Biochemical and functional characterization of CpMuc4, a Cryptosporidium surface antigen that binds to host epithelial cells

Abstract

Cryptosporidium spp. are intracellular apicomplexan parasites that cause outbreaks of waterborne diarrheal disease worldwide. Previous studies had identified a C. parvum sporozoite antigen, CpMuc4, that appeared to be involved in attachment and invasion of the parasite into intestinal epithelial cells. CpMuc4 is predicted to be O- and N-glycosylated and the antigen exhibits an apparent molecular weight 10kDa larger than the antigen expressed in E. coli, indicative of post-translational modifications. However, lectin blotting and enzymatic and chemical deglycosylation did not identify any glycans on the native antigen. Expression of CpMuc4 in T. gondii produced a recombinant protein of a similar molecular weight to the native antigen. Both purified native CpMuc4 and T. gondii recombinant CpMuc4, but not CpMuc4 expressed in E. coli, bind to fixed Caco-2A cells in a dose dependent and saturable manner, suggesting that this antigen bears epitopes that bind to a host cell receptor, and that the T. gondii recombinant CpMuc4 functionally mimics the native antigen. Binding of native CpMuc4 to Caco2A cells could not be inhibited with excess CpMuc4 peptide, or an excess of E. coli recombinant CpMuc4. These data suggest that CpMuc4 interacts directly with a host cell receptor and that post-translational modifications are necessary for the antigen to bind to the host cell receptor. T. gondii recombinant CpMuc4 may mimic the native antigen well enough to serve as a useful tool for identifying the host cell receptor and determining the role of native CpMuc4 in host cell invasion.

Immunolocation and enzyme activity analysis of Cryptosporidium parvum enolase

“…Immunolocalization of enolase in C. parvum oocysts was analysed as previously described by Paluszynski et al [ 27 ] with minor modifications. Briefly, 50 μl of the purified oocysts (approximately 10 5 oocysts/ml) were added to pre-cleaned glass slides and air-dried.…”

Genomic reconstruction and features of glycosylation pathways in the apicomplexan Cryptosporidium parasites

“…The importance of glycoproteins in Cryptosporidium has been indicated by their presence in all stages of the parasite, including the oocyst walls (e.g., the cryptosporidial oocyst wall proteins (COWPs) and molecules tethering sporozoites on the inner side of the oocyst wall), the surface of sporozoites (e.g., gp40/gp15, CpMuc4) and the PVM (recognizable by certain lectins such as VVL) ( Petersen et al, 1992 ; Winter et al, 2000 ; Priest et al, 2001 ; Wanyiri and Ward, 2006 ; Paluszynski et al, 2014 ; Haserick et al, 2017a ; Haserick et al, 2017b ; Tandel et al, 2019 ; Li et al, 2022 ). Structural glycoproteins play protective and lubricative roles, such as those in the oocyst walls and those on the plasma membranes of zoites and PVM.…”