DataSheet_4_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.

疟原虫由按蚊传播。在其蚊媒生命周期中，疟原虫子孢子逃逸了餐后中肠的蛋白水解环境，通过穿越中肠壁来实现。此过程需要穿透中肠上皮衬里的含几丁质的营养基质，部分依赖于子孢子分泌的几丁质酶。恶性疟原虫子孢子具有一种几丁质酶（PfCHT1），而禽类感染寄生虫P. gallinaceum的子孢子则具有两种，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可以形成一个高分子量（HMW）的敏感复合物；其中一个结合伙伴是由子孢子产生的含有冯·威勒布兰德A结构域的蛋白质，WARP。本研究所报告的排阻色谱数据显示，禽疟原虫PgCHT2及其同源物恶性疟原虫PfCHT1是高分子量含几丁质酶复合物（>1,300 kDa）的共价连接组分。使用新的几丁质珠下拉法分离子孢子分泌蛋白的质谱分析，在恶性疟原虫和禽疟原虫子孢子条件培养基中鉴定出与几丁质酶相关的蛋白质。该复合物的质谱分析显示存在多种微绒毛蛋白，包括含有冯·威勒布兰德因子A结构域相关蛋白（WARP）、子孢子表面烯醇化酶和分泌的子孢子粘附蛋白（SOAP）。为了测试子孢子产生的PfCHT1能否形成高分子量的同源多聚体，或者，它是否与伯氏疟原虫子孢子产生的蛋白质相互作用以产生高分子量的异源多聚体，我们创建了表达PfCHT1以取代PbCHT1的嵌合伯氏疟原虫，从而能够产生大量的表达PfCHT1的子孢子。我们的研究表明，嵌合伯氏疟原虫子孢子表达单体PfCHT1，但含有PfCHT1的高分子量复合物并不存在。对含几丁质酶的高分子量复合物的深入了解，有望促进下一代针对疟疾传播阶段的疫苗或药物的研发。