Genetic modification of the diarrhoeal pathogen cryptosporidium parvum

Sumiti Vinayak, Mattie C. Pawlowic, Adam Sateriale, Carrie F. Brooks, Caleb J. Studstill, Yael Bar-Peled, Michael J. Cipriano & Boris Striepen

Nature. 2015 Jul 15;523(7561):477-80. doi: 10.1038

Speaker: Ming-Yang Wu (吳明陽)                               Time: 14:00~15:00, Nov 4, 2015

Commentator: Dr. Wei-Chen Lin (林威辰 老師)     Place: Room 601

Abstract:

Cryptosporidium parvum is one of several protozoal species that cause diarrhoeal disease. These protozoan parasites are known to be among the second most important diarrhoeal pathogens after rotavirus[1], accounting for 10.5% of global child mortality and often infecting people who have compromised immune systems. There is no vaccine and a paucity of treatment options.. The limitations of studying with C. parvum, which don’t have easily continuous culture system, facile animal models, and molecular genetic tools[1][2]. In this study, the authors want to generate stable transgenic C. parvum parasites. First, they used  electroporation to transfection a nanoluciferase reporter construct to C. parvum sporozoites in tissue culture, and established that incorpora­tion of the neomycin resistance marker reduced parasite susceptibility to paromo­mycin. To isolate stable transgenic parasites, a method for direct cultured sporozoites in the mice intes­tines was developed. In parallel, they used the C. parvum U6 RNA pro­mote to build a CRISPR–Cas9 system to drive guide RNA expression and the Streptococcus pyogenes cas9 gene flanked by C. parvum regulatory sequences to increase the efficiency and stability of genetic modification. Taken together, these advances set up the transfection, propagation and paromomycin-based selec­tion of nanoluciferase-positive parasites. This system was used to  nanoluciferase reporter parasites to establish a drug screening platform and this approach was more sensitive than previous PCR-based techniques to quantitate parasite survival, and to demonstrate the basis of C. parvum resistance to antifolates. Here, the authors successfully deleted thymidine kinase (TK) gene, which is absent in other apicomplexan genera, to show that TK provides an alterna­tive pathway for thymidine monophosphate synthesis. Therefore, C. parvum can tolerate high doses of antifolate drugs. Together, the appli­cation of Cryptosporidium genetic modifica­tion will greatly increase our understanding of the pathogen’s basic biology and virulence, and provide key information and validation for the development of improved vaccines and therapeutics.

Reference:

1. Checkley, W. et al. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for cryptosporidium. Lancet Infect. Dis. 15, 85–94 (2015).

2. Striepen, B. Parasitic infections: time to tackle cryptosporidiosis. Nature 503, 189–191 (2013).