Recent studies into the global causes of severe diarrhoea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrhoeal pathogen after rotavirus1,2,3. Diarrhoeal disease is estimated to be responsible for 10.5% of overall child mortality4. Cryptosporidium is also an opportunistic pathogen in the contexts of human immunodeficiency virus (HIV)-caused AIDS and organ transplantation5,6. There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger: malnourished children and immunocompromised patients7,8. Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes a lack of systems for continuous culture, facile animal models, and molecular genetic tools3,9. Here we describe an experimental framework to genetically modify this important human pathogen. We established and optimized transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we developed a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, and in vivo selection for aminoglycoside resistance. We derived reporter parasites suitable for in vitro and in vivo drug screening, and we evaluated the basis of drug susceptibility by gene knockout. We anticipate that the ability to genetically engineer this parasite will be transformative for Cryptosporidium research. Genetic reporters will provide quantitative correlates for disease, cure and protection, and the role of parasite genes in these processes is now open to rigorous investigation.
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We thank L. Sharling for initial contributions and L. Hedstrom, J. Mead, S. Vaishnava, L. Xiao and Y. Belkaid for discussion. This work was funded in part by the National Institutes of Health (NIH; R01AI112427) to B.S. and by a pilot grant from the Centers for Disease Control and the University of Georgia Research Foundation to B.S. and L. Xiao. M.J.C. was supported by training grant NIH T32AI060546 and B.S. is a Georgia Research Alliance Distinguished Investigator.
Competing interests: S.V., C.F.B. and B.S. are listed as inventors on the Patent Cooperation Treaty (PCT) application entitled “Cryptosporidium transfection methods and transfected Cryptosporidium cells”, filed by the University of Georgia Research Foundation.
Extended data figures and tables
a, Ten-million sporozoites prepared in either cytomix (BTX) or Lonza Buffers SE, SF or SG (4D Nucleofection) were combined with 10 μg DNA (Eno_Nluc-GS-Nluc_Eno). Samples were electroporated using previously determined settings for BTX (1,500 V, 25 Ω, 25 μF) or various program settings for 4D Nucleofection as indicated. Parasites were added to cultures of HCT-8 cells and luciferase activity was read after 48 h. Bars represent average of two technical replicates. b, Transfection was further optimized by comparing the best preliminary settings (buffers SF and SG; programs EH 100 and EO 100) with additional pulse programs as indicated. Transfection was carried out as in a. Bars represent average of two technical replicates. c, Electroporation systems (BTX and 4D Nucleofection) were compared using the same number of C. parvum sporozoites and quantities of DNA using buffers and conditions optimized in a and b. Bars represent average of three technical replicates. Note about tenfold enhancement of transient transfection using 4D Nucleofection. The impact of electroporation on stable transformation cannot be assessed in this setup and may be higher. Experiments in a and b were done once for the purpose of optimization, while c was repeated three times; a single representative experiment is shown.
Extended Data Figure 2 Direct surgical injection of transfected C. parvum sporozoites into the small intestine.
Mice are shaved and anaesthetized with isofluorane (3% initially, then maintained at 1.5% for the surgery). The abdominal skin is disinfected with Betadine and a small incision is made into the peritoneum. Forceps are used to grasp the small intestine and 100 µl of PBS containing 107 transfected C. parvum sporozoites is injected into the lumen. The peritoneum and the abdominal skin are each sutured with 4-0 polydioxanone and mice are injected with meloxicam (1 mg kg−1) subcutaneously. Each procedure takes around 15 min, and mice recover rapidly.
a, Dosing of mice accounting for drug concentration, animal weight, and measured daily water consumption. At 16 mg ml−1 each mouse received 40 mg paromomycin daily (dotted line). b, This dose was found to be sufficient to decrease oocyst shedding in treated mice to background. By day 7 mice without paromomycin treatment shed large amounts of oocysts when compared to untreated mice. Treated mice showed no shedding above background. Oocysts were enumerated by high-throughput imaging assay. Five mice were analysed individually with two technical replicates.
Repeat of the experiment described in Fig. 3b. a, Measurement of C. parvum infection using faecal PCR. b, Luminescence measurements. Note increasing luminescence from day 6 in parasites that received resistance and Cas9 plasmids. Mice were infected in groups of four per cage and pooled faeces was analysed for each cage (each measurement represents three technical replicates).
Extended Data Figure 5 C. parvum maintains the stable transgene when passed serially in mice without paromomycin treatment.
a, Mice were infected orally with 100,000 transgenic oocysts. b, c, Infected mice were then treated with paromomycin (b) or left untreated (c). Oocysts were purified from faecal collections by sucrose flotation and CsCl centrifugation, and used to infect a second cohort of mice. Again, each mouse received 100,000 transgenic oocysts and mice were treated or not. Faeces were tested for luminescence every 3 days. Each reading represents the pooled faecal sample from five mice with three technical replicates.
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Vinayak, S., Pawlowic, M., Sateriale, A. et al. Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum. Nature 523, 477–480 (2015) doi:10.1038/nature14651
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