Abstract
The apicomplexan parasites, Cryptosporidium spp. are a global health threat, with devastating effects for children under five years of age, particularly in developing nations. Cryptosporidium spp. is also an important cause of disease in neonatal calves and lambs, leading to poor health of animals and economic loss for farms. Despite its clinical and economic importance, research into Cryptosporidium spp. remains neglected which is further hindered by the lack of a reproducible continuous in vitro culture model. As a result, there are only two approved drugs for the disease, both of which have limited efficacy against vulnerable populations, including children and the immunocompromised.
Firstly, this research has investigated the published COLO-680N host cell monolayer model for its ability to propagate a continuous culture of Cryptosporidium parvum. Although we detected some growth of the parasite in this culture, this was not at the level of the original publications and optimisation with various media supplements failed to improve the culture. Furthermore, lack of expression of dmc1 a known marker of sexual cycle progression in the parasite indicated that the culture did not support the switch to the sexual cycle of the parasite. Consequently, we investigated other mammalian cell lines, both adherent and suspension, for their ability to grow the parasite. One cell line, CT26 was identified as a potential candidate, however it was not able to produce parasites in high numbers as with the gold-standard discontinuous HCT-8 culture.
Additionally, we investigated the ability of Cryptosporidium parvum to grow in the absence of host cells (axenic culture). Growth of the parasite in an axenic culture was detected using qPCR over a period of one week. To validate these results, we used transmission electron microscopy to compare the life cycle stages of the parasite in HCT-8 and axenic culture, however only specific life cycle stages in HCT-8 could be identified. We then performed a proteomic analysis on the axenically cultured parasites and identified several proteins, including cgd4_4160 predicted to play a role in calcium ion binding, that were upregulated during the weeklong experiment.
Utilising the HCT-8 culture we screened a library of 18 synthetic peptoids, peptidomimetics of peptides for their anti-cryptosporidial activity. We identified nine peptoids with anti-cryptosporidial effects and limited cell toxicity. Two peptoids, TM9 and TM19, were selected for further cell toxicity assays which demonstrated that only TM9 did not affect cell viability. TM9 was then tested against different life cycle stages of Cryptosporidium parvum however the peptoid did not appear to affect specific stages. We then used scanning electron microscopy to visualize the effect of the peptoid on the oocyst and sporozoite stages of the parasite, which revealed that the peptoid may act with a membrane disrupting mechanism.
Overall, these findings provide valuable insights into the development of various culture models for Cryptosporidium parvum and highlights the limitations associated with this. We also present a promising new avenue for the treatment of cryptosporidiosis with synthetic peptoids. It is hoped that this study will aid other research groups and contribute to the global efforts to fight this parasite.