DataSheet_3_A Hetero-Multimeric Chitinase-Containing Plasmodium falciparum and Plasmodium gallinaceum Ookinete-Secreted Protein Complex Involved in Mosquito Midgut Invasion.pdf

Malaria parasites are transmitted by Anopheles mosquitoes. During its life cycle in the mosquito vector the Plasmodium ookinete escapes the proteolytic milieu of the post-blood meal midgut by traversing the midgut wall. This process requires penetration of the chitin-containing peritrophic matrix lining the midgut epithelium, which depends in part on ookinete-secreted chitinases. Plasmodium falciparum ookinetes have one chitinase (PfCHT1), whereas ookinetes of the avian-infecting parasite, P. gallinaceum, have two, a long and a short form, PgCHT1 and PgCHT2, respectively. Published data indicates that PgCHT2 forms a high molecular weight (HMW) reduction-sensitive complex; and one binding partner is the ookinete-produced von Willebrand A-domain-containing protein, WARP. Size exclusion chromatography data reported here show that P. gallinaceum PgCHT2 and its ortholog, P. falciparum PfCHT1 are covalently-linked components of a HMW chitinase-containing complex (> 1,300 kDa). Mass spectrometry of ookinete-secreted proteins isolated using a new chitin bead pull-down method identified chitinase-associated proteins in P. falciparum and P. gallinaceum ookinete-conditioned culture media. Mass spectrometry of this complex showed the presence of several micronemal proteins including von Willebrand factor A domain-related protein (WARP), ookinete surface enolase, and secreted ookinete adhesive protein (SOAP). To test the hypothesis that ookinete-produced PfCHT1 can form a high molecular homo-multimer or, alternatively, interacts with P. berghei ookinete-produced proteins to produce an HMW hetero-multimer, we created chimeric P. berghei parasites expressing PfCHT1 to replace PbCHT1, enabling the production of large numbers of PfCHT1-expressing ookinetes. We show that chimeric P. berghei ookinetes express monomeric PfCHT1, but a HMW complex containing PfCHT1 is not present. A better understanding of the chitinase-containing HMW complex may enhance development of next-generation vaccines or drugs that target malaria transmission stages.

疟原虫通过按蚊进行传播。在蚊子宿主的生命周期中，疟原虫的 ookinete 通过穿越中肠壁逃逸血餐后中肠的蛋白水解环境。这一过程需要穿透覆盖中肠上皮的含几丁质的围食膜，部分依赖于 ookinete 分泌的几丁质酶。恶性疟原虫的 ookinete 拥有一种几丁质酶（PfCHT1），而感染鸟类的寄生虫 P. gallinaceum 的 ookinete 则有两种，分别为长型和短型，分别称为 PgCHT1 和 PgCHT2。已发表的数据表明，PgCHT2 形成一个高分子量（HMW）对还原敏感的复合物；其中一种结合伙伴是 ookinete 产生的含有 von Willebrand A 结构域的蛋白，WARP。本研究所报告的排阻色谱数据表明，家鸡疟原虫 PgCHT2 及其同源物恶性疟原虫 PfCHT1 是含有 HMW 几丁质酶复合物（> 1,300 kDa）的共价连接成分。利用一种新的几丁质珠下拉法分离的 ookinete 分泌蛋白的质谱分析，鉴定出恶性疟原虫和家鸡疟原虫 ookinete 培养基中的几丁质酶相关蛋白。该复合物的质谱分析显示，其中存在多种微绒毛蛋白，包括 von Willebrand 因子 A 结构域相关蛋白（WARP）、ookinete 表面烯醇化酶和分泌的 ookinete 粘附蛋白（SOAP）。为了测试 ookinete 产生的 PfCHT1 可以形成高分子量同源多聚体，或者，可选择地与 P. berghei ookinete 产生的蛋白相互作用以产生 HMW 异源多聚体的假设，我们创建了表达 PfCHT1 以取代 PbCHT1 的嵌合 P. berghei 虫株，从而能够产生大量表达 PfCHT1 的 ookinete。我们发现嵌合 P. berghei ookinete 表达单体的 PfCHT1，但并不存在含有 PfCHT1 的 HMW 复合物。对含有几丁质酶的 HMW 复合物的深入了解，有望促进针对疟疾传播阶段的新型疫苗或药物的开发。