Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X

Abstract

Background: Plasmodium invasion of the mosquito midgut is a population bottleneck in the parasite lifecycle. Interference with molecular mechanisms by which the ookinete invades the mosquito midgut is one potential approach to developing malaria transmission-blocking strategies. Plasmodium aspartic proteases are one such class of potential targets: plasmepsin IV (known to be present in the asexual stage food vacuole) was previously shown to be involved in Plasmodium gallinaceum infection of the mosquito midgut, and plasmepsins VII and plasmepsin X (not known to be present in the asexual stage food vacuole) are upregulated in Plasmodium falciparum mosquito stages. These (and other) parasite-derived enzymes that play essential roles during ookinete midgut invasion are prime candidates for transmission-blocking vaccines.

Methods: Reverse transcriptase PCR (RT-PCR) was used to determine timing of P. falciparum plasmepsin VII (PfPM VII) and plasmepsin X (PfPM X) mRNA transcripts in parasite mosquito midgut stages. Protein expression was confirmed by western immunoblot and immunofluorescence assays (IFA) using anti-peptide monoclonal antibodies (mAbs) against immunogenic regions of PfPM VII and PfPM X. These antibodies were also used in standard membrane feeding assays (SMFA) to determine whether inhibition of these proteases would affect parasite transmission to mosquitoes. The Mann-Whitney U test was used to analyse mosquito transmission assay results.

Results: RT-PCR, western immunoblot and immunofluorescence assay confirmed expression of PfPM VII and PfPM X in mosquito stages. Whereas PfPM VII was expressed in zygotes and ookinetes, PfPM X was expressed in gametes, zygotes, and ookinetes. Antibodies against PfPM VII and PfPM X decreased P. falciparum invasion of the mosquito midgut when used at high concentrations, indicating that these proteases play a role in Plasmodium mosquito midgut invasion. Failure to generate genetic knockouts of these genes limited determination of the precise role of these proteases in parasite transmission but suggests that they are essential during the intraerythrocytic life cycle.

Conclusions: PfPM VII and PfPM X are present in the mosquito-infective stages of P. falciparum. Standard membrane feeding assays demonstrate that antibodies against these proteins reduce the infectivity of P. falciparum for mosquitoes, suggesting their viability as transmission-blocking vaccine candidates. Further study of the role of these plasmepsins in P. falciparum biology is warranted.

Fig. 1

Plasmepsin VII and Plasmepsin X mRNA was detected in Plasmodium falciparum sexual stage parasites. a Total RNA isolated from in vitro-cultivated asexual stages (A), gametocytes (G), zygotes (Z), ookinetes (O), and uninfected human erythrocytes (uB). Samples were reverse transcribed and amplified using primers specific for PfPM VII (+RT). Samples that were not reverse transcribed (−RT) and amplified with PfPM VII-specific primers did not generate PCR product. b Total RNA isolated ex vivo from ookinete-containing mosquito midguts (O) or uninfected human blood (uB), in vitro-cultivated gametocytes (G) and mixed zygotes and ookinetes (Z/O), as well as DNA from P. falciparum (NF) was isolated. Samples were reverse transcribed and amplified using primers specific for PfPM X and pfs25 (+RT). Samples that were not reverse transcribed (−RT) and amplified with PfPM X-specific primers did not generate PCR product

Fig. 2

Peptide monoclonal antibodies directed against Plasmodium falciparum Plasmepsin VII and Plasmepsin X. a Schematic representation of Plasmepsin showing the predicted signal peptide (blank), pro-enzyme domains (pro), and catalytic domain (cat). Peptide monoclonal antibodies designed against three regions of PfPM VII and PfPM X are designated prodomain (PD, black), catalytic domain 1 (CD1, red) and catalytic domain 2 (CD2, purple). b Protein sequence of PfPM VII and c. PfPM X showing the predicted signal peptide (blue) and the predicted pro-enzyme domain (italics); the two active aspartic acid residues (red) are found within conserved regions (bold). Monoclonal antibody targets to prodomain (black), CD1 (red), and CD 2 (purple). d Alignment of 3G6 peptide target between all P. falciparum plasmepsins shows moderate conservation between PfPM X and PfPM IX at this site

Fig. 3

PfPM VII and PfPM X expression in Plasmodium falciparum sexual stage parasites demonstrated by IFA and western immunoblot. IFA of in vitro-cultivated parasites was performted using primary antibodies 1B4, 2B1, 6A9, 3G6 or isotype IgG2b and subsequently labelled with Alexa Fluor 488-labelled anti-mouse antibodies (green); nuclei are visualized with DAPI (blue). a IFA of in vitro-cultivated P. falciparum demonstrated PfPM VII expression in zygotes (Z) and ookinetes (O). b IFA of in vitro-cultivated P. falciparum demonstrated PfPM X expression in zygotes (Z) and ookinetes (O) but not gametocytes (G). c Western immunoblot analysis of total protein isolated from P. falciparum sexual stage parasites. Antibodies directed against PfPM VII recognized a ~46 kDa protein expressed in ookinetes (O) but not gametocytes (G). This protein is between the predicted sizes of full length PfPM VII at 52 kDa and the catalytic domain at 43 kDa. d Western immunoblot analysis of total protein isolated from P. falciparum sexual stage parasites. Both 6A9 and 3G6 recognized protein expressed in mixed gametes and zygotes (m/Z) and ookinetes (O) but not gametocytes (G). Bands are consistent with the predicted sizes of full length PfPM X at 61 kDa (←F), the PfPM X catalytic domain at 36 kDa (←C), and the PfPM X proenzyme domain at 23 kDa (←P). IgG isotype control did not recognize the 61 kDa or 23 kDa bands on western immunoblot but continued to recognize the bands at 42–55 kDa and 27–30 kDa