Fig 1.
U1 gene silencing strategy in T. gondii.
(A) Alignment of the first 50 nucleotides of the U1 snRNAs of the indicated organisms. Shaded in green are nucleotides 2–11 complementary to the artificial U1 snRNA recognition sequence in (B). Nucleotides 3–11 are identical across the aligned species. H. sapiens, Homo sapiens; A. thaliana, Arabidopsis thaliana; T. gondii, Toxoplasma gondii; P. falciparum, Plasmodium falciparum. (B) Schematic of strategy. Modification of the target gene’s 3’ terminal exon by insertion of a U1 snRNA recognition sequence leads to recruitment of the U1 snRNP to the pre-mRNA of the target gene. Base pairing between these 10 complementary nucleotides of the U1 site and the U1 snRNA of the U1 snRNP occurs and the resulting U1 snRNP pre-mRNA complex inhibits polyadenylation and therefore pre-mRNA maturation. For simplification nucleotides are illustrated as black bars and the target gene is intron-less. UTR, untranslated region.
Fig 2.
U1 mediated inhibition of reporter gene expression in T. gondii.
(A) Schematic of reporter gene expression constructs engineered for U1 knockdown strategy. In all three constructs mycGFP expression is driven by the constitutive p5RT70 promoter. In mycGFP-U1 and mycGFP-mutU1 two U1 sites were placed between the STOP codon and the 3’ UTR. In contrast to the wild type U1 site the mutated U1 site (mutU1) confers to gene expression. Respective U1 sequences are indicated above the brackets and matching or mismatching with U1 snRNA are illustrated in the box with mutated nucleotides shown in red. In mycGFP-floxU1 the two U1 sites are placed upstream of the loxP flanked 3’ UTR. Upon addition of 50 nM rapamycin DiCre expression and henceforward site specific recombination is induced. Excision of the floxed linker leads to U1 transposition and reduction of mature mycGFP mRNA levels. For fluorescence (B) and immunoblot analysis (C) of RH BloxP DiCre parasites were stable transfected with the expression constructs shown in (A). Clonal parasite lines were cultivated for 48 hr in absence and presence of 50 nM rapamycin prior analyses. Microscopy was performed on intracellular parasites and DAPI was used to visualise the host cell nuclei. (B) Green, GFP; blue, DAPI; scale bar represents 100 μM. GFP expression is only detectable in mycGFP-mutU1 and mycGFP-floxU1 parasites in absence of rapamycin. (C) Immunoblot probed with anti-c-Myc and anti-Catalase antibodies. Catalase was used as loading control. Only mycGFP-floxU1 parasites show rapamycin dependent mycGFP expression. Note the very faint band obtained for mycGFP-floxU1 under rapamycin conditions is similar in intensity to the ones obtained for mycGFP-U1 with or without rapamycin treatment. (D) Quantification of GFP silencing efficiency. The graph shows the percentage of GFP positive and negative parasitophorous vacuoles (PVs) determined by examination of 200 vacuoles per condition based on fluorescence analyses of mycGFP expressing clonal mycGFP-floxU1 parasites grown for 48 h in absence or presence of 50 nM rapamycin. Values are means ±SD (n = 3). Under rapamycin conditions a significant GFP downregulation of 97.5 ± 1.6% was obtained (***, p<0.001, unpaired two-tailed Student’s T-test). (E) Relative expression of GFP for indicated parasite strains as determined by qPCR following normalisation against GFP-mutU1. floxGFP-U1 expression is shown with and without 48 h induction of DiCre by 50 nM rapamycin. Values are means ±SE (n = 3; ** p<0.005, **** p<0.0001, unpaired two-tailed Student’s T-test).
Fig 3.
Targeted silencing of chc1 in ku80::diCre T. gondii parasites.
(A) Schematics of the genomic locus of chc1 after single homologous integration of the BamHI linearised endogenous ha tagging and U1 gene silencing construct. In absence of rapamycin the four U1 sites are separated from the STOP codon of the inserted tag by a floxed spacer consisting of the 3’ UTR of sag1 and the hxgprt selection cassette. Addition of rapamycin induces Cre recombinase activity. The floxed spacer is excised by site specific recombination and the U1 sites placed adjacent to the STOP codon promoting silencing of chc1. (B) Analytical PCR on genomic DNA of clonal CHC1-HA-U1 parasites using oligos indicated as orange arrows in (A). The obtained band of 1484 bp confirms construct integration. (C) Confirmation of Cre/loxP site specific recombination by analytical PCR of genomic DNA. Genomic DNA has been extracted from clonal CHC-HA-U1 parasites grown for 24 h in absence and presence of 50 nM rapamycin. Binding sites of primers used for analysis are indicated with purple arrows in (A). The presence of a specific 2303 bp fragment in absence of rapamycin reflects the genomic composition before Cre/loxP site specific recombination. An additional specific 129 bp fragment in presence of rapamycin verifies successful Cre/loxP site specific recombination. L, ladder. (D) Immunoblot analysis of clonal CHC1-HA-U1 parasites cultured for 24 h with or without 50 nM rapamycin. Membrane probed with anti-HA and anti-Aldolase as loading control. Intensities of detected bands show no difference (E) Giemsa stain. Growth analysis over a 7 days period in absence and presence of 50 nM rapamycin shows no difference in plaque formation. Scale bar represents 500 μm. (F) Immunofluorescence analysis of clonal CHC1-HA-U1 parasites grown for 24 h in presence of 50 nM rapamycin. Green, HA; red, IMC1; blue, Dapi; scale bar represents 10 μM. Endogenous HA-tagged CHC1 localises apical to the nucleus to the trans-Golgi network (yellow asterisks, positive vacuoles). In parasites with silenced chc1 no specific signal for HA is detectable (yellow arrow heads, HA negative vacuoles). In comparison to HA positive parasites, HA negative parasites show abnormal IMC formation. (G) Quantification of chc1 silencing efficiency. The graph shows the percentage of HA positive vacuoles (yellow asterisks in (F)) and negative vacuoles (yellow arrow heads in F) determined by examination of 200 vacuoles per condition based on immunofluorescence analyses of clonal CHC1-HA-U1 parasites grown for 24 h in absence or presence of 50 nM rapamycin. Values are means ±SD (n = 9). Under rapamycin conditions a significant chc1 downregulation of 25.4 ± 2.1 was obtained (***, p<0.001, unpaired two-tailed Student’s T-test). (H) Confirmation of specific CHC1 depletion phenotype by immunofluorescence analysis. CHC1-HA-U1 parasites were cultured for 24 h with or without rapamycin. The fate of the inner membrane complex (IMC), micronemes, rhopties, apicoplast and mitochondrion under rapamycin conditions were visualised by using anti-IMC1, anti-MIC3, and anti-ROP5 antibodies and transient transfection of FNR-RFP and HSP60-RFP respectively. In contrast to normal vacuoles (asterisks) silenced vacuoles (arrow heads) show abnormal IMC formation with MIC3 retention in the ER and mislocalisation of ROP5. FNR mislocalises and HSP60 manifests collapsed interlaced mitochondria. Scale bars represent 10 μm.
Fig 4.
Targeted silencing of vps26 in Δku80-DHFR T. gondii parasites.
(A) Schematics of the genomic locus of vps26 after single homologous integration of the XbaI linearised endogenous ha tagging and U1 gene silencing construct in absence and presence of rapamycin. A codon optimised DiCre cassette is cloned into the SpeI restriction site between the hxgprt selection cassette and the second loxP site. (B) and (C) Analytical PCR on genomic DNA extracted from clonal VPS26-HA-U1 parasites and the parental line Δku80-DHFR grown for 24 h in absence and presence of 50 nM rapamycin. (B) Construct integration was confirmed by using oligos indicated as orange arrows in (A). Theoretical fragment sizes of 2200 bp and 2500 bp for 5’ and 3’ integration were amplified respectively in VPS26-HA-U1 parasites independent of rapamycin. (C) Confirmation of Cre/loxP site specific recombination. Binding sites of primers used for analysis are indicated with purple arrows in (A) with the theoretical fragment sizes. Even though it was impossible to amplify the very large spacer (5900 bp) in absence of rapamycin the PCR product of 600 bp in presence of rapamycin confirms Cre/loxP site specific recombination. L, ladder. (D) Immunoblot analysis of clonal VPS26-HA-U1 parasites cultured for 48 h with or without 50 nM rapamycin. Membrane probed with anti-HA and anti-IMC1 as loading control. In presence of rapamycin no downregulation of VPS26-HA expression was observed. (E) Giemsa stain. Growth analysis over a 7 days period in absence and presence of 50 nM rapamycin shows no difference in plaque formation. Scale bar represents 500 μm. (F) Immunofluorescence analysis of clonal VPS26-HA-U1 parasites grown for 48 h in presence of 50 nM rapamycin. Green, HA; red, IMC1; blue, DAPI; scale bar represents 10 μM. Endogenous HA-tagged VPS26 localises apical to the nucleus in Golgi region. Parasites with silenced vps26 are encircled in yellow. (G) Quantification of vps26 silencing efficiency. The graph shows the percentage of HA positive and negative vacuoles determined by examination of 100 vacuoles per condition based on immunofluorescence analyses of clonal VPS26-HA-U1 parasites grown for 48 h in absence or presence of 50 nM rapamycin. Values are means ±SD (n = 3). Under rapamycin conditions a vps26 downregulation of 12.6 ± 5.32 was obtained (***, p<0.001, Mann-Whitney test).
Fig 5.
Generation of optimised DiCre expressing recipient and reporter strains DiCreΔku80 and DiCre ku80::KRedflox-YFP.
(A) Schematics of parental line RH DiCre-DHFR. In this strain the DiCre cassette is randomly integrated into the genome and not targeted into the ku80 gene locus. (B) Schematics of DiCre ku80::hxgprt generation. The ku80 gene is replaced with the hxgprt selection cassette by double homologous recombination and positively selected for HXGPRT using mycophenolic acid and xanthine. Yellow crosses indicate homologous regions in (A) and (B) between which crossovers take place. (C) Schematics of DiCreΔku80 generation. The hxgprt selection cassette is removed by double homologous recombination with an empty knock out construct and negatively selected for HXGPRT using 6-thioxanthine. Red crosses indicate homologous regions in (B) and (C) between which crossovers occur. (C) Schematics of DiCre ku80::KRedflox-YFP generation. The hxgprt selection cassette is replaced with the reporter cassette [7] by double homologous and negatively selected for HXGPRT using 6-thioxanthine. Red crosses indicate homologous regions in (B) and (D) between which crossovers occur. In absence of rapamycin the constitutive p5RT70 promoter drives Killer Red (KRed) expression. Upon addition of rapamycin the floxed open reading frame of kred is excised by Cre/loxP site specific recombination and replaced by yfp. The shift from red fluorescent to green fluorescent parasites can be used as a measure of Cre recombinase activity which allows discrimination of single parasites. (E-E”) Analytical PCRs on genomic DNA extracted from the indicated parasite strains. Oligonucelotides used are indicated as arrowheads next to the gel or on top of the lanes. Oligonucleotide binding sites are indicated with the same symbols in (A-D). Predicted PCR product sizes are displayed on black dashed lines between the respective forward and reverse oligonucleotides. Diagnostic PCR for the DiCre cassette was positive in all strains (E). Diagnostic PCRs with different oligonucleotide combinations show successful replacement of the ku80 locus with the hxgprt selection cassette in the DiCre ku80::hxgprt strain (E’ and E”). Diagnostic PCRs confirm cleanup of the hxgprt selection cassette in the ku80 locus of DiCreΔku80 strain and replacement with the reporter cassette in the DiCre ku80::KRedflox-YFP strain (E”). (F) Flourescence analysis of DiCre ku80::KRedflox-YFP parasites in presence and absence of 50 nM rapamycin for 48 h. Upon rapamycin induction parasite fluorescence shifts from red to green. Scale bar represents 50 μm. (G) Quantification of Cre/loxP site specific recombination efficiency of the DiCre strains. Except for DiCre ku80::KRedflox-YFP all strains were transiently transfected with the reporter cassette. The graph shows fluorescence analyses of parasites grown for 48 h in presence of 50 nM of rapamycin, the percentage of KRed positive and YFP positive vacuoles were determined by examination of 200 vacuoles in three fields of view. Values are means ±SD (n = 4). *, P-Value is <0.0001 ku80::diCre vs RH DiCre-DHFR/ DiCreΔku80/ DiCre ku80::KRedflox-YFP (indicated exemplary for the first pair only), **, P-Value is <0.00001 in a 2-tailed Student’s t-test switch from red to green. (H) Immunoblot analysis of indicated parasite strains cultured for 48 h with or without 50 nM rapamycin. Membrane probed with anti-FRB and anti-Catalase antibodies. The latter was used as loading control. FRB-Cre59 subunits show greater abundance in strains with randomly integrated DiCre cassette in comparison to the ku80::diCre strainwith a single DiCre cassette in the ku80 locus.
Fig 6.
Targeted silencing of drpC in ku80::diCre, DiCreΔku80, and DiCre ku80::KRedflox-YFP parasites.
(A) Schematic of the genomic locus of drpC after single homologous integration of the NsiI linearised endogenous ha tagging and U1 gene silencing construct in absence of rapamycin. Oligos used for analytical PCRs are illustrated with orange and purple arrows. Theoretical product sizes dependent on rapamycin are indicated below antisense oligos in the colour of the respective sense oligo. Stable construct integration into ku80::diCre ([7], B-F), DiCreΔku80 (B’-F’), and DiCre ku80::KRedflox-YFP (B”-F”). (B-B”) Analytical integration PCR (orange and mixed oligo combination) and Cre/loxP site specific recombination PCR (all oligo combinations) on genomic DNA extracted from clonal parasite lines after 48 h incubation with or without rapamycin. Resulting PCR product sizes reveal correct integration into all recipient strains, but only complete Cre/loxP site specific recombination in the new recipient strains (B’ and B”). L, ladder. (C-C”) Immunoblot analysis of total parasite lysates of clonal lines cultured for 48 h with or without 50 nM rapamycin. Membrane probed with anti-HA, anti-Aldolase, and anti-GFP antibodies. Aldolase was used as loading control. Only new recipient strains show clear rapamycin dependent DrpC-HA expression (C’ and C”). As expected GFP expression is only detectable in presence of rapamycin in the reporter recipient strain (C”). (D-D”) Quantification of drpC silencing efficiency. Graphs show the percentage of HA positive vacuoles and negative vacuoles determined by examination of 200 vacuoles per condition based on immunofluorescence analyses as shown in S1 Fig). Values are means ±SD (n = 3). In contrast to a partial drpC downregulation of 10.5 ± 0.8 (**, p<0.01, unpaired two-tailed Student’s T-test) in ku80::diCre parasites (D), drpC was almost completely silenced in DiCreΔku80 (D’; 98.5 ± 0.4; ***, p<0.001, unpaired two-tailed Student’s T-test) and DiCre ku80::KRedflox-YFP (D”; 98.3 ± 0.6; ***, p<0.001, unpaired two-tailed Student’s T-test) parasites under rapamycin conditions. (E-E”) Giemsa stain. Growth analysis over a 7 days period in absence and presence of 50 nM rapamycin shows no difference in plaque formation in ku80::diCre parasites, while in DiCreΔku80 and ku80::KRedflox-YFP parasites no plaque formation was observed in the presence of rapamycin. Scale bar represents 500 μm. (F’-F”) qRT-PCR. Relative expression of DrpC in the indicated parasite strains normalised against DrpC expression in RH parasites. DrpC expression is shown after 72 h incubation with and without rapamycin (50 nM). Values are means ±SE (n = 3; ** p<0.001, * p<0.005, unpaired two-tailed Student’s T-test).
Fig 7.
The U1 mediated knockdown strategy is not functional in P. falciparum.
(A) Schematic showing the strategy used to replace the endogenous pfsub1 3’ UTR with a floxed heterologous PbDT 3’ UTR with a downstream sequence comprising 10 U1 recognition sequences. The single cross-over homologous recombination event also results in the fusion of a HA3 epitope tag to PfSUB1. The predicted results of DiCre mediated recombination induced by treatment with rapamycin are shown. The positions of hybridization of PCR primers designed to specifically amplify sequences from the intact and excised locus are indicated with red and blue arrows. (B) IFA of mature schizonts of integrant clone D3, probed with mAb NIMP.M7 (anti PfSUB1) and mAb 3F10 (anti HA3). (C) Efficient DiCre-mediated recombination between the integrated loxP sites, as shown by diagnostic PCR analysis of parasite genomic DNA extracted 44 h following rapamycin treatment of two independent parasite clones D3 and B11. The blue and red arrows correspond to primer pairs marked in (A). (D) Upper panel: Western blot analysis with mAb 3F10 shows no down-regulation of PfSUB1 protein expression protein 44 h or 6 days after rapamycin treatment. Lower panel: loading controls, showing total protein SDS PAGE gels detected by UV fluorescence on the BioRad Chemidoc XRS system prior to Western transfer.
Table 1.
Correlation of U1 gene silencing efficiency to genomic locus and copy numbers of DiCre.
Table 2.
Oligonucleotides used for cloning of T. gondii and P. falciparum constructs, confirmation of construct integration and site-specific recombination.
LIC, ligation independent cloning; for, forward; rev, reverse; mut, mutated; ssr, site specific recombination; integr, integration