Recent Breakthroughs and Ongoing Limitations in Cryptosporidium Research

Abstract

The intestinal apicomplexan parasite Cryptosporidium is a major cause of diarrheal disease in humans worldwide. However, treatment options are severely limited. The search for novel interventions is imperative, yet there are several challenges to drug development, including intractability of the parasite and limited technical tools to study it. This review addresses recent, exciting breakthroughs in this field, including novel cell culture models, strategies for genetic manipulation, transcriptomics, and promising new drug candidates. These advances will stimulate the ongoing quest to understand Cryptosporidium and the pathogenesis of cryptosporidiosis and to develop new approaches to combat this disease.

Keywords: Cryptosporidium; cell culture; drug discovery; genetic modification; transcriptomics.

Figure 1.. Bioengineered three-dimensional human intestinal tissue model.

Silk cocoons ( A) were processed to yield a viscous silk solution ( B). ( C) The silk solution was poured into cylindrical molds, and a wire was inserted to develop a lumen equivalent. Caco-2 and HT29-MTX cells ( D) were seeded into the lumen ( E), while the porous bulk space was seeded with H-InMyoFibs. ( F) The Caco-2 and HT29-MTX cells in the lumen were infected with Cryptosporidium parvum oocysts or purified sporozoites. Intracellular development through asexual and sexual cycles occurred to complete the life cycle with the formation of oocysts. 3D, three-dimensional; PDMS, polydimethylsiloxane. Figure reproduced with permission from the American Society for Microbiology .

Figure 2.. Genetic modification of Cryptosporidium.

Cryptosporidium parvum oocysts isolated from the feces of infected calves can be excysted to release sporozoites which can infect mammalian epithelial cells in culture but only for one or two rounds of replication before they die. Vinayak et al. used CRISPR/Cas9 technology to genetically modify C. parvum sporozoites . Selection and replication of modified parasites requires direct injection into surgically isolated intestines of interferon-gamma knockout mice. Modified oocysts collected from mouse feces can be analyzed in culture or used to inoculate new mice to maintain the transgenic line. Figure reproduced with permission from Springer .

Figure 3.. A drug-discovery screening pipeline for Cryptosporidium.

(1) A high content imaging infection assay in vitro was used to identify Cryptosporidium parvum inhibitory compounds. (2) Secondary screening using a cytopathic effect-based assay was used to identify imidazopyrazines and pyrazolopyridines with inhibitory activity against C. parvum and Cryptosporidium hominis. (3) Identification, expression, and enzymatic activity of the C. parvum phosphatidylinositol-4-OH kinase (PI4K). (4) Pharmacokinetics and toxicity testing of KDU731. (5) Activity of KDU731 against transgenic C. parvum infection in interferon-gamma knockout mice. (6) Activity of KDU731 against native C. parvum infection in the neonatal calf model. (7) Additional preclinical evaluation is needed before initiation of human clinical trials. Figure reproduced with permission from Elsevier .