Evidence for mucin-like glycoproteins that tether sporozoites of Cryptosporidium parvum to the inner surface of the oocyst wall

Abstract

Cryptosporidium parvum oocysts, which are spread by the fecal-oral route, have a single, multilayered wall that surrounds four sporozoites, the invasive form. The C. parvum oocyst wall is labeled by the Maclura pomifera agglutinin (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. parvum sporozoites have on their surface multiple mucin-like glycoproteins with Ser- and Thr-rich repeats (e.g., gp40 and gp900). Here we used ruthenium red staining and electron microscopy to demonstrate fibrils, which appear to attach or tether sporozoites to the inner surface of the C. parvum oocyst wall. When disconnected from the sporozoites, 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, were COWP1, COWP6, and COWP8, while COWP2, COWP3, and COWP4 were present in trace amounts. In contrast, MPA affinity-purified glycoproteins from C. parvum oocysts, which are composed of walls and sporozoites, included previously identified mucin-like glycoproteins, a GalNAc-binding lectin, a Ser protease inhibitor, and several novel glycoproteins (C. parvum MPA affinity-purified glycoprotein 1 [CpMPA1] to CpMPA4). By immunoelectron microscopy (immuno-EM), we localized mucin-like glycoproteins (gp40 and gp900) to the ruthenium red-stained fibrils on the inner surface wall of oocysts, while antibodies to the O-linked GalNAc 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 sporozoites to the inner surface of oocyst walls.

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 of the oocyst wall. (C) Low-magnification image of a partially excysted oocyst reveals loss of the outer veil due to purification of oocysts (prior to excystation) by centrifugation through a cesium chloride cushion. Radially arranged, ruthenium red-stained fibrils or tethers extend between the outer surface of a vacuolated sporozoite and the inner surface of the oocyst wall. (D) At higher magnification, the wall of an excysted oocyst, which is also missing the outer veil, includes an outer glycocalyx (Glx), an electron-dense bilayer (Bil) surrounding an electron-translucent layer, an inner moderately electron-dense layer (Inn), numerous tethers, and large ruthenium red-stained globules. The glycocalyx on the outer surface of the oocyst wall is visualized only when the outer veil has been removed, suggesting the possibility that the outer veil is preventing staining of the electron-translucent layer. Bars, 500 nm (A and C) and 100 nm (B and D).

FIG. 2.

TEMs of excysted and sonicated C. parvum oocyst walls. (A) The oocyst wall (OW) of a sonicated oocyst, which has not been purified on cesium chloride gradient, contains the outer veil (OV). Some dense globules (Glob) are present on the inner surface of the oocyst wall, but they are 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) and a sonicated C. parvum (C), which were purified using centrifugation through a cesium chloride cushion. The fibrillar tethers (Teth) on the inner surface 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 the oocyst wall (Inn) are removed by protease treatment of the oocyst walls. After protease treatment, the rigid double bilayer (Bil) is all that remains of oocyst walls. Bars, 100 nm (A and B) and 250 nm (C and D).

FIG. 3.

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

FIG. 4.

(A) Blotting shows that the GalNAc-binding lectin MPA binds to numerous C. parvum glycoproteins, and this binding is not affected by PNGase F treatment. (B) The fucose-binding lectin UEA1 also binds to numerous C. parvum glycoproteins, some of which are affected by PNGase F treatment. (C) In contrast, the mannose-binding, anti-retroviral lectin cyanovirin-N no longer binds to C. parvum glycoproteins treated with PNGase F. The structure of the predicted N-glycan of C. parvum, which binds cyanovirin-N, is Man₅GlcNAc₂ (after removal of a terminal Glc) (29). The positions of molecular size markers (in kilodaltons) are shown to the left of the blot in panel A.

FIG. 5.

(A) Membranes isolated from C. parvum sporozoites transport UDP-GalNAc, UDP-Gal, GDP-Fuc, and GDP-Man but fail to transport UDP-Glc, UDP-glucuronic acid (UDP-GlcA), and UDP-GlcNAc. Negative controls are membranes permeabilized with detergent, which fail to concentrate nucleotide sugars. (B) The sugars (UDP-GalNAc, UDP-Gal, GDP-Fuc, and GDP-Man), which are transported by C. parvum membranes, are transferred to O-linked glycans, which are released with strong base. In contrast, only GDP-Fuc and GDP-Man are transferred to N-glycans, which are released with PNGase F.

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 inner surface of broken oocyst walls (Set A), the surface of intact sporozoites (B), and to vesicles within permeabilized sporozoites (C). The broken oocyst 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 protein A-gold binding to 4E9 antibody, which reacts with globules (Glob) on the inner surface of the oocyst walls (OW) and to small electron-dense vesicles (arrows) in sporozoites. Bars, 2 μm (Set A), 1 μm (B and C), and 200 nm (D).

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 (Set A) 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 μm (Set A), 1 μm (B), and 250 nm (C).

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 red with 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 and C are shown at the same magnification. Bars, 2 μm (Set A), 1 μm (C), and 200 nm (D).

FIG. 9.

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

FIG. 10.

A revised model for the C. parvum oocyst walls includes fibrillar tethers, which appear to attach sporozoites to the inner surface of the oocyst wall. The outer veil (khaki) stains with anti-gp15 antibodies, while the rigid protease-resistant bilayer (blue-green) remains uncharacterized for the most part. The weakly glycosylated, protease-sensitive, moderately electron-dense inner layer of the wall (dark blue) contains COWPs, as previously shown (33). The fibrillar tethers (red), which appear to connect the sporozoite to the oocyst wall, include two mucins (gp900 and gp40). These fibrils likely collapse into electron-dense globules, which have also been called “knobs” (11) that stain with antibodies to O-linked GalNAc.