Stable expression of mucin glycoproteins GP40 and GP15 of Cryptosporidium parvum in Toxoplasma gondii

Cryptosporidium spp. are common protozoa causing diarrhea in humans and animals. There are currently only one FDA-approved drug and no vaccines for cryptosporidiosis, largely due to the limited knowledge of the molecular mechanisms involved in the invasion of the pathogens. Previous studies have shown that GP60, which is cleaved into GP40 and GP15 after expression, is an immunodominant mucin protein involved in the invasion of Cryptosporidium. The protein is highly O-glycosylated, and recombinant proteins expressed in prokaryotic systems are non-functional. Therefore, few studies have investigated the function of GP40 and GP15.

The establishment of a robust and efficient heterologous expression system of GP40 in T. gondii represents a novel approach and concept for investigating Cryptosporidium mucins, overcoming the limitations of previous studies that relied on unstable transient transfection, which involved complex steps and high costs.

To obtain recombinant GP40 with correct post-translational modifications, we used CRISPR/Cas9 technology to insert GP40 and GP15 into the UPRT locus of Toxoplasma gondii, allowing heterologous expression of Cryptosporidium proteins. In addition, the Twin-Strep tag was inserted after GP40 for efficient purification of GP40.

Western blotting and immunofluorescent microscopic analyses both indicated that GP40 and GP15 were stably expressed in T. gondii mutants. GP40 localized not only in the cytoplasm of tachyzoites but also in the parasitophorous vacuoles, while GP15 without the GPI anchor was expressed only in the cytoplasm. In addition, a large amount of recTgGP40 was purified using Strep-TactinXT supported by a visible band of ~ 50 kDa in SDS-PAGE. Conclusions: The establishment of a robust and efficient heterologous expression system of GP40 in T. gondii represents a novel approach and concept for investigating Cryptosporidium mucins, overcoming the limitations of previous studies that relied on unstable transient transfection, which involved complex steps and high costs.