Apical surface expression of aspartic protease Plasmepsin 4, a potential transmission-blocking target of the plasmodium ookinete

Abstract

To invade its definitive host, the mosquito, the malaria parasite must cross the midgut peritrophic matrix that is composed of chitin cross-linked by chitin-binding proteins and then develop into an oocyst on the midgut basal lamina. Previous evidence indicates that Plasmodium ookinete-secreted chitinase is important in midgut invasion. The mechanistic role of other ookinete-secreted enzymes in midgut invasion has not been previously examined. De novo mass spectrometry sequencing of a protein obtained by benzamidine affinity column of Plasmodium gallinaceum ookinete axenic culture supernatant demonstrated the presence of an ookinete-secreted plasmepsin, an aspartic protease previously only known to be present in the digestive vacuole of asexual stage malaria parasites. This plasmepsin, the ortholog of Plasmodium falciparum plasmepsin 4, was designated PgPM4. PgPM4 and PgCHT2 (the P. gallinaceum ortholog of P. falciparum chitinase PfCHT1) are both localized on the ookinete apical surface, and both are present in micronemes. Aspartic protease inhibitors (peptidomimetic and natural product), calpain inhibitors, and anti-PgPM4 monoclonal antibodies significantly reduced parasite infectivity for mosquitoes. These results suggest that plasmepsin 4, previously known only to function in the digestive vacuole of asexual blood stage Plasmodium, plays a role in how the ookinete interacts with the mosquito midgut interactions as it becomes an oocyst. These data are the first to delineate a role for an aspartic protease in mediating Plasmodium invasion of the mosquito and demonstrate the potential for plasmepsin 4 as a malaria transmission-blocking vaccine target.

FIGURE 1.

Identification of P. gallinaceum ookinete-secreted plasmepsin 4. SDS-polyacrylamide gel of unfractionated ookinete conditioned medium (lane 1) and the eluate (lane 2) from benzamidine affinity chromatography are shown. The band indicated was excised from the gel and submitted for de novo mass spectrometry sequencing. Four peptide sequences of P. gallinaceum plasmepsin 4 (as indicated by underlining/shading in supplemental Fig. 1B) were obtained. Molecular mass indicated at left in kDa.

FIGURE 2.

Western immunoblot demonstrating the recognition of recombinant PgPM4 and plasmepsin 4 (PgPM4) in P. gallinaceum ookinete conditioned medium and lysate by mAb 1H10. −, unreduced condition; +, reducing conditions with dithiothreitol.

FIGURE 3.

Localization of PgPM4 in P. gallinaceum zygote, ookinete, and asexual blood stages as demonstrated by deconvolution immunofluorescence microscopy. A, C, and E, ookinete. B and D, zygote. F and G, asexual stage parasite (trophozoite). A and B, fixed, permeabilized ookinetes and zygotes stained with mAb 2G4. C and D, surface staining of live ookinetes and zygotes using mAb 2G4. E, negative control using isotype IgG2b as primary antibody. F and G, fixed, permeabilized trophozoite. F, stained by mAb 2G4. G, negative control using isotype IgG2b as primary antibody. Blue, 4′,6-diamidino-2-phenylindole staining of nucleus. Scale bar, 1 μm in all panels.

FIGURE 4.

Western immunoblot demonstrating the presence of plasmepsin 4 (PgPM4) in P. gallinaceum zygotes, ookinetes, and asexual blood stage probed with mAb 1H10 that recognizes PgPM4. Ookinete lysate (1st lane, 2.5 × 10³ parasites loaded), zygote lysate (2nd lane, 2.5 × 10³ parasites loaded), P. gallinaceum-infected chicken blood lysate (3rd lane, 10 μl of blood loaded), uninfected chicken blood lysate (4th lane, 10 μl of blood loaded), A. aegypti midgut lysate at 0 h after blood meal (5th lane, infected; 6th lane, uninfected; 1 midgut/lane) and at 24 h (7th lane, infected; 8th lane, uninfected; 1 midgut/lane).

FIGURE 5.

Surface localization of plasmepsin 4 in relation to the chitinase PgCHT2 on the apical surface of the P. gallinaceum ookinete. Live, nonpermeabilized ookinetes were subjected to live IFA with dual staining of PgPM4 and PgCHT2. A, PgPM4 detected by mAb 1H10; B, PgCHT2 detected by mAb 1C3; C, PgPM4 stained with QDot 655 streptavidin-conjugated mAb 1H10 (red) and PgCHT2 recognized by mAb 1C3 stained with QDot 565 goat anti-mouse IgG (green). Blue, 4′,6-diamidino-2-phenylindole staining of nucleus. This pattern of staining was reproducibly observed in the vast majority of mature, elongated P. gallinaceum ookinetes. Scale bar, 1 μm in all panels.

FIGURE 6.

Micronemal localization of PgPM4 in the P. gallinaceum ookinete by immunoelectron microscopy. Each panel represents progressively higher magnification. Arrowheads indicate surface localization of the protein on the apical end of the parasite. Scale bar, 1 μm in all panels.

FIGURE 7.

Effect of inhibitors and monoclonal antibodies on in vitro P. gallinaceum ookinete development. A, effect of calpain-like protease inhibitors ALLN and ALLM and pepstatin A on P. gallinaceum ookinete formation in vitro. B, effect of anti-P. gallinaceum plasmepsin IV monoclonal antibodies 1H10 and 2G4 on P. gallinaceum ookinete formation in vitro.