Journal: Cell reports

Article Title: The Cryptosporidium signaling kinase CDPK5 plays an important role in male gametogenesis and parasite virulence

doi: 10.1016/j.celrep.2024.114263

Figure Lengend Snippet: (A) Domain architecture of CDPK5 showing the serine/threonine kinase catalytic domain and four calcium-binding (EF-hand) motifs. AA, amino acids. (B) Median transcript expression of cgd2_1300 gene over 2, 6, 12, 24, 36, and 48 h infection time points. Gene expression was normalized to C. parvum 18S rRNA. Data shown here have been replotted from Mauzy et al. (C) Maximum likelihood phylogenetic tree showing the relationship of Cryptosporidium CDPK5 with selected apicomplexan kinases. Tip labels indicate the gene IDs of the kinases. Dots outside the ring represent annotated CDPKs. Abbreviated species names for Cryptosporidium parvum , C. hominis , C. andersoni , C. muris , C. meleagridis , C. tyzzeri , Gregarina niphandrodes , Toxoplasma gondii , Plasmodium falciparum , P. yoelli , P. berghei , Hammondia hammondi , Neospora caninum , Eimeria tenella , Cyclospora cayetanensis , Cystoisospora suis , Babesia bovata , Besnoitia besnoiti , Sarcosystis neurona , and Theileria annulata are shown, and detailed annotations are provided in . Bootstrap support values for 1,000 replicates are indicated on the branches. (D) Schematic for endogenous tagging of cdpk5 gene locus with 3X-HA epitope tag. The locations of the single guide RNA (sgRNA) in the 3ʹ UTR, the Cas9 cut site (arrow), and the repair template for homologous recombination are shown. Nluc, nanoluciferase; Neo, neomycin resistance marker; Eno, enolase promoter and 3ʹ UTR. (E) PCR mapping of the correct integration events in the CDPK5-HA transgenic parasite. Fecal genomic DNA extracted from wild-type (WT) and CDPK5-HA transgenic parasites were used for PCRs. The locations of primers for checking 5ʹ and 3ʹ integrations are indicated in (A), and their sequences are provided in . (F) Western blot detection of 80 kDa C. parvum CDPK5 protein in CDPK5-HA strain using anti-HA antibody. WT, wild type C. parvum . CP23 antibody was used to detect the presence of protein in both WT and transgenic strains. (G) Immunofluorescence assay and super-resolution microscopy of CDPK5-HA transgenic parasites showing localization only in the male (microgamont stage). Asexual stages (trophozoite and 8-nuclei meront) were visualized after 24 h of HCT-8 infection using tryptophan B (TrpB) and 5E3 antibodies. Female and male sexual stages at 48 h post-infection were detected using H3K9ac and α-tubulin (tub) antibodies, respectively, and are shown in red. HA, anti-HA antibody (green). Parasite nuclei were detected with Hoechst stain (blue). hc, host cell. Maximum intensity projection images are shown. Scale bars, 2 μm. Representative data from three independent experiments are shown.

Article Snippet: Cryptosporidium parvum AUCP-1 oocysts were subjected to excystation, and sporozoites were electroporated with the Cas9/guide plasmid (50 μg) and repair DNA template (50 μg) using Lonza Nucleofector 4D device., , Transfected sporozoites were delivered to IFN-γ KO mice via oral gavage after buffering the acidic stomach with 8% sodium bicarbonate.

Techniques: Binding Assay, Expressing, Infection, Gene Expression, Homologous Recombination, Marker, Transgenic Assay, Western Blot, Immunofluorescence, Super-Resolution Microscopy, Staining

Journal: Cell reports

Article Title: The Cryptosporidium signaling kinase CDPK5 plays an important role in male gametogenesis and parasite virulence

doi: 10.1016/j.celrep.2024.114263

Figure Lengend Snippet: (A) Schematic showing C. parvum CDPK5-KO transgenic line. The cdpk5 gene was replaced with an Nluc-Neo cassette by homologous recombination. The locations of the sgRNA within the gene and the Cas9 cut site (arrow) are shown. Nluc, nanoluciferase; Neo, neomycin resistance marker; Eno, enolase promoter and 3ʹ UTR. (B) PCR mapping of the CDPK5-KO transgenic to confirm the correct 5ʹ and 3ʹ integration events. Fecal genomic DNA extracted from WT and CDPK5-KO transgenic parasites were used for PCRs. The locations of primers for integrations are indicated in (A), and their sequences are provided in . (C) Quantification of microgamont stages in CDPK5-HA and CDPK5-KO transgenics at 48 and 56 h post-infection of HCT-8 cells. (D) Violin plot showing distribution of free male gamete counts after egress in CDPK5-HA and CDPK5-KO parasites at 48 and 56 h post-infection of HCT-8 cells. The inset shows a representative image of Hoechst-stained free male gametes. Scale bar, 2 μm. Data represented are from two independent experiments. Mann-Whitney test: two-tailed ** p = 0.063 for 48 h and ** p = 0.0016 for 56 h comparisons. (E) Quantification of microgamont stages in CDPK5-HA and CDPK5-KO parasites treated with or without 4 μM A23187. Data represented are mean ± SEM (standard error of mean) from two independent experiments. (F) Violin plot showing distribution of free male gamete counts after egress in CDPK5-HA and CDPK5-KO at 48 h post-infection of HCT-8 cells incubated for 30 min with or without A23187. Mann-Whitney test: two-tailed **** p < 0.0001; ns, not significant. Data represented are from two independent experiments. (G) Quantification of microgamont stages in CDPK5-HA and CDPK5-KO parasites treated with or without 50 μM BIPPO. Data represented are mean ± SEM from two independent experiments. For (C)–(G), images captured and quantified were from three technical replicates representing 30 fields of view for each strain. Representative images for (C) and (D) are provided in and .

Article Snippet: Cryptosporidium parvum AUCP-1 oocysts were subjected to excystation, and sporozoites were electroporated with the Cas9/guide plasmid (50 μg) and repair DNA template (50 μg) using Lonza Nucleofector 4D device., , Transfected sporozoites were delivered to IFN-γ KO mice via oral gavage after buffering the acidic stomach with 8% sodium bicarbonate.

Techniques: Transgenic Assay, Homologous Recombination, Marker, Infection, Staining, MANN-WHITNEY, Two Tailed Test, Incubation

Journal: Cell reports

Article Title: The Cryptosporidium signaling kinase CDPK5 plays an important role in male gametogenesis and parasite virulence

doi: 10.1016/j.celrep.2024.114263

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cryptosporidium parvum AUCP-1 oocysts were subjected to excystation, and sporozoites were electroporated with the Cas9/guide plasmid (50 μg) and repair DNA template (50 μg) using Lonza Nucleofector 4D device., , Transfected sporozoites were delivered to IFN-γ KO mice via oral gavage after buffering the acidic stomach with 8% sodium bicarbonate.

Techniques: Plasmid Preparation, Virus, Recombinant, Luciferase, SYBR Green Assay, Software