Apicomplexan F-actin is required for efficient nuclear entry during host cell invasion

The obligate intracellular parasites Toxoplasma gondii and Plasmodium spp. invade host cells by injecting a protein complex into the membrane of the targeted cell that bridges the two cells through the assembly of a ring‐like junction. This circular junction stretches while the parasites apply a traction force to pass through, a step that typically concurs with transient constriction of the parasite body. Here we analyse F‐actin dynamics during host cell invasion. Super‐resolution microscopy and real‐time imaging highlighted an F‐actin pool at the apex of pre‐invading parasite, an F‐actin ring at the junction area during invasion but also networks of perinuclear and posteriorly localised F‐actin. Mutant parasites with dysfunctional acto‐myosin showed significant decrease of junctional and perinuclear F‐actin and are coincidently affected in nuclear passage through the junction. We propose that the F‐actin machinery eases nuclear passage by stabilising the junction and pushing the nucleus through the constriction. Our analysis suggests that the junction opposes resistance to the passage of the parasite's nucleus and provides the first evidence for a dual contribution of actin‐forces during host cell invasion by apicomplexan parasites

Targeted silencing of <i>chc1</i> in <i>ku80</i>::<i>diCre T</i>. <i>gondii</i> parasites.

<p>(A) Schematics of the genomic locus of <i>chc1</i> after single homologous integration of the BamHI linearised endogenous <i>ha</i> 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 <i>sag1</i> and the <i>hxgprt</i> 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 <i>chc1</i>. (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 <i>chc1</i> 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 <i>chc1</i> 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 <i>chc1</i> 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.</p

The U1 mediated knockdown strategy is not functional in <i>P</i>. <i>falciparum</i>.

<p>(A) Schematic showing the strategy used to replace the endogenous <i>pfsub1</i> 3’ UTR with a floxed heterologous <i>PbDT</i> 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.</p

Targeted silencing of <i>drpC</i> in <i>ku80</i>::<i>diCre</i>, DiCreΔ<i>ku80</i>, and DiCre <i>ku80</i>::<i>KRed</i>_<i>flox</i><i>-YFP</i> parasites.

<p>(A) Schematic of the genomic locus of <i>drpC</i> after single homologous integration of the NsiI linearised endogenous <i>ha</i> 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 <i>ku80</i>::<i>diCre</i> ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130356#pone.0130356.ref007" target="_blank">7</a>], B-F), DiCreΔ<i>ku80</i> (B’-F’), and DiCre <i>ku80</i>::<i>KRed</i>_{<b><i>flox</i></b>}<i>-YFP</i> (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 <i>drpC</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130356#pone.0130356.s001" target="_blank">S1 Fig</a>). Values are means ±SD (n = 3). In contrast to a partial <i>drpC</i> downregulation of 10.5 ± 0.8 (**, p<0.01, unpaired two-tailed Student’s T-test) in <i>ku80</i>::<i>diCre</i> parasites (D), <i>drpC</i> was almost completely silenced in DiCreΔ<i>ku80</i> (D’; 98.5 ± 0.4; ***, p<0.001, unpaired two-tailed Student’s T-test) and DiCre <i>ku80</i>::<i>KRed</i>_{<b><i>flox</i></b>}<i>-YFP</i> (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 <i>ku80</i>::<i>diCre</i> parasites, while in DiCreΔ<i>ku80</i> and <i>ku80</i>::<i>KRed</i>_{<b><i>flox</i></b>}<i>-YFP</i> 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).</p

Generation of optimised DiCre expressing recipient and reporter strains DiCreΔ<i>ku80</i> and DiCre <i>ku80</i>::<i>KRed</i>_<i>flox</i><i>-YFP</i>.

<p>(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 <i>ku80</i> gene locus. (B) Schematics of DiCre <i>ku80</i>::<i>hxgprt</i> generation. The <i>ku80</i> gene is replaced with the <i>hxgprt</i> 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 DiCreΔ<i>ku80</i> generation. The <i>hxgprt</i> 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 <i>ku80</i>::<i>KRed</i>_{<b><i>flox</i></b>}<i>-YFP</i> generation. The <i>hxgprt</i> selection cassette is replaced with the reporter cassette [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130356#pone.0130356.ref007" target="_blank">7</a>] 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 <i>kred</i> is excised by Cre/loxP site specific recombination and replaced by <i>yfp</i>. 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 <i>ku80</i> locus with the <i>hxgprt</i> selection cassette in the DiCre <i>ku80</i>::<i>hxgprt</i> strain (E’ and E”). Diagnostic PCRs confirm cleanup of the <i>hxgprt</i> selection cassette in the <i>ku80</i> locus of DiCreΔ<i>ku80</i> strain and replacement with the reporter cassette in the DiCre <i>ku80</i>::<i>KRed</i>_{<b><i>flox</i></b>}<i>-YFP</i> strain (E”). (F) Flourescence analysis of DiCre <i>ku80</i>::<i>KRed</i>_{<b><i>flox</i></b>}<i>-YFP</i> 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 <i>ku80</i>::<i>KRed</i>_{<b><i>flox</i></b>}<i>-YFP</i> 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 <i>ku80</i>::<i>diCre</i> vs RH DiCre-DHFR/ DiCreΔ<i>ku80/</i> DiCre <i>ku80</i>::<i>KRed</i>_{<b><i>flox</i></b>}<i>-YFP</i> (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 <i>ku80</i>::<i>diCre</i> strainwith a single DiCre cassette in the <i>ku80</i> locus.</p

Targeted silencing of <i>vps26</i> in Δ<i>ku80-</i>DHFR <i>T</i>. <i>gondii</i> parasites.

<p>(A) Schematics of the genomic locus of <i>vps26</i> after single homologous integration of the XbaI linearised endogenous <i>ha</i> 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 <i>hxgprt</i> 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 Δ<i>ku80-</i>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 <i>vps26</i> are encircled in yellow. (G) Quantification of <i>vps26</i> 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 <i>vps26</i> downregulation of 12.6 ± 5.32 was obtained (***, p<0.001, Mann-Whitney test).</p

Oligonucleotides used for cloning of <i>T</i>. <i>gondii</i> and <i>P</i>. <i>falciparum</i> constructs, confirmation of construct integration and site-specific recombination.

<p>LIC, ligation independent cloning; for, forward; rev, reverse; mut, mutated; ssr, site specific recombination; integr, integration</p

U1 gene silencing strategy in <i>T</i>. <i>gondii</i>.

<p>(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. <i>H</i>. <i>sapiens</i>, <i>Homo sapiens</i>; <i>A</i>. <i>thaliana</i>, <i>Arabidopsis thaliana</i>; <i>T</i>. <i>gondii</i>, <i>Toxoplasma gondii</i>; <i>P</i>. <i>falciparum</i>, <i>Plasmodium falciparum</i>. (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.</p

Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics.

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation