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

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.

A P. falciparum NF54 Reporter Line Expressing mCherry-Luciferase in Gametocytes, Sporozoites, and Liver-Stages

“…Gametocytes of mCherry-luc@gapdh were mCherry positive, again in agreement with our previous observations of the GFP@ gapdh parasites (Marin-Mogollon et al, 2018) (Figure 2B; Supplementary Figure 3B). However, mCherry signals in gametocytes were relatively weak in all stages during their development (stage II-IV).…”

“…We selected the P. falciparum p47 gene (PF3D7_1346800) locus for introduction of the transgene expression cassette into the genome as it has been shown to be suitable for transgene expression without compromising parasite development in blood- and liver-stages as well as in A. stephensi mosquitoes (Talman et al, 2010; Vaughan et al, 2012; Vos et al, 2015). We had previously generated various transgenic reporter P. falciparum lines, where GFP-expression cassettes were introduced into the p230p gene locus (PF3D7_0208900), but disruption of this gene resulted in parasites that could not complete mosquito stage development (Marin-Mogollon et al, 2018). Using CRISPR/Cas9 methodology a transgenic P. falciparum (Pf) parasite line was created that encodes an mCherry-luciferase fusion gene under the control of 1.7 kb of 5' UTR of the etramp10.3 gene.…”

Plasmodium vivax HAP2/GCS1 gene exhibits limited genetic diversity among parasite isolates from the Greater Mekong Subregion

“…Although the molecular mechanisms of fertilization are poorly understood, this process is a promising target for the interruption of transmission [13]. Several proteins involved in the fertilization process have been identified, including P47, P48/45, P230 and HAP2 [14,15]. HAP2/Generative Cell Specific 1 (GCS1), first identified in the plants Arabidopsis thaliana and Lilium longflorum, is a class II viral fusion protein with a cysteine-rich extracellular region [16][17][18].…”

The NTP generating activity of pyruvate kinase II is critical for apicoplast maintenance in Plasmodium falciparum

“…BamHI and HindIII were used to remove the mevalonate gene from p15-Mev-aSFG and insert the tetR-DOZI coding region, a p2A skip peptide and BSD, as found in pMG74 ( Ganesan et al, 2016 ). We then added homology arms (HA) to facilitate site-specific integration into the p230p gene (PF3D7_0208900), which is dispensable in blood stage parasites ( Marin-Mogollon et al, 2018 ). HA1 and HA2 were sewn together with PCR primers p230HA1.F and p230HA1.NotI.R (for HA1) and primers p230HA2.NotI.F and p230HA2.R (for HA2) and inserted into the NotI site in the backbone of p15-Mev-aSFG ( Supplementary file 1 ).…”