Plasmodium berghei Gamete Egress Protein is required for fertility of both genders

Abstract

Male and female Plasmodium gametocytes ingested by the Anopheles mosquitoes during a blood meal egress from the red blood cells by rupturing the two surrounding membranes, the parasitophorous vacuole and the red blood cell membranes. Proteins of the so-called osmiophilic bodies, (OBs), secretory organelles resident in the cytoplasm, are important players in this process. Once gametes emerge, the female is ready to be fertilized while the male develops into motile flagellar gametes. Here, we describe the function(s) of PBANKA_1115200, which we named Gamete Egress Protein (GEP), a protein specific to malaria parasites. GEP is restricted to gametocytes, expressed in gametocytes of both genders and partly localizes to the OBs. A mutant lacking the protein shows aberrant rupture of the two surrounding membranes, while OBs discharge is delayed but not aborted. Moreover, we identified a second function of GEP during exflagellation since the axonemes of the male flagellar gametes were not motile. Genetic crossing experiments reveal that both genders are unable to establish infections in mosquitoes and thus the lack of GEP leads to a complete block in Plasmodium transmission from mice to mosquitoes. The combination of our results reveals essential and pleiotropic functions of GEP in Plasmodium gametogenesis.

Keywords: Anopheles; gametocyte egress; malaria; osmiophilic bodies.

Figure 1

(a) Scheme of the gep locus (PBANKA_1115200) showing the coding region of 1,138 amino acids in 10 exons (red boxes). Violet and pink indicated, respectively, the 5’ upstream and the 3’ downstream regulatory regions. (b) Growth rate of gep(‐) parasites compared with WT parasites. Three independent experiments were done. No significant difference was detected (t test, p‐value = .4024). (c) The number of exflagellation events per field in the mutant line compared with WT. No exflagellation was detected in the mutant in 13 independent experiments. (d) Immunofluorescence assay on WT and gep(‐) male and female gametocytes activated for 15–20 min. Anti‐SEP1 antibodies were used to label the PVM (red) while TER‐119 (green) was used as RBCM marker. Mutant parasites constitutively express cytoplasmic GFP. Scale bar is 5 μm. (e) Percentage of activated males (upper panel) and females (lower panel) gametocytes in the WT and the gep(‐) cl1 with ruptured PVM or PVM and RBCM. Differences between WT and mutant are all statistically significant (Student's t test: male ruptured PVM: p‐value = .0074; female ruptured PVM: p‐value = .0041 male ruptured RBCM: p‐value = .0019; female ruptured RBCM: p‐value = .0022).

Figure 2

(a) Western blot analysis on purified HPE parasites, WT gametocytes (2 independent preparations), and gep(‐) gametocytes (clones 1 and 2). GEP serum recognizes a specific band at 133 kDa in the WT gametocyte samples, while no signal was detected both in asexual parasites (HPE) and in gametocytes of the gep(‐) cloned lines. Samples were normalized using anti‐Pb14‐3‐3 detected in both asexual and sexual stages (Lalle et al., 2011). (b) Immunofluorescence assay using GEP‐specific serum. A signal is detected in WT gametocytes but not in mutant parasites. Scale bar 5 μm. (c) Double IFA on WT gametocytes using immune sera against GEP serum and the nuclear protein SET, highly abundant in male gametocytes. Scale bar 5 μm. (d) Double IFA using anti‐GEP and anti‐G377 as a marker of OBs. Scale bars 5 μm

Figure 3

(a) Ultrastructural analysis of male and female gametocytes of gep(‐) parasites. The cells display the typical appearance of gametocytes. In males, an eccentric and very large nucleus (Nu), a lot of typical club‐shaped male osmiophilic bodies (Mobs) and of hemozoin crystals (Hz), both randomly distributed in the cytoplasm, and the inner membrane. In females pronounced rough endoplasmic reticulum (ER), hemozoin granules and abundant oval‐shaped osmiophilic bodies (OBs) scattered in the cytoplasm. Scale bar 1 μm. (b) WT ultrastructure section of male and female gametocyte. Nu: Nucleus; arrowhead: MOBs in male and OBs in female gams; Hz: hemozoin; (c) IFA on gep(‐) parasites: a female gametocyte (upper panel) stained with the immune serum against the OB marker G377 and a male gametocyte stained with the immune serum against the OB marker MDV. Scale bar 5 μm

Figure 4

(a) Double IFA of activated WT and gep(‐) gametes labeled with the PVM marker SEP1 and the OB marker MDV1. In the WT, OBs discharged their content 5–8 min postactivation with the concomitant disruption of the PVM. In the gep(‐) cl1 gametocytes, an intact PVM and OBs still inside the cells are visible. Scale bar 5 μm. (b) Ultrastructure of gep(‐) female gametocyte activated 20 min (upper panel); the PVM and erythrocyte membrane (EM) are intact and OBs (asterisks) are present in the cytoplasm. WT female gametocyte activated 8 min (bottom panel) fully egressed. (c) Western blot analysis using GEP, MDV1, and G377 sera on samples prepared from purified WT and gep(‐) gametocytes (lane a), gametes (lane b), and exflagellation supernatant (lane c). The samples correspond to 10⁷ gametocytes purified from synchronous infections. Gametes and supernatant were from samples activated for an extended time (30 min). GEP is partly secreted during gamete activation as demonstrated in the upper panel of the WB. At this time point, OBs protein content is normally secreted in gep(‐) parasites, as revealed in the blots probed with MDV1 serum. G377 is partly retained in the gametes and partly secreted

Figure 5

(a) Morphological EM section of an activated gep(‐) male gametocyte 20 min after induction of gametogenesis. The RBCM is intact as well as the PVM. In the male cell, normally structured axonemes (arrowhead) are seen. (b) Morphological EM section of activated gep(‐) male gametocyte. In this section, a center of the microtubular organization is showed (MTOC) as well as nuclear lobes (asterisks). (c) IFA on WT 10 and 20 min activated and gep(‐) activated gametocytes at 20 min after induction stained with anti‐tubulin and SEP1 sera. In red, tubulin is detected on the flagella of WT and gep(‐) gametes. In the mutant samples, PVM is detected (labeled with SEP1) and tubulin is visible in a pattern consistent with nonmotile axonemes. Enlarged nuclei are also visible suggesting

Figure 6

(a) Ookinete conversion from genetic crosses of gep(‐) parasites and parasite lines producing sterile male (Δ45/48) and female (Δ47) gametocytes. As a control, Δ45/48 was also crossed to Δ47. Dotted line: average WT ookinete conversion. b) Oocyst formation of WT and gep(‐). In mosquitoes infected with gep(‐) parasites, no oocysts were detected in dissected mosquito midguts 13 days after blood feeding

Figure A1

Alignment of GEP in Plasmodium ssp P. berghei, P. falciparum, P. yoelii P. malariae, and P. vivax. GEP is highly conserved through the main Plasmodium species affecting humans. The sequences are P. falciparum (PF3D7_0515600), P. berghei (PBANKA_1115200), Plasmodium vivax (PVP01_1018300), Plasmodium malariae (PmUG01_10029000), and P. yoelii (PY17X_02013). The sequences were obtained from the PLASMODB database (Aurrecoechea et al., 2009).

Figure A2

(a) Schematic representation of the strategy used to generate gep(‐) parasites. The 3’UTR region selected for plasmid design is about 900 bp upstream the adjacent gap40 stop codon. (b) PCR genotyping of gep(‐) parasites clones 1 and 2. Transfected parasites were cloned to remove genomic WT contamination and the cloning verified by PCR. The primers are shown schematically in Figure 1a, and their sequences are found in Table A2. In neither gep(‐) clone WT contamination was detected. Expected sizes in diagnostic PCRs: left side of the gene, primers a‐c for WT, 1,866 bp and primers a–e for KO, 1,029 bp; right side: primers b–d for WT, 1,428 bp and primers b–f for KO, 1,524 bp.