Additional file 1 of Stable expression of mucin glycoproteins GP40 and GP15 of Cryptosporidium parvum in Toxoplasma gondii

Additional file 1: Table S1. Primers used in this study

TPI2 depletion does not affect fatty acids synthesis.

The iTPI2 strain was pretreated with or without rapamycin for 3 days and then incubated in medium containing 8 mM 13C6-glucose and cultured under the same pretreatment condition for another two days. Then the parasites were harvested and the incorporation of 13C into each fatty acid species was determined by LC-MS. The percentage of fatty acid that contained one or more 13C atoms was plotted for each fatty acid species. (PDF)</p

Raw data of DOXP/MEP/IPP/DMAPP levels measured by MS.

(DOCX)</p

The messenger RNA levels of TPI2 or GAPDH2 before and after gene depletion, as determined by RT-PCR.

The iTPI2 and iGAPDH2 mutants were treated with or without rapamycin or ATc for 5 days, respectively. Then, the parasites were collected and the messenger RNA levels of TPI2 or GAPDH2 were quantified by RT-PCR, using β-tubulin as a reference. The relative mRNA level of the target gene in each sample was expressed as fold of the mRNA level of β-tubulin. Means ± SEM of three independent experiments. ***P (PDF)</p

The cytosolic TPI1 and apicoplast TPI2 are both required for <i>T</i>. <i>gondii</i> growth.

A, the DiCre system was used to conditionally knock out the TPI genes. A loxP flanked TPI1 or TPI2 construct was used to replace the corresponding TPI gene using CRISPR/Cas9 assisted homologous recombination in the DiCre-T2A strain. The TPI genes in the resulting iTPI strains could be excised after rapamycin treatment. B-E, diagnostic PCRs (B, D) and Western blotting (C, E) on representative iTPI clones, to demonstrate the correct integration of corresponding constructs and rapamycin dependent depletion of TPI proteins. F, intracellular replication comparing the proliferation rates of indicated strains in the presence and absence of rapamycin. Means ± SEM of three independent experiments, each with three replicates. ***P of more than 100 plaques, ***P in vitro and in vivo. The iTPI2 strain was treated with rapamycin for three days to obtain TPI2- parasites (indicated by YFP+), which were then mixed with non-treated iTPI2 parasites (which were TPI2+ and YFP-) at the ratio of 1:1. Then, the mixed culture was either used to infect mice (in vivo) or grown in HFF monolayers (in vitro) and the ratio between YFP+ (TPI2-) and YFP- (TPI2+) populations was determined by fluorescent microscopy at indicated time point. Means ± SEM of three independent experiments.</p

Depletion of GAPDH2 leads to impaired parasite growth.

A, strategy used to construct the iGAPDH2 strain, which contained an anhydrotetracycline (ATc) regulatable promoter pS1O7 to regulate the expression of GAPDH2. The endogenous GAPDH2 gene was replaced with a construct that contained Ty tagged GAPDH2 expressed from pS1O7 and the selection marker DHFR. B, diagnostic PCR on an iGAPDH2 clone. C, Western blotting demonstrating the reduction of GAPDH2 expression after ATc treatment, as probed by a Ty antibody. D, IFA checking the expression of GAPDH2 in the presence or absence of ATc. Note that after five days ATc treatment, about 40% of parasites still expressed GAPDH2 (as determined by visible Ty staining signal in IFA), although the expression level might not be as high as that in parasites without ATc treatment. E, intracellular replication assay comparing the proliferation rates of iGAPDH2 in the presence or absence of ATc treatment. ***P of more than 100 plaques (G), Means ± SD of three replicates (H), ***P < 0.001, student’s t-test.</p

Plasmids used in this study.

(DOCX)</p

The iTPI2 strain turned on YFP expression after rapamycin treatment, which indicated TPI2 deletion.

Parasites were treated with rapamycin for five days and then imaged under a fluorescence microscope. Nearly 100% of the parasites became YFP positive after rapamycin treatment. (PDF)</p

TPI2 depletion reduces isoprenoid precursors synthesis through the MEP pathway.

Relative abundance of DOXP, MEP and IPP/DMAPP in the iTPI2 (A) and iTPI1 (B) strains cultured in the presence (+rapa) or absence (-rapa) of rapamycin, as determined by mass spectrometry.</p

Primers used in this study.

(DOCX)</p