Key resources table.
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Cryptosporidium parvum) | MEDLE2 | Li et al., 2017 | cgd5_4590 | |
| Gene (C. parvum) | MEDLE1 | Fei et al., 2018 | cgd5_4580 | |
| Gene (C. parvum) | MEDLE6 | This paper | cgd6_5490 | Named according to the spatial localization in the genome similarly to MEDLE2 and MEDLE1 |
| Gene (C. parvum) | WYLE4 | This paper | cgd8_3560 | Named for the gene family and according to the spatial localization in the genome |
| Gene (C. parvum) | SKSR7 | This paper | Cgd8_30 | Named for the gene family and according to the spatial localization in the genome |
| Strain, strain background (C. parvum) | C. parvum oocysts, IOWAII strain (WT) | Bunchgrass | ||
| Strain, strain background (Mus musculus) | Ifng-/-, C57BL/6J | The Jackson Laboratory | Jax 002287; RRID:IMSR_JAX:002287 |
|
| Strain, strain background (M. musculus) | Ddit3 tm2.1Dron | The Jackson Laboratory | Jax 005530; RRID:IMSR_JAX:005530 |
|
| Strain, strain background (M. musculus) | C57BL/6J | The Jackson Laboratory | Jax 000664; RRID:IMSR_JAX:000664 |
|
| Genetic reagent (C. parvum) | MEDLE2-HA | This paper | cgd5_4590 modified | Stable transgenic parasite line expressing HA |
| Genetic reagent (C. parvum) | WYLE4-HA | This paper | cgd8_3570 modified | Stable transgenic parasite line expressing HA |
| Genetic reagent (C. parvum) | SKSR7-HA | This paper | cgd8_30 modified | Stable transgenic parasite line expressing HA |
| Genetic reagent (C. parvum) | MEDLE2 KO | This paper | Cgd5_4590 | Stable transgenic parasite line with one copy of MEDLE2 knocked out |
| Genetic reagent (C. parvum) | MEDLE1-HA | This paper | cgd5_4580 modified | Stable transgenic parasite line expressing HA |
| Genetic reagent (C. parvum) | MEDLE6-HA | This paper | cgd6_5490 modified | Stable transgenic parasite line expressing HA |
| Genetic reagent (C. parvum) | Medle2 MEDLE1-HA | This paper | cgd5_4440 modified | Stable transgenic parasite line expressing HA |
| Genetic reagent (C. parvum) | MEDLE2-HA-tdNeon | This paper | cgd5_4590 modified | Stable transgenic parasite line expressing HA and tdNeon |
| Genetic reagent (C. parvum) | MEDLE2-mScarlet | This paper | cgd5_4590 modified | Stable transgenic parasite line expressing mScarlet |
| Genetic reagent (C. parvum) | MEDLE2-Bla-2A-tdTomato | This paper | cgd5_4590 modified | Stable transgenic parasite line expressing BLA and tdTomato |
| Genetic reagent (C. parvum) | MEDLE2-Cre | This paper | cgd5_4590 modified | Stable transgenic parasite line expressing Cre recombinase |
| Genetic reagent (C. parvum) | Ectopic MEDLE2-HA | This paper | cgd5_4440 modified | Stable transgenic parasite line expressing extra copy of MEDLE2 |
| Genetic reagent (C. parvum) | ΔSP | This paper | cgd5_4440 modified | Stable transgenic parasite line expressing extra copy of MEDLE2 (aa 21–209) |
| Genetic reagent (C. parvum) | KDVSLI/6A | This paper | cgd5_4440 modified | Stable transgenic parasite line expressing extra copy of MEDLE2 with KDVSLI (aa 31–36) mutated to six alanines |
| Genetic reagent (C. parvum) | KPVLKN/6A | This paper | cgd5_4440 modified | Stable transgenic parasite line expressing extra copy of MEDLE2 with KPVLKN (aa 73–78) mutated to six alanines |
| Genetic reagent (C. parvum) | KNVNLS/6A | This paper | cgd5_4440 modified | Stable transgenic parasite line expressing extra copy of MEDLE2 with KDVSLI (aa 77–82) mutated to six alanines |
| Genetic reagent (C. parvum) | RGLLRGLSG/9A | This paper | cgd5_4440 modified | Stable transgenic parasite line expressing extra copy of MEDLE2 with KDVSLI (aa 191–199) mutated to six alanines |
| Genetic reagent (C. parvum) | K31A | This paper | cgd5_4440 modified | Stable transgenic parasite line expressing extra copy of MEDLE2 with K31 mutated to alanine |
| Genetic reagent (C. parvum) | D32A | This paper | cgd5_4440 modified | Stable transgenic parasite line expressing extra copy of MEDLE2 with D32 mutated to alanine |
| Genetic reagent (C. parvum) | V33A | This paper | cgd5_4440 modified | Stable transgenic parasite line expressing extra copy of MEDLE2 with V33 mutated to alanine |
| Genetic reagent (C. parvum) | S34A | This paper | cgd5_4440 modified | Stable transgenic parasite line expressing extra copy of MEDLE2 with S34 mutated to alanine |
| Genetic reagent (C. parvum) | L35A | This paper | cgd5_4440 modified | Stable transgenic parasite line expressing extra copy of MEDLE2 with L35 mutated to alanine |
| Genetic reagent (C. parvum) | I36A | This paper | cgd5_4440 modified | Stable transgenic parasite line expressing extra copy of MEDLE2 with I36 mutated to alanine |
| Cell line (human) | HCT-8 | ATCC | CCL-224; RRID:CVCL_2478 |
|
| Cell line (human) | HEK293T | ATCC | CRL-3216; RRID:CVCL_0063 |
|
| Cell line (Escherichia coli) | GC5 | Genesee Scientific |
42-653 | Electrocompetent cells |
| Cell line (E. coli) | One Shot Topo10, |
Invitrogen | C404003 | Electrocompetent cells |
| Transfected construct (human) | loxP GFP/RFP color switch lentivirus | GenTarget Inc | Cat#: LVP460-Neo | Transfected construct (human) |
| Biological sample (M. musculus) | Isolated sections of ileum | Ifng-/- mice | Jax 002287; RRID:IMSR_ JAX:002287 |
6-week-old male mice |
| Antibody | Anti-HA (rat monoclonal) | MilliporeSigma | Cat# 11867431001; RRID:AB_390919 | IF (1:500), WB (1:500), IHC (1:500) |
| Antibody | Anti-Cp23 (mouse monoclonal) | LS Bio | Cat# LS-C137378; RRID:AB_10947007 | IF (1:100) |
| Antibody | Anti-alpha tubulin (mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat#12G10; RRID:AB_1157911 | IF (1:1000) |
| Antibody | Anti-COWP1 (rat monoclonal) | This paper, produced by GenScript | This paper | IF (1:100) |
| Antibody | Anti-neomycin phosphotransferase II (rabbit polyclonal) | MilliporeSigma | Millipore Cat# 06-747; RRID:AB_310234 | WB (1:1000) |
| Antibody | Anti-mouse IFN gamma | Bio X Cell | Clone: XMG1.2; Cat# BE0055; RRID:AB_1107694 |
In vivo 100 µg |
| Antibody | Goat anti-rat polyclonal Alexa Fluor 594 | Thermo Fisher Scientific | Cat# A-21213; RRID:AB_2535799 | IFA (1:500) |
| Antibody | Goat anti-mouse polyclonal Alexa Fluor 488 | Thermo Fisher Scientific | Cat# A-11001; RRID:AB_2534069 | IFA (1:500) |
| Strain, strain background | Alexa Fluor 647 Phalloidin | Thermo Fisher Scientific | A22287; RRID:AB_2620155 |
IFA (1:1000) |
| Antibody | IRDye 800CW goat anti-rat IgG | LI-COR | 926-32219; RRID:AB_1850025 |
WB (1:10,000) |
| Antibody | IRDye 680RD goat anti-rabbit IgG | LI-COR | 926-68071; RRID:AB_2721181 |
WB (1:10,000) |
| Recombinant DNA reagent | Cas9 cgd5_4590 (plasmid) | This paper | Guide targeting C terminus of MEDLE2 | |
| Recombinant DNA reagent | Cas9 cgd8_3560 (plasmid) | This paper | Guide targeting C terminus of WYLE4 | |
| Recombinant DNA reagent | Cas9 cgd8_30 (plasmid) | This paper | Guide targeting C terminus of SKSR7 | |
| Recombinant DNA reagent | Cas9 cgd5_4580 (plasmid) | This paper | Guide targeting C terminus of MEDLE1 | |
| Recombinant DNA reagent | Cas9 Cgd6_5490 (plasmid) | This paper | Guide targeting C terminus of MEDLE6 | |
| Recombinant DNA reagent | Cas9 Tk guide int (plasmid) | Tandel et al., 2019 | Guide targeting internal cgd5_4440 | |
| Recombinant DNA reagent | Cas9 MEDLE2 KO (plasmid) |
This paper | Guide targeting internal MEDLE2 | |
| Recombinant DNA reagent | Lic HA (plasmid) |
This paper | Crypto expression vector for HA tagging | |
| Recombinant DNA reagent | Lic tdTomato KO (plasmid) |
This paper | Crypto expression vector for replacing gene KO with tdTomato | |
| Recombinant DNA reagent | Lic medle2-MEDLE1 HA (plasmid) | This paper | Crypto expression vector with medle2 promoter driving MEDLE1-HA expression | |
| Recombinant DNA reagent | Lic HA-2A- TdNeon (plasmid) |
This paper | Crypto expression vector for HA tagging and cytoplasmic tdNeon | |
| Recombinant DNA reagent | Lic mScarlet (plasmid) | This paper | Crypto expression vector for mScarlet tagging | |
| Recombinant DNA reagent | Lic Bla-2A-TdTomato (plasmid) |
This paper | Crypto expression vector for BLA tagging and cytoplasmic tdTomato | |
| Recombinant DNA reagent | Lic Cre (plasmid) |
This paper | Crypto expression vector for Cre tagging | |
| Recombinant DNA reagent | Lic Extra MEDLE2-HA (plasmid) |
This paper | Crypto expression vector for extra copy of MEDLE2-HA (MEDLE2 promoter) | |
| Recombinant DNA reagent | Lic ΔSP MEDLE2-HA (plasmid) | This paper | Crypto expression vector for extra copy of MEDLE2-HA (aa 21–209) | |
| Recombinant DNA reagent | Lic KDVSLI/6A-HA (plasmid) | This paper | Crypto expression vector for extra copy of MEDLE2-HA with KDVSLI (aa 31–36) mutated to six alanines | |
| Recombinant DNA reagent | Lic KPVLKN/6A-HA (plasmid) | This paper | Crypto expression vector for extra copy of MEDLE2-HA with KPVLKN (aa 73–78) mutated to six alanines | |
| Recombinant DNA reagent | Lic KNVNLS/6A-HA (plasmid) | This paper | Crypto expression vector for extra copy of MEDLE2-HA with KNVNLS (aa 77–82) mutated to six alanines | |
| Recombinant DNA reagent | Lic RGLLRGLSG/9A-HA (plasmid) | This paper | Crypto expression vector for extra copy of MEDLE2-HA with RGLLRGLS (aa 191–199) mutated to six alanines | |
| Recombinant DNA reagent | Lic K31A-HA (plasmid) | This paper | Crypto expression vector for extra copy of MEDLE2-HA with K31 mutated to alanine | |
| Recombinant DNA reagent | Lic D32A-HA (plasmid) | This paper | Crypto expression vector for extra copy of MEDLE2-HA with D32 mutated to alanine | |
| Recombinant DNA reagent | Lic V33A-HA (plasmid) | This paper | Crypto expression vector for extra copy of MEDLE2-HA with V33 mutated to alanine | |
| Recombinant DNA reagent | Lic S34A-HA (plasmid) | This paper | Crypto expression vector for extra copy of MEDLE2-HA with S34 mutated to alanine | |
| Recombinant DNA reagent | Lic L35A-HA (plasmid) | This paper | Crypto expression vector for extra copy of MEDLE2-HA with L35 mutated to alanine | |
| Recombinant DNA reagent | Lic I36A-HA (plasmid) | This paper | Crypto expression vector for extra copy of MEDLE2-HA with I36 mutated to alanine | |
| Recombinant DNA reagent | mEGFP-Lifeact-7 (plasmid) | Addgene | # 54610 | Used as a mammalian expression vector to clone codon optimized MEDLE2 into |
| Recombinant DNA reagent | GFP-only | This paper | Removed Lifeact domain from Addgene plasmid #54610 for a GFP-only control plasmid | |
| Recombinant DNA reagent | Recod MEDLE2-GFP (plasmid) | This paper | Human codon optimized MEDLE2 (aa 21–209) with a GFP tag | |
| Recombinant DNA reagent | GRA16-GFP | This paper | T. gondii GRA16 (aa 24–505) with GFP tag. | |
| Recombinant DNA reagent | Recod MEDLE2-HA (plasmid) | This paper | Human codon optimized MEDLE2 (aa 21–209) with a HA tag. | |
| Recombinant DNA reagent | Recod KDVSLI/6A-HA (plasmid) | This paper | Human codon optimized MEDLE2 (aa 21–209) with a HA tag and KDVSLI (aa 31–36) mutated to six alanines | |
| Recombinant DNA reagent | Recod KPVLKN/6A-HA (plasmid) | This paper | Human codon optimized MEDLE2 (aa 21–209) with a HA tag and KPVLKN (aa 73–78) mutated to six alanines | |
| Recombinant DNA reagent | Recod KNVNLS/6A-HA (plasmid) | This paper | Human codon optimized MEDLE2 (aa 21–209) with a HA tag and KNVNLS (aa 77–82) mutated to six alanines | |
| Recombinant DNA reagent | Recod RGLLRGLSG/9A-HA (plasmid) | This paper | Human codon optimized MEDLE2 (aa 21–209) with a HA tag and RGLLRGLS (aa 191–199) mutated to six alanines | |
| Sequence-based reagent |
Recodonized MEDLE2 | Integrated DNA Technologies | MEDLE2 (aa 21–209) codon optimized for human expressionSee Supplementary file 1 for sequence | |
| Sequence-based reagent |
PCR primers | This paper | Please see Supplementary file 1 | |
| Commercial assay or kit | DNeasy Blood & Tissue Kit | QIAGEN | Cat# 69504 | |
| Commercial assay or kit | ZymoPureII Plasmid Maxiprep Kit | Zymo Research | Cat# 11-555B | |
| Commercial assay or kit | Nano-Glo Luciferase Assay System | Promega | Cat# N1130 | |
| Commercial assay or kit | SF Cell Line 4D X Kit L | Lonza | Cat# V4XC-2024 | |
| Commercial assay or kit | LiveBLAzer FRET-B/G Loading Kit | Thermo Fisher | Cat# K1095 | |
| Commercial assay or kit | ZeroBlunt TopoTA Kit | Invitrogen | Cat# 450245 | |
| Commercial assay or kit | RNeasy Microkit | QIAGEN | Cat# 74004 | |
| Commercial assay or kit | SMART cDNA synthesis kit | Takara | Cat# 635040 | |
| Commercial assay or kit | Nextera XT DNA Library Prep Kit | Illumina | Cat# FC-131-1096 | |
| Commercial assay or kit | RNeasy MiniKit | QIAGEN | Cat# 74104 | |
| Commercial assay or kit | QIAshredder | QIAGEN | Cat# 79656 | |
| Commercial assay or kit | SuperScript First Strand Synthesis kit | Thermo Fisher | Cat#18091050 | |
| Commercial assay or kit | Lipofectamine 3000 | Thermo Fisher | Cat# L3000015 | |
| Chemical compound, drug | Paromomycin | Gemini | Cat# 400-155P | Used 16 g/L water |
| Chemical compound, drug | Brefeldin A (BFA) | BioLegend | Cat# 420601 | Used 10 µg/mL |
| Chemical compound, drug | Thapsigargin | MedChemExpress | HY-13433 | Used 1 µM |
| Chemical compound, drug | GSK2606414 | MedChemExpress | HY-18072 | Used 30 nm |
| Chemical compound, drug | KIRA6 | MedChemExpress | HY-19708 | Used 500 nm |
| Software, algorithm | Prism 8 | GraphPad | RRID:SCR_002798 | |
| Software, algorithm | ImageJ | Fiji | RRID:SCR_003070 | |
| Software, algorithm | FlowJo v10, LLC | TreeStar | RRID:SCR_008520 | |
| Software, algorithm | Kallisto v0.44.0 | BioConductor (Bray et al., 2016) | Pachter Lab | |
| Software, algorithm | Limma-Voom | BioConductor (Law et al., 2014; Ritchie et al., 2015) | ||
| Software, algorithm | Bioconductor tximport | BioConductor (Robinson et al., 2010) | DOI: 10.18129/B9.bioc.tximport | |
| Software, algorithm | Molecular Signatures Database (MSigDB) | UC San Diego and Broad Institute (Mootha et al., 2003; Subramanian et al., 2005) | https://www.gsea-msigdb.org/gsea/msigdb | |
| Software, algorithm | CryptoDB | VEuPathDB | cryptodb.org | |
| Other | Fluorescin Vicia villosa lectin stain | Vector Labs | Cat# FL-1231-2 | IF (1:1000) |
| Other | DAPI stain | Invitrogen | Cat# D1306 | Flow cytometry (1 µg/mL) |
| Other | Hoechst 33342 | Thermo Fisher | Cat# H3570 | IF (1:10,000) |
| Other | Alexa Fluor 647 Phalloidin | Thermo Fisher | Cat# A22287 | IF (1:1000) |