PMC

Repeat of the experiment described in . a, Measurement of C. parvum infection using fecal PCR. b, Luminescence measurements. Note increasing luminescence from day 6 in parasites that received resistance and Cas9 plasmids. Mice were infected in groups of 4 per cage and pooled feces was analysed for each cage (each measurement represents 3 technical replicates).

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 100ul 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 (1mg/kg) subcutaneously. Each procedure takes around 15 minutes, and mice recover rapidly.

a, Mice were infected orally with 100,000 transgenic oocysts. Infected mice were then treated with paromomycin (b) or left untreated (c). Oocysts were purified from fecal 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. Feces were tested for luminescence every 3 days. Each reading represents the pooled fecal sample from 5 mice with three technical replicates.

a, Dosing of mice accounting for drug concentration, animal weight, and measured daily water consumption. At 16mg/ml each mouse received 40mg 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.

a, 10 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 (1500 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 hrs. Bars represent average of 2 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 2 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.

a, Schematic overview. C. parvum sporozoites were prepared from oocysts purified from infected calves and electroporated in the presence of plasmid DNA prior to infection of HCT-8 cells. Luminescence measurements (the means of 3 technical replicates, s.d. shown as error bar) of T. gondii (f), human HCT-8 cells (g), or C. parvum (all other panels, blue) transfected with nanoluciferase (Nluc) expression plasmids. C. parvum transfection requires electroporation (b) of DNA (c) into parasites (d). Transfection also requires plasmids to carry parasite specific promoter sequences (e, f, testing C. parvum and T. gondii promoters in both parasites), and is susceptible to the Cryptosporidium drug nitazoxanide (h). Lipofection of HCT-8 cells with the original Nluc plasmid pNL1.1 (Promega), but not derived parasite vectors results in luciferase activity in the host alone (g). Choice of promoter (i, Eno, enolase; Aldo, aldolase; Tub, alpha-tubulin 5′ regions, the 3′ UTR was uniformly from the enolase gene) or codon composition (j, oNluc, optimized to 35% GC) influences expression level in C. parvum. Note automatic gain adjustment of luminescence measurements, units are not comparable between panels. Independent experiments were repeated 3 times, and representative data are shown.

a, Due to a horizontal gene transfer C. parvum has two pathways to synthesize dTMP: thymidine kinase (TK) and dihydrofolate reductase-thymidylate synthase (DHFR-TS). DHF and THF, dihydro- and tetrahydrofolic acid. b, Map of the C. parvum TKlocus, the targeting plasmid and the predicted modified locus. Primers and amplicon sizes of diagnostic PCR products are indicated. c, PCR analysis using genomic DNA from wild type (WT) and transgenic parasites (Nluc-Neo, oocysts purified from feces of infected mice shown in ). Primer sequences are provided in a . e, Quantification of EdU labelling experiments (meronts with 4 or more nuclei were scored, two biological repeats, n=105 each sample, error bar is s.d.), d shows representative fluorescence micrographs. Antibody to C. parvum tryptophan synthase B was used to identify parasites (green). f, Trimethoprim treatment of WT (blue) and Nluc-Neo transgenic (red) parasites. WT parasites were measured in transient transfection assays with Nluc plasmid (n=3, technical replicates, error bar is s.d.). Assay shown was conducted in the presence of 10 μM thymidine to avoid indirect host cell toxicity (experiments without thymidine produced indistinguishable results). Experiments were repeated 3 times and representative data are shown.

a, HCT-8 cells were infected with Nluc transfected sporozoites and grown for 2 days in presence of paromomycin. b, Translational fusions were constructed placing Neo to the N- or C-terminus of Nluc. Nluc-Neo shows luciferase activity, albeit at reduced level when compared to Nluc alone. c, C. parvum transfected with Nluc (blue) or Nluc-Neo (red) were grown in different concentrations of paromomycin. Luciferase activity for each plasmid was normalized to its drug free level. d, CRISPR/Cas9 plasmid for C. parvum (u6, newly annotated promoter CM000433:553110–553472; nls, nuclear localization signal; flag, epitope tag; ribo, ribosomal protein L13a 3′ UTR). e, Outline and g, sequences for Nluc repair assay (guide RNA target, blue; protospacer adjacent motif, green; mutagenized codon 18, red). f, Sporozoites were transfected with Nluc or a codon 18 termination mutant (Dead Nluc), note ablation of signal. In addition to the Dead Nluc plasmid, some parasites also received a 125 bp double-stranded repair DNA fragment, and the Cas9 plasmid with the indicated guide RNAs (no target, empty gRNA cassette; off target, GFP gRNA; on target, Nluc gRNA). Statistical analysis compares Dead Nluc alone with Dead Nluc and Cas9 and specific gRNA. Note significant Cas9 mediated restoration of luciferase activity (***p=0.0006, unpaired t test). n=3 technical replicates for a, b, c, and controls from f; n=6 technical replicates for On target samples in f. Error bar is s.d. and all experiments depicted here were repeated 3 times and representative data are shown.

a, Outline. Transfected sporozoites were injected into the small intestine by surgery (Extendend Data ) and mice were treated with paromomycin. Oocysts were purified from the feces and used to infect cultures or mice by oral gavage. b, Quantitative PCR of C. parvum DNA isolated from feces of mice infected with transfected sporozoites (four mice per group) and treated as indicated. Emergence of paromomycin resistance required Nluc-Neo and a Cas9 plasmid. Upon reinfection parasites show strong drug resistance (c) and luciferase activity (d). In repeat experiments we noted that luciferase is detectable as early as 6 days after transfection in the feces of the first infected mouse (Extendended Data ). e, Protein extracts from oocysts were analysed by SDS-PAGE and Western blot using an antibody against Neo (rabbit anti-neomycin phosphotransferase II, EMD Millipore). Predicted molecular mass of the Nluc-Neo fusion protein is 48.3 kDa. f, Immunofluorescence staining using anti Neo (mouse anti Neo, Alpha Diagnostic Inc.) and C. parvum (tryptophan synthase B) antibodies. Note multiple nuclei in DAPI stain typical for C. parvum meronts. No anti Neo staining was observed in wild type parasites. g, Luciferase assays for HCT-8 cultures infected with wild type (WT, blue) and transgenic (Nluc-Neo, red) parasites. Y-axis is split to show level of background. n=3 technical replicates, error bar is s.d., experiment done twice. h, 96-well luciferase drug assay using 1000 oocysts per well. Note significant growth inhibition on treatment with 10 μM nitazoxanide (**p=0.0036, unpaired t test). n=3 technical replicates, error bar is s.d., experiment was repeated two times and representative data are shown.