, 28), are also com-ponents of the fibrillar tethers that attach sporozoites to the innersurface of the oocyst wall. In support of this idea, the 4E9 mono-clonal antibody (7) binds to O-linked GalNAc on the surface of C.parvum sporozoites and within small vesicles within permeabil-ized sporozoites (Fig. 6). The 4E9 antibody also binds in a punc-tate manner to the inside C. parvum oocyst walls. By immuno-EM, the 4E9 antibody bound to globules on the inside of theoocyst wall and to small vesicles within sporozoites.

Also in support of this idea, 4G12 monoclonal antibodies tothe mucin gp900 bind to large vesicles within sporozoites andbind in a punctate manner to the inner surface of the oocystwall (Fig. 7) (43). By immuno-EM, 4G12 antibodies to gp900bind to fibrillar tethers and to large relatively electron-trans-lucent vesicles within sporozoites.

Similarly, mono-specific antibodies to the recombinant gp40(7) densely stain the surface of intact sporozoites and bind torelatively large vesicles within permeabilized sporozoites (Fig.8). Anti-gp40 antibodies also bind in a punctate manner to theinner aspect of broken oocyst walls and bind to fibrillar tetherson the inner aspect of the oocyst wall (Fig. 8). Because we donot have antibodies to MPA1 to MPA6, we were unable todetermine whether these C. parvum glycoproteins contributeto fibrillar tethers.

An exception to this idea is the binding of antibodies togp15, which is cleaved from a precursor protein that also con-tains gp40 (6, 26, 42). Antibodies to gp15, which bind to theiranterior ends and agglutinate the sporozoites, also bind to thesurface of intact and broken oocysts (Fig. 9). By TEM, anti-gp15 antibodies bind to the outer veil, which is removed withhigh salt.

A revised model for the Cryptosporidium oocyst wall includesthe fibrillar tethers. Starting from the outside and workinginwards (Fig. 10), the outer veil, which is labile to bleach,cesium chloride, and protease, contains gp15 and gp40 (6, 18,26, 42). The inner, electron-translucent layer of the outer veil,as well as the rigid protease-resistant bilayer, remains for themost part uncharacterized. The weakly glycosylated, protease-sensitive, moderately electron-dense inner layer of the wallcontains COWPs, as previously shown (33). The fibrillartethers, which appear to connect the sporozoite to the oo-cyst wall, collapse into electron-dense globules that havealso been called “knobs” (11). Antibodies to two mucinsbind to the tethers (anti-gp900 and anti-gp40), while anti-bodies to O-linked GalNAc bind to globules.

This model does not explain how the various proteins andcarbohydrates are assembled in the C. parvum oocyst wall, andthis model does not invalidate these mucins as vaccine candi-dates against C. parvum. The relatively simple protein compo-sition of the C. parvum oocyst wall (composed mostly ofCOWPs) is similar to that of Entamoeba cyst walls, which

FIG. 10. A revised model for the C. parvum oocyst walls includes fibrillar tethers, which appear to attach sporozoites to the inner surface ofthe oocyst wall. The outer veil (khaki) stains with anti-gp15 antibodies, while the rigid protease-resistant bilayer (blue-green) remains uncharac-terized for the most part. The weakly glycosylated, protease-sensitive, moderately electron-dense inner layer of the wall (dark blue) containsCOWPs, as previously shown (33). The fibrillar tethers (red), which appear to connect the sporozoite to the oocyst wall, include two mucins (gp900and gp40). These fibrils likely collapse into electron-dense globules, which have also been called “knobs” (11) that stain with antibodies to O-linkedGalNAc.

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contain just three types of chitin-binding lectins, or to that ofGiardia cyst walls, which contain a small set of Leu-rich repeatproteins (called CWPs) (36, 40). In contrast, fungal and plantwalls are much more complex than those of C. parvum oocysts(9, 16).

This model provides a novel tethering function for mucins inC. parvum oocysts, so that sporozoites are not “loose” withinoocyst walls. Because we were unable to separate oocyst wallsfrom sporozoites without removing the outer veil and most ofthe fibrillar tethers from the walls, the glycoprotein composi-tion of the outer veil and fibrillar tethers is limited to thoseantigens, for which we have monoclonal antibodies or mono-specific polyclonal antibodies. We expect that as more antibod-ies become available (particularly to CpMPA1 to CpMPA6 orto C. parvum POWP1 [CpPOWP1] to CpPOWP5) or as bettermethods for purifying C. parvum oocyst walls are developed,other components of the outer veil, oocyst wall, and fibrillartethers will be identified.

ACKNOWLEDGMENTS

This work was supported in part by NIH grants AI44070 (J.S.),GM31318 (P.W.R.), AI52786 (H.D.W.), and DK62816 (R.M.O.).

Thanks to Maria Ericsson for expert work with electron microscopyand Dick Cook for help with mass spectroscopy. Thanks to Joe Crabbfor use of the anti-CWP1 monoclonal antibody.

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