Fig 1.
Endogenous epitope tagging of INS6.
(A) Schematic representation of the structural domains of INS6 and its homologue gene, toxolysin 4 (TLN4). Both INS6 and TLN4 have three structural domains associated with classical insulinase peptidase, including the M16 N-terminus, the M16 middle/third terminus, and the M16 C-terminus, in addition to a signal peptide in TLN4. (B) Violin plots demonstrate the transcript expression of C. parvum genes in sporozoites and different development stages of parasites grown on HCT-8 cells, as indicated by TPM values from RNA-seq analysis of the transcriptome. Colored dots represent microneme genes, with INS6 indicated in purple. (C) Schematic showing the tagging strategy of INS6 locus. The location of the sgRNA, Cas9 break site, and the repair template for homologous recombination are shown. 3 × HA, triple hemagglutinin epitope tag; Nluc, nanoluciferase; NeoR, neomycin resistance marker; Eno, enolase promoter. (D) Fecal luminescence measurements of mice infected with INS6-3HA sporozoites. Ifng−/− mice were infected with INS6-3HA electroporated sporozoites, relative luminescence was measured from feces after infection at indicated days. Each data point represents one mouse. In total, four mice were used in this experiment. (E) PCR analysis of INS6-3HA strain. Fecal genomic DNA from wild-type (WT) and INS6-3HA parasites were used as template, and primers for checking 5’ insertion (5’ Ins), 3’ insertion (3’ Ins), and the 3’ UTR sequence of INS6 as a positive control (+ve) are indicated in panel C. (F) Western blot analysis of INS6 expression in oocysts. INS6-3HA transgenic and WT oocysts were probed with anti-HA antibody (top panel) and a polyclonal antibody against the NFDQ1 as a loading control (bottom panel). NFDQ1 is a protein that contains the characteristic NFDQ amino acid repeat motifs [26].
Fig 2.
Localization and expression of C. parvum INS6.
(A) Immunofluorescence assay of INS6-3HA expression in sporozoites. Parasites were fixed and stained with anti-HA (red) to label INS6 protein, anti-CpEF1α antibody (green) to label the entire parasite, and Hoechst to visualize parasite nuclei (blue). The INS6 protein was primarily localized to the mid-sporozoite region. Scale bars = 5 μm. (B) Immunofluorescence analysis of INS6-3HA expression in meronts, male gamonts and female gametes. Parasites were stained with anti-HA (red) to detect INS6 protein and VVL (green) to label the parasite. INS6 forms punctate aggregates in the meront, is not expressed in the male gamonts (indicated by the white arrow) and shows low expression in the female gametes (indicated by the orange arrow). Scale bars = 5 μm. (C) Ultrastructure expansion microscopy (U-ExM) of INS6-3HA in sporozoites. Sporozoites were fixed, expanded in gel, and stained with rat anti-HA (green), NHS-ester (red) and Hoechst (blue). The distribution pattern of INS6 exhibits partial spatial overlap with microneme proteins, but does not fully align with canonical microneme localization. Scale bars = 5 μm. (D) Immunoelectron microscopy of INS6-3HA. Mice were infected with INS6-3HA oocysts and the ileum tissues were fixed at 18 dpi and stained with rabbit anti-HA followed by 10-nm colloidal gold goat anti-rabbit IgG. Black dots indicate the distribution of gold particles, and red arrows indicate the distribution of INS6 protein.
Fig 3.
INS6 is not essential for parasite survival.
(A) Schematic representation of the INS6 knockout strategy. The location of the sgRNA, the Cas9 cleavage site, and the repair template for homologous recombination are indicated. pAct, actin promoter. (B) Fecal luminescence measurements in mice infected with Δins6 sporozoites. Ifng−/− mice infected with electroporated Δins6 sporozoites have their fecal samples analyzed for relative luminescence at the indicated time points post-infection. Each data point represents one mouse. In total, three mice were used in this experiment. (C) Fluorescence imaging of Δins6 oocysts. Δins6 oocysts expressing mEGFP were visualized using microscopy. Scale bars = 2 µm. (D) PCR analysis of the Δins6 strain. Genomic DNA extracted from fecal samples of WT and Δins6 parasites was used as a template. The primers used to verify the 5’ Ins, 3’ Ins, open reading frame (ORF) and 3’ UTR sequences of INS6, which were used as a positive control are indicated in panel A.
Fig 4.
Genetic ablation of INS6 affects C. parvum proliferation.
(A) Relative luminescence measurements of INS6-3HA and Δins6 parasites grown on HCT-8 cells. The cells were infected with either INS6-3HA or Δins6 parasites and the growth of the parasites was assessed by measuring luciferase activity at the indicated time points. Data are presented as the mean ± SD from eight replicates across two independent experiments. Differences between the INS6-3HA and Δins6 groups were analyzed using a two-way ANOVA followed by a Sidak’s multiple comparisons test. (B) Fecal luminescence measurements (left) and area under the curve analysis (right) in mice infected with INS6-3HA and Δins6 oocysts. Ifng−/− mice were orally gavaged with either INS6-3HA or Δins6 oocysts, and parasite burden was quantified by luciferase activity at different time points. Data points represent mean ± SD from six or five individually housed infected mice (n = 6 for INS6-3HA and n = 5 for Δins6). Differences between the INS6-3HA and Δins6 groups were analyzed using a two-way ANOVA followed by a Sidak’s multiple comparisons test. (C) Hematoxylin and eosin (H&E) staining of ileum sections from mice infected with INS6-3HA or Δins6 parasites. Black arrows indicate sites of C. parvum infection. (D) Quantification of parasite numbers per villus in the small intestine of infected mice. Each point on the graph represents the number of parasites on a single villus. Different shapes indicate individual data points. Select ten villi at random from each group and count the number of parasites on each one. Differences between the INS6-3HA and Δins6 groups were analyzed using two-tailed Mann-Whitney U test. (E) Scanning electron microscopy (SEM) images of the ileum from mice infected with INS6-3HA (top) or Δins6 (bottom) parasites.
Fig 5.
INS6 affects the pathogenicity of C. parvum.
(A) Hematoxylin and eosin staining showing pathological damage to the ileum of Ifng−/− mice infected with INS6-3HA or Δins6 strains. Uninfected mice were used as a control. (B) The ratio of villus height to crypt depth in mice infected with INS6-3HA, Δins6 strains or uninfected (two mice per group). (C) Body weight gain (g) in Ifng−/− mice infected with INS6-3HA or Δins6 strains, with uninfected mice used as the control. Data were normalized to the day before infection. (D) Body weight gain (%). Data were collected from the day before infection until the end of the experiment. Five mice per group are shown in panels C and D. Differences among the INS6-3HA, Δins6 and uninfected groups were analyzed using two-way ANOVA followed by a Tukey’s multiple comparisons test in panels B and D. (E) Survival curves of Ifng−/− mice infected with the INS6-3HA or Δins6 strains, or left uninfected as a control group. Data were obtained from five individually housed infected mice. Statistical significance was tested using the log-rank test.