DataSheet_2_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。在蚊媒的生命周期中，疟原虫的子孢子通过穿越中肠壁逃离血餐后中肠的蛋白水解环境。此过程需要穿透含有几丁质的中肠上皮周围形成的营养层，这一过程部分依赖于子孢子分泌的几丁质酶。恶性疟原虫的子孢子拥有一种几丁质酶（PfCHT1），而感染鸟类的寄生虫P. gallinaceum的子孢子则拥有两种，分别为长形和短形的几丁质酶，分别命名为PgCHT1和PgCHT2。已发表的数据表明，PgCHT2形成了一种高分子量（HMW）的敏感复合物；其中一种结合伙伴是由子孢子产生的包含冯·维勒布兰德因子A结构域的蛋白质，WARP。在此，我们报道了通过新的几丁质珠沉淀方法从子孢子分泌的蛋白质中分离出的质量排除层析数据，显示P. gallinaceum的PgCHT2及其同源物P. falciparum的PfCHT1是高分子量几丁质酶复合物（> 1,300 kDa）的共价连接组分。使用新的几丁质珠沉淀方法对子孢子分泌的蛋白质进行质谱分析，在P. falciparum和P. gallinaceum的子孢子条件培养基中鉴定了几丁质酶相关的蛋白质。该复合物的质谱分析显示了包括冯·维勒布兰德因子A结构域相关蛋白（WARP）、子孢子表面酶醇和分泌的子孢子粘附蛋白（SOAP）在内的多种微绒毛蛋白的存在。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，作为替代，与P. berghei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以替代PbCHT1的嵌合P. berghei寄生虫，从而能够产生大量的PfCHT1表达子孢子。我们发现嵌合P. berghei子孢子表达单体的PfCHT1，但并未检测到含有PfCHT1的高分子量复合物。对含有几丁质酶的高分子量复合物的深入了解可能有助于开发针对疟疾传播阶段的新型疫苗或药物。