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.

Strategies To Discover the Structural Components of Cyst and Oocyst Walls

“…The rigidity of the Cryptosporidium oocyst wall is shown by the uniform, oval shape of intact oocysts, the cup-like appearance of excysted walls, and the tight scrolls formed by mechanically broken walls ( Fig. 2K) (10). Atomic force microscopy shows that the oocyst wall resembles common plastic materials (23).…”

“…In contrast, oocyst walls of Toxoplasma and Cryptosporidium, as well as walls of nematode eggs, have multiple layers (Fig. 2G) (10,16). Since different structural components will likely bind different proteins, one can expect that the protein composition of oocyst walls is much more complicated than that of cyst walls.…”

“…Monoclonal antibodies also identified Ser-and Thr-rich glycoproteins which are teth- ers that attach to the surface of sporozoites on the interior of the Cryptosporidium oocyst wall (Fig. 2G) (10,28).…”

“…These O-linked glycans, which are not stage specific, can be detected with the plant lectin MPA, which binds avidly to oocyst and sporocyst walls (9,10). Finally, Asn-linked glycans (N-glycans) of coccidia contain 7 to 10 sugars (Glc 0 -3 Man 5 GlcNAc 2 ) (47).…”

“…Proteins and sugars are the major components of cyst walls of Entamoeba and Giardia, which cause amebic dysentery and diarrhea, respectively (6)(7)(8). In addition to proteins and carbohydrates, lipids are important structural components of oocyst walls of Cryptosporidium and Toxoplasma, which cause diarrhea and disseminated infections, respectively (9)(10)(11).…”