Functional Conservation of P48/45 Proteins in the Transmission Stages of Plasmodium vivax (Human Malaria Parasite) and P. berghei (Murine Malaria Parasite).
Functional Conservation of P48/45 Proteins in the Transmission Stages of Plasmodium vivax (Human Malaria Parasite) and P. berghei (Murine Malaria Parasite).
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DOI：
10.1128/mbio.01627-18
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发表时间：
2018-09-04
期刊：
影响因子：
6.4
通讯作者：
Kumar N
中科院分区：
文献类型：
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作者：
Cao Y;Hart RJ;Bansal GP;Kumar N
疟疾传播取决于脊椎动物宿主体内寄生虫的成功性分化和成熟以及蚊子中肠的进一步发育。性阶段的阶段特异性蛋白质已被证明在按蚊媒介的发育和成功传播中发挥着关键作用。当前手稿中提出的研究评估了一种此类蛋白质 P48/45 在两种不同物种（伯氏疟原虫和间日疟原虫）中的功能保护。用 pvs48/45（间日疟原虫同源物）替换伯氏疟原虫中的内源性 pbs48/45 不会影响寄生虫的生存能力，并且表达 Pvs48/45 的转基因寄生虫仍具有传播能力。这些研究不仅确定了伯氏疟原虫和间日疟原虫中 P48/45 的功能保守性，而且还为目前正在开发的基于 Pvs48/45 的间日疟原虫传播阻断疫苗开发体内测试模型提供了机会。性阶段蛋白在蚊子传播过程中具有独特的功能，也是开发阻断疟疾传播疫苗的目标。 P48/45 是一种领先的候选疫苗，对于雄配子的生育能力至关重要，并且在不同疟原虫物种之间显示出 > 50% 的相似性。我们评估了间日疟原虫和伯氏疟原虫中 P48/45 的功能保守性，目的是在体内测定中建立表达间日疟原虫 P48/45 (Pvs48/45) 的转基因伯氏疟原虫菌株，以评估 Pvs48/45 引发的抗体的传播阻断活性。采用同源重组来靶向伯氏疟原虫 s48/45 (pbs48/45)，以进行敲除 (KO) 或用两种不同形式的间日疟原虫 s48/45 (pvs48/45)（全长基因和由 pbs48/45 5' 信号和 pvs48/45 侧翼 3' 锚定序列组成的嵌合基因）进行替换。通过逆转录-PCR (RT-PCR) 和蛋白质印迹证实转基因寄生虫中 Pvs48/45 的表达以及 KO 寄生虫中任何 P48/45 的表达缺失。转基因和敲除寄生虫在小鼠中都显示出与野生型寄生虫相当的无性生长动力学。当用于蚊子感染实验时，两种转基因寄生虫菌株均保持传播能力并发育成传染性子孢子，而基因敲除寄生虫则无法建立蚊子阶段感染。这些结果表明 P48/45 蛋白在传播过程中的功能保守性，并且本研究中产生的转基因寄生虫代表了一种有价值的工具，可用于评估基于 Pvs48/45 的疫苗引发的传播阻断抗体的保护功效，使用体内小鼠动物测定，而不是依赖于获取受感染患者的间日疟原虫配子细胞的离体膜喂养测定 (MFA)。
Malaria transmission depends upon successful sexual differentiation and maturation of parasites in the vertebrate host and further development in the mosquito midgut. Stage-specific proteins in the sexual stages have been shown to play a critical role in development and successful transmission through the anopheline mosquito vector. Studies presented in the current manuscript evaluated functional conservation of one such protein, P48/45, in two diverse species (P. berghei and P. vivax). Replacement of endogenous pbs48/45 in P. berghei with pvs48/45 (P. vivax homologue) did not affect the viability of the parasites, and the transgenic parasites expressing Pvs48/45 remained transmission competent. These studies establish not only the functional conservation of P48/45 in P. berghei and P. vivax but also offer an opportunity to develop an in vivo test model for Pvs48/45-based P. vivax transmission-blocking vaccines, currently under development. Sexual-stage proteins have a distinct function in the mosquito vector during transmission and also represent targets for the development of malaria transmission-blocking vaccine. P48/45, a leading vaccine candidate, is critical for male gamete fertility and shows >50% similarity across various species of Plasmodium. We evaluated functional conservation of P48/45 in Plasmodium vivax and P. berghei with the motivation to establish transgenic P. berghei strains expressing P. vivax P48/45 (Pvs48/45) in an in vivo assay to evaluate the transmission-blocking activity of antibodies elicited by Pvs48/45. Homologous recombination was employed to target P. berghei s48/45 (pbs48/45) for knockout (KO) or for its replacement by two different forms of P. vivax s48/45 (pvs48/45) (the full-length gene and a chimeric gene consisting of pbs48/45 5′ signal and 3′ anchor sequences flanking pvs48/45). Expression of Pvs48/45 in transgenic parasites and lack of expression of any P48/45 in KO parasites were confirmed by reverse transcription-PCR (RT-PCR) and Western blotting. Both transgenic and knockout parasites revealed asexual growth kinetics in mice comparable to that seen with wild-type parasites. When employed in mosquito infection experiments, both transgenic parasite strains remained transmission competent and developed into infectious sporozoites, whereas the knockout parasites were incapable of establishing mosquito-stage infection. These results indicate the functional conservation of P48/45 protein during transmission, and the transgenic parasites generated in this study represent a valuable tool to evaluate the protective efficacy of transmission-blocking antibodies elicited by Pvs48/45-based vaccines using an in vivo mouse animal assay instead of ex vivo membrane feeding assays (MFA) relying on access to P. vivax gametocytes from infected patients.