DataSheet_5_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。在其作为蚊媒的生命周期中，疟原虫的顶体通过穿越中肠壁逃逸出餐后中肠的蛋白水解环境。此过程需要穿透覆盖在中肠上皮细胞上的含几丁质的围食膜，这部分依赖于顶体分泌的几丁质酶。恶性疟原虫的顶体具有一种几丁质酶（PfCHT1），而侵袭鸟类的寄生虫P. gallinaceum的顶体则具有两种，分别为长型和短型，分别命名为PgCHT1和PgCHT2。已发表的数据表明，PgCHT2能够形成一种高分子量（HMW）的敏感复合物；其中一种结合伴侣为顶体产生的含冯·维勒布兰德因子A结构域蛋白，WARP。本研究所报道的排阻色谱数据表明，P. gallinaceum的PgCHT2及其同源物P. falciparum的PfCHT1是高分子量含几丁质酶复合物（> 1,300 kDa）的共价连接成分。利用一种新的几丁质珠捕获方法从顶体分泌蛋白中分离出的质谱分析识别了在P. falciparum和P. gallinaceum顶体条件培养基中的几丁质酶相关蛋白。该复合物的质谱分析显示存在多种微绒毛蛋白，包括含冯·维勒布兰德因子A结构域蛋白（WARP）、顶体表面烯醇化酶和分泌的顶体粘附蛋白（SOAP）。为测试顶体产生的PfCHT1能够形成高分子量的同源多聚体，或者，另一种可能性，与P. berghei顶体产生的蛋白相互作用以产生高分子量的异源多聚体的假设，我们创建了表达PfCHT1以替代PbCHT1的嵌合P. berghei寄生虫，从而能够大量生产表达PfCHT1的顶体。我们发现嵌合P. berghei顶体表达单体PfCHT1，但并不存在含有PfCHT1的高分子量复合物。对含几丁质酶的高分子量复合物的深入理解，有望促进针对疟疾传播阶段的下一代疫苗或药物的研发。