Fig 1.
Expression of 7-Helix-1 in P. falciparum gametocytes.
(A) Schematic depicting the domain structure of 7-Helix-1. The seven predicted transmembrane domains (red boxes) are highlighted. The black bars underneath the structure of 7-Helix-1 denote regions of the recombinant proteins. AA, amino acids. (B) Transcription profile of 7-helix-1 in the WT NF54 blood and sexual stages. Transcript of 7-helix-1 (527 bp) was amplified by diagnostic RT-PCR from ring stages (R), trophozoites (TZ), schizonts (SZ), immature (GCII-IV) and mature (GCV) gametocytes and gametocytes at 15 min p.a. (aGC). Transcript analysis of pfaldolase (378 bp) was used for loading control. Transcript analyses of pfama1 (189 bp) and pfccp2 (198 bp) were used to demonstrate purity of the asexual blood stage and gametocyte samples, respectively. (C) Expression of 7-Helix-1 in the blood and sexual stages. WB analysis of lysates from asexual blood stages (ABS), immature (GCII-IV), mature (GCV) and activated gametocytes (aGC; at 30 min p.a.) of WT NF54 using the mouse anti-7-Helix-1rp2 antisera was employed for detection of 7-Helix-1 (~50 kDa). Equal loading was confirmed using a polyclonal mouse anti-Pf39 antiserum (~39 kDa). (D) Expression of HA-tagged 7-Helix-1 in the 7-Helix-1-HA line. Gametocyte (GC) lysates of WT NF54 and the 7-Helix-1-HA line were subjected to WB and immunolabeled with rabbit anti-HA antibodies to detect 7-Helix-1-HA (~60 kDa). Lysate of uninfected RBCs was used for negative control; equal loading was confirmed using a polyclonal mouse anti-Pf39 antiserum (~39 kDa). Results (in B-D) are representative of two to three independent experiments.
Fig 2.
Granular localization of 7-Helix-1 in female P. falciparum gametocytes.
(A) Immunolocalization of 7-Helix-1 in the sexual stages. Gametocytes (GC stage II–V) and activated gametocytes (aGC; at 2, 10 and 15 min p.a.) of WT NF54 were immunolabeled with mouse anti-7-Helix-1rp1 antisera (green) and counterlabeled with rabbit antibodies directed against Pfs230 and Pfs25 as indicated (red). (B) Female-specific expression of 7-Helix-1. WT NF54 gametocytes were immunolabeled with mouse anti-7-Helix-1rp1 antisera (green) and counterlabeled with anti-Pfs25 antisera (red). Nuclei (in A and B) were highlighted by Hoechst33342 nuclear stain (blue). Bar, 5 μm. Results are representative of five independent experiments.
Fig 3.
Impaired transmission of 7-Helix-1-KO gametocytes to mosquitoes.
(A) Mosquito infection efficiency of 7-Helix-1-KO. Enriched mature gametocytes of WT NF54 or the 7-Helix-KO line 2E6 were fed to An. stephensi mosquitoes via SMFAs. The numbers of oocysts per midgut were counted at day 10 p.i. in five independent feeds. * p ≤ 0.05; *** p ≤ 0.001 (Mann-Whitney-U test). (B) Efficiency of 7-Helix-1-KO to develop into mosquito midgut stages. Following SMFAs as described above, midgut smears at 24 h p.i. were subjected to IFA, the ookinetes and retorts were immunolabeled with rabbit anti-Pfs28 antisera and counted in 30 optical fields for four times (mean ± SD). (C-E) Efficiency of 7-Helix-1-KO to undergo sexual reproduction. Mature WT NF54 and 7-Helix-1-KO 2E6 gametocytes were activated in vitro. Samples were taken at 4 h p.a. (zygotes), 30 min p.a. (macrogametes) or 15 min p.a. (exflagellation centers). Zygotes (C) and macrogametes (D) were subjected to IFA for immunolabelling with anti-Pfs25 antibody and counted in 30 optical fields in triplicate; exflagellation centers (E) were counted in 30 optical fields for four times using light microscopy (mean ± SD). The numbers of WT parasites were set to 100% (for B-E). * p ≤ 0.05; **** p ≤ 0.0001 (Student‘s t-test). Results (in B-E) are representative of three to eight independent experiments.
Fig 4.
Ultrastructural analysis of activated 7-Helix-1-KO gametocytes and phenotype rescue.
(A) Ultrastructure of 7-Helix-1-KO gametocytes. Transmission electron microscopic analyses of WT NF54 and 7-Helix-1-KO 2E6 gametocytes at 0 and 30 min p.a.. EM, erythrocyte membrane; IMC, inner membrane complex; PPM, parasite plasma membrane; PVM, parasitophorous vacuole membrane. Bar, 2 μm; enlargement, 0.2 μm. (B, C) 7-Helix-1-KO phenotype rescue by episomal complementation. Gametocytes of WT NF54, 7-Helix-1-KO 1D12 and 7-Helix-1-KO(+) were activated in vitro. Samples were taken at 30 min (macrogametes, B) and 4 h (zygotes, C) p.a. and immunolabeled with anti-Pfs25 antibody. The numbers of parasites were counted in 30 optical fields in triplicate (mean ± SD). The numbers of WT parasites were set to 100%. n.s., not significant; * p ≤ 0.05; ** p ≤ 0.01 (One-Way ANOVA with Post-Hoc Bonferroni Multiple Comparison test). Results (in B and C) are representative of three independent experiments.
Fig 5.
Transcriptional deregulation in 7-Helix-1-KO gametocytes and loss-of-function mimicry by chemical inhibition of translation.
(A) Pie charts depicting genes with deregulated transcripts in activated 7-Helix-1-KO gametocytes grouped by function. Mature gametocytes of strain WT NF54 and 7-Helix-1-KO 2E6 were activated and samples were collected at 30 min p.a. Total RNA was isolated and cDNA synthesized to be employed in microarray assays. Genes expressed in gametocytes (defined by transcript levels > 50% of peak transcript levels) with relative transcript levels greater (left pie) and lower (right pie) than 1.5-fold were grouped based on the predicted functions. Microarray analyses were performed once. (B) Loss-of-function analysis in gametocytes following chemical inhibition of translation. Mature gametocytes of WT NF54 or 7-Helix-1-KO line 2E6 were treated with emetine or cycloheximide at IC50 concentrations (as determined by Malstat assay) or solvent alone for 30 min at 37°C, followed by in vitro activation. Samples were taken at 30 min (macrogametes) and 4 h (zygotes) p.a. and immunolabeled with anti-Pfs25 antibody. The numbers of parasites per 1,000 RBCs were counted five times (mean ± SD). Gametes/zygotes formed in the solvent-treated WT line were set to 100%. *** p ≤ 0.001 (One-Way ANOVA with Post-Hoc Bonferroni Multiple Comparison test). Results are representative for two independent experiments.
Fig 6.
Localization of 7-Helix-1 in SGs and interaction with translational repressors.
(A) Co-localization of 7-Helix-1 with mRNA-aggregates. Mature WF NF54 gametocytes were subjected to mRNA-FISH-IFA and mRNA was labeled with a biotinylated oligo-dT25 probe (red); counterlabeling was performed using mouse anti-7-Helix-1rp2 antisera (green). Nuclei were highlighted by Hoechst33342 nuclear stain (blue). Frame indicates the area chosen for enlargement. DIC, differential interference contrast. Bar, 5 μm; enlargement, 1 μm. (B) Accumulation of 7-Helix-1 in SG fractions. Gametocytes of line 7-Helix-1-HA were stressed by treatment with sodium arsenite for 1 h and a SG core fraction enrichment was conducted. Lysates of 7-Helix-1-HA gametocytes (-) and of enriched SGs (+) were subjected to WB, using mouse antisera directed against Pf39 (~39 kDa) or PfM1-AP (~126 and 68 kDa, black arrows) or rabbit antibodies against HSP70-1 (~70 kDa) or the HA-tag to detect 7-Helix-1-HA (~60 kDa). (C) Co-immunoprecipitation of 7-Helix-1 with CITH, PABP1 and DOZI. Lysates of WT NF54 or 7-Helix-1-HA gametocytes were subjected to co-immunoprecipitation assays using polyclonal mouse anti-7-Helix-1rp2 antisera or polyclonal rabbit anti-DOZI antisera, followed by WB using rabbit anti-CITH, anti-PABP1 and anti-HA antibodies or mouse anti-Pf39 antibody to detect precipitated proteins. Grey arrow indicates the expected running line in the negative control. Asterisk indicates a band corresponding to the precipitation antibody. (D) Co-localization of 7-Helix-1 with CITH, PABP1 and DOZI. WT NF54 gametocytes were immunolabeled with mouse anti-7-Helix-1rp2 antisera (green) and rabbit anti-CITH, anti-PABP1, or anti-DOZI antibodies (red). Nuclei were highlighted by Hoechst33342 nuclear stain (blue). DIC, differential interference contrast. Bar, 5 μm. Results are representative of three independent experiments.
Fig 7.
Complex formation of 7-Helix-1 with Puf2 and repressed pfs25 mRNA and Pfs25 synthesis.
(A) Co-immunoprecipitation of 7-Helix-1 with Puf2. Co-immunoprecipitation assays on lysates of 7-Helix-1-HA or WT NF54 gametocytes were employed using either polyclonal mouse anti-Puf2 or anti-Pf39 antisera, followed by WB using rabbit anti-HA antibodies or mouse anti-Pf39 antibody to detect precipitated proteins. Black arrows indicate precipitated proteins, grey arrows indicate expected running lines in the negative controls. Asterisks indicate bands corresponding to the precipitation antibodies. Results are representative of three independent experiments. (B) Association of repressed transcript with 7-Helix-1. RIP assays on lysates of 7-Helix-1-HA gametocytes were employed using either rabbit anti-CITH or rabbit anti-HA antibodies, followed by RNA isolation. RT-PCR was performed amplifying co-eluted transcripts of ccp2 (198 bp), pfs25 (459 bp) and pfs28 (494 bp). cDNA preparation lacking the reverse transcriptase (-RT) was used to prove absence of gDNA contamination. (C, D) Quantitative expression analysis of gametocyte-specific adhesion proteins. (C) Expression of Pfs25 and Pfs230 in activated gametocytes. WT NF54 and 7-Helix-1-KO 2E6 gametocytes at 30 min p.a. were immunolabeled with rabbit anti-Pfs25 (green) and mouse anti-Pfs230 antibodies (red). Nuclei were highlighted by Hoechst33342 nuclear stain (blue). Bar, 5 μm. (D) Quantitative IFA analysis of Pfs25 and Pfs230. IFAs were employed as described above and the fluorescence intensities for Pfs25 and Pfs230 of 20 immunolabeled activated gametocytes were measured in triplicate using ImageJ (mean ± SD). The respective signal in WT lysates was set to 1. *** p ≤ 0.001 (Student‘s t-test). Results (in A-C) are representative of two to three independent experiments.