Pyp25α is required for male gametocyte exflagellation

Abstract

Malaria, a mosquito-borne infectious disease, is caused by the unicellular apicomplexan protozoa of the genus Plasmodium. For malaria parasite transmission, the essential sexual stage includes production of gametocytes through gametocytogenesis in vertebrate hosts and formation of gametes from gametocytes through gametogenesis in mosquito vectors. Whereas each female gametocyte forms a single immotile macrogamete, a male gametocyte produces eight flagella-like microgametes in a process called exflagellation. We identified a conserved protein named as Py05543 (Pyp25α), required for male gametocyte exflagellation in Plasmodium yoelii, which is the ortholog of PFL1770c (PF3D7_1236600). Interestingly, PF3D7_1236600 was previously phenotypically screened to be gametocyte-essential genes during gametocytogenesis of Plasmodium falciparum, using piggyBac transposon-mediated insertional mutagenesis. In this study, using CRISPR/Cas9-mediated genome editing, the Pyp25α¯ (KO) parasite line was successfully established. We found that the KO parasites proliferated asexually in mouse blood normally. In addition, compared with that of the parental parasites, the KO parasites displayed similar levels of gametocytes formation. Unexpectedly, the KO parasites showed considerable deficiency in exflagellation of male gametes, by observing exflagellation centre formation. Taken together, our data suggested that Pyp25α gene, the ortholog of PF3D7_1236600, was nonessential for the growth of asexual parasites, required for male gametocyte exflagellation in P. yoelii.

Tubulin Polymerization Promoting Proteins (TPPPs) of Aphelidiomycota: Correlation between the Incidence of p25alpha Domain and the Eukaryotic Flagellum

“…An essential role of TPPP in the formation of flagella was demonstrated in Chlamydomonas reinhardtii , biflagellated green algae, through the use of null mutant of FAP265, its TPPP ortholog [ 7 ]. Very recently, it has also been shown that TPPP (Py05543) is required for male gametocyte exflagellation in Plasmodium yoelli [ 8 ].…”

The dCas9-based genome editing in Plasmodium yoelii

“…To measure the gene editing activity of dCas9-SSAP in malaria parasites, we first attempted to delete the parasite gene Py05543 , which is nonessential for the growth of blood stages ( 16 ). The plasmid pDEF-dCas9-MCP-RecT- Py05543 containing a 46 bp tag DNA flanked by two homologous regions of Py05543 (403 bp of the 5′-flanking region and 499 bp of the 3′-flanking region) was constructed ( Fig.…”

“…According to the previously reported method ( 7 , 16 ), blood cell samples were collected from the orbital sinus of infected mice and lysed using 1% saponin in phosphate-buffered saline (PBS) to release parasites. These parasites were collected through centrifugation with 12,000 rpm for 3 min, and then the parasite genomic DNAs were isolated using DNeasy Blood kits (QIAGEN), as the template for PCR amplification (TaKaRa).…”

The molecular mechanisms driving Plasmodium cell division

“…Deletion of a gene encoding a P. falciparum protein with an AT-rich DNA interaction domain (PfARID) resulted in down-regulation of genes important for gametogenesis, with complete ablation of exflagellation [ 56 ]. Other players include Pb22, APC3 and radial spoke protein 9 (RSP9) in P. berghei [ 57–59 ], p25α in Plasmodium yoelii [ 60 ] and the patatin-like phospholipase, PLP2 in P. falciparum [ 61 ]. For a complete overview of essential regulators of gametogenesis, see [ 44 ].…”