The Plasmodium falciparum male gametocyte protein P230p, a paralog of P230, is vital for ookinete formation and mosquito transmission

Catherin Marin-Mogollon,Jai Ramesar,Shahid M Khan,Chris J Janse,Liwang Cui,Hans Kroeze,Geert-Jan Van Gemert,Robert W Sauerwein,Jun Miao,Fiona J A Van Pul,Kim C Williamson,Ahmad Syibli Othman,Marga Van De Vegte-Bolmer

doi:10.1038/s41598-018-33236-x

Abstract

Two members of 6-cysteine (6-cys) protein family, P48/45 and P230, are important for gamete fertility in rodent and human malaria parasites and are leading transmission blocking vaccine antigens. Rodent and human parasites encode a paralog of P230, called P230p. While P230 is expressed in male and female parasites, P230p is expressed only in male gametocytes and gametes. In rodent malaria parasites this protein is dispensable throughout the complete life-cycle; however, its function in P. falciparum is unknown. Using CRISPR/Cas9 methodology we disrupted the gene encoding Pfp230p resulting in P. falciparum mutants (PfΔp230p) lacking P230p expression. The PfΔp230p mutants produced normal numbers of male and female gametocytes, which retained expression of P48/45 and P230. Upon activation male PfΔp230p gametocytes undergo exflagellation and form male gametes. However, male gametes are unable to attach to red blood cells resulting in the absence of characteristic exflagellation centres in vitro. In the absence of P230p, zygote formation as well as oocyst and sporozoite development were strongly reduced (>98%) in mosquitoes. These observations demonstrate that P230p, like P230 and P48/45, has a vital role in P. falciparum male fertility and zygote formation and warrants further investigation as a potential transmission blocking vaccine candidate.

Highlights

Two members of 6-cysteine (6-cys) protein family, P48/45 and P230, are important for gamete fertility in rodent and human malaria parasites and are leading transmission blocking vaccine antigens
We demonstrate that P. falciparum P230p plays a vital role in parasite transmission through mosquitoes
We show that the PfΔp230p male gametes have lost the capacity to bind to red blood cells (RBC) and could not form the characteristic exflagellation centres

Summary

Introduction

Two members of 6-cysteine (6-cys) protein family, P48/45 and P230, are important for gamete fertility in rodent and human malaria parasites and are leading transmission blocking vaccine antigens. In mutants lacking P230 expression the characteristic clusters of uninfected red blood cells that form around male gametes, so-called exflagellation centres, are absent[7]. P. falciparum P47 does not play such a crucial role in gamete fertilization[11] These observations indicate that differences exist in the precise function of the sex-specific 6-Cys members between human and rodent malaria species. We generated transgenic P. falciparum parasites where we disrupted the p230p gene by introducing transgenes into this locus using adapted CRISPR/Cas[9] methodology[20] These PfΔp230p parasites show normal blood stage growth and are able produce gametocytes. In the absence of P230p expression, ookinete and oocyst development in Anopheles stephensi mosquitoes is almost absent These observations identify P230p as one of a limited number of gamete-specific proteins critical for P. falciparum transmission

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