Release of Plasmodium sporozoites requires proteins with histone-fold dimerization domains

Abstract

The sporozoite, the stage of the malaria parasite transmitted by the mosquito, first develops for ∼2 weeks in an oocyst. Rupture of the oocyst capsule is required for release of sporozoites, which then transfer to the salivary gland where they are injected into a new host. Here we identify two parasite proteins that we call oocyst rupture proteins 1 (ORP1) and ORP2. These proteins have a histone-fold domain (HFD) that promotes heterodimer formation in the oocyst capsule at the time of rupture. Oocyst rupture is prevented in mutants lacking either protein. Mutational analysis confirms the HFD as essential for ORP1 and ORP2 function, and heterodimer formation was verified in vitro. These two proteins are potential targets for blocking transmission of the parasite in the mosquito.

Figure 1. NF-YB/NF-YC proteins are essential for oocyst rupture.

(a) Schematic representation of the HFD containing proteins in P. berghei (ORP1, PBANKA_0902500 and ORP2, PBANKA_1303400) compared with the human NF-YB and NF-YC. HFD is indicated in blue in the upper part and in red in the bottom part. (b) Multiple sequence alignment of N-terminal portion of NFY-B from human (aa 34–146), Drosophila melanogaster (aa 17–130) and Dictyostelium discoideum (aa 28–140), HAP3 of Saccharomyces cerevisae (aa 16–129) and aa 750–863 of ORP1. The peptide used for antibody generation is marked with a green rectangle. Accession numbers: P13434.1HAP3_YEAST, Q54WV0.1NFYB_DICDI, P63140.1, P25208.2 NFYB_HUMAN and NP_609997.1 (D. melanogaster). (c) Multiple sequence alignment of HFD of NFY-C from human (aa 22–125), D. melanogaster (aa 133–236) and D. discoideum (aa 252–353), HAP5 of S. cerevisae (aa 133–236) and aa 9–115 of ORP2. Accession numbers: Q557I1.1 NFYC_DICDI, NP_572354.1 (D. melanogaster), Q13952.3 NFYC_HUMAN, Q02516.1 HAP5_YEAST. In b,c, the α-helices of the HFD (α1–α3) are denoted in red on top of the sequence alignment and also the NF-Y-specific αC domain. The domains are derived from a published alignment. (d,e) Oocyst loads of WT (red circles), orp1(−) cl1 and cl2 (blue squares), and orp2(−) cl1 and cl2 (green triangles) mutants. The data represent pooled data from two experiments of each clone. The complete data set is presented in Supplementary Fig. 4 and Supplementary Table 1. ***P<0.0001, **P<0.01 and *P<0.05, Mann–Whitney test. Error bars denote s.e.m. (d) Oocyst loads counted at day 11 p.b.f. (e) Oocyst loads counted at day 21 p.b.f. (f) Representative pictures of orp1(−) cl1 (left) and orp2(−) cl 1 (right) oocysts containing sporozoites in midguts dissected at day 20 p.b.f. The oocysts were labelled with Cap380 (red). The magnification of one oocyst shows also the nuclei of the sporozoites (4,6-diamidino-2-phenylindole (DAPI), blue) and the bright field image (BF) sporozoites within the oocysts. Scale bar, 15 μm. WT infected mosquitoes contained very few degenerated oocysts and are not shown.

Figure 2. ORP1 is expressed in the oocyst capsule and localization is independent of ORP2.

(a) Localization of ORP1 using an antibody directed against a peptide of the HFD in ookinetes, oocysts at 10 days p.b.f. and sporozoites. Scale bars, 2.5, 15 and 10 μm, respectively. The ookinete was also labelled with an antibody directed against the P28 surface protein (red). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; blue). BF, bright field image. (b) Schematic depiction of a construct encoding a GFP fusion of the N-terminal 365 aa fragment of ORP1. (c) Localization of ORP::GFP in ookinetes, oocysts at 6 days p.b.f. and sporozoites using immunolabelling. Of note is the localization of ORP1::GFP in proximity to Cap380 in the oocyst periphery. Scale bars, 2.5, 10 and 5 μm, respectively. (d) ORP1 is localized to the oocyst capsule revealed by double labelling with antibodies recognizing ORP1 and CSP, at day 7 bpf (scale bars, 10 μm (top row) and 2.5 μm (bottom row)). (e) Immunolabelling of an orp2(−) mutant oocyst reveals localization of ORP1 (red) at the oocyst periphery. Scale bar, 15 μm.

Figure 3. ORP2 is re-localized from the cytoplasm to the oocyst capsule concomitantly with oocyst rupture.

(a) ORP2::mCherry construct. (b) Double labelling of ORP2::mCherry (red) and ORP1 (green) in oocysts 7 and 10 days p.b.f. Sporozoites have not yet formed. ORP2 is localized in the cytoplasm. Scale bars, 15 and 20 μm. (c) A mature oocyst at day 13 p.b.f. ORP2 is detected at the periphery partly overlapping with the Cap380 marker of the oocyst capsule. Scale bar, 10 μm. (d) Higher magnification of the oocyst in c reveals ORP2 (red) localization close to the capsule highlighted with Cap380 (green). Scale bar, 2.5 μm.

Figure 4. ORP1 and ORP2 HFDs form a dimer in vitro.

(a) Structural modelling of ORP1/ORP2 dimer together with human NF-YB and NF-YC chains used as templates. Thick ribbons show ORP1 in red and ORP2 in blue. The thin orange and middle blue ribbons present the template chains B and C of 1N1J.pdb, respectively, and the thin yellow and light blue ribbons the template chains B and C of 4AWL.pdb, respectively. (b) Pull-down assay of His-ORP2 with Ni-NTA beads reveal binding of ORP1. Gene sequences encoding peptides corresponding to each HFD with short flanking sequence were cloned in the pET-Duet-1 vector and the proteins expressed together in E. coli. ORP1 was tagged C-terminally to the S-tag and ORP2 N-terminally to a poly-His tag. The western blotting was loaded with cleared lysate (lanes L) and eluate from the Ni-NTA bead (lane E) and the proteins visualized by commercial antibodies directed against the tags. M, molecular weight marker in kDa. (c) Co-immunoprecipitation using the antibody against the S-tag to capture the complex from bacterial lysate. His-ORP2 was detected in the immuno-precipitated fraction. INP, input, bacterial lysate; PC, pre-cleaned lysate; IP, precipitated fraction; M, molecular weight marker in kDa.

Figure 5. The HFD is necessary for ORP1 function.

(a) Schematic depiction of the construct encoding ORP1 in which the α2 to αC helices (aa 797–860) of the HFD were deleted leaving the epitope for the ORP1 antibody intact (green box). (b) Oocyst load in mosquitoes infected with orp1-hfd⁻compared with WT at day 11 (left) and day 21 p.b.f. (right). Pooled data from two independent experiments of each clone. NS, nonsignificant, ***P<0.0001, Mann–Whitney test. The complete data set is presented in Supplementary Fig. 9 and Supplementary Table 1. Error bars denote s.e.m. (c) ORP1 lacking the HFD is localized at the oocyst capsule at both day 14 and 20 p.b.f. Sporozoites are visible inside the oocysts. Nuclei stained with 4,6-diamidino-2-phenylindole (DAPI; blue) and the bright field (BF) image. Scale bars, 20 μm.