<em>Plasmodium falciparum</em> Field Isolates from South America Use an Atypical Red Blood Cell Invasion Pathway Associated with Invasion Ligand Polymorphisms

Studies of Plasmodium falciparum invasion pathways in field isolates have been limited. Red blood cell (RBC) invasion is a complex process involving two invasion protein families; Erythrocyte Binding-Like (EBL) and the Reticulocyte Binding-Like (PfRh) proteins, which are polymorphic and not fully characterized in field isolates. To determine the various P. falciparum invasion pathways used by parasite isolates from South America, we studied the invasion phenotypes in three regions: Colombia, Peru and Brazil. Additionally, polymorphisms in three members of the EBL (EBA-181, EBA-175 and EBL-1) and five members of the PfRh (PfRh1, PfRh2a, PfRh2b, PfRh4, PfRh5) families were determined. We found that most P. falciparum field isolates from Colombia and Peru invade RBCs through an atypical invasion pathway phenotypically characterized as resistant to all enzyme treatments (NrTrCr). Moreover, the invasion pathways and the ligand polymorphisms differed substantially among the Colombian and Brazilian isolates while the Peruvian isolates represent an amalgam of those present in the Colombian and Brazilian field isolates. The NrTrCr invasion profile was associated with the presence of the PfRh2a pepC variant, the PfRh5 variant 1 and EBA-181 RVNKN variant. The ebl and Pfrh expression levels in a field isolate displaying the NrTrCr profile also pointed to PfRh2a, PfRh5 and EBA-181 as being possibly the major players in this invasion pathway. Notably, our studies demonstrate the uniqueness of the Peruvian P. falciparum field isolates in terms of their invasion profiles and ligand polymorphisms, and present a unique opportunity for studying the ability of P. falciparum parasites to expand their invasion repertoire after being reintroduced to human populations. The present study is directly relevant to asexual blood stage vaccine design focused on invasion pathway proteins, suggesting that regional invasion variants and global geographical variation are likely to preclude a simple one size fits all type of vaccine.针对恶性疟原虫（Plasmodium falciparum）野外分离株入侵通路的研究目前仍较为有限。红细胞（RBC）入侵是一个复杂的过程，涉及两类入侵蛋白家族：红细胞结合样蛋白（Erythrocyte Binding-Like, EBL）与网织红细胞结合样蛋白（Reticulocyte Binding-Like, PfRh），这两类蛋白均具有多态性，且在野外分离株中尚未得到完全表征。为明确南美洲来源的疟原虫分离株所采用的各类恶性疟原虫入侵通路，我们对哥伦比亚、秘鲁与巴西三个地区的入侵表型展开了研究。此外，我们还对EBL家族的三个成员（EBA-181、EBA-175与EBL-1）以及PfRh家族的五个成员（PfRh1、PfRh2a、PfRh2b、PfRh4、PfRh5）的多态性进行了检测。研究结果显示，哥伦比亚与秘鲁的绝大多数恶性疟原虫野外分离株通过一种非典型入侵通路侵入红细胞，该通路在表型上表现为对所有酶处理均具有抗性（NrTrCr）。进一步分析发现，哥伦比亚与巴西分离株的入侵通路及配体多态性存在显著差异，而秘鲁分离株则兼具哥伦比亚与巴西野外分离株的入侵特征。NrTrCr入侵表型与PfRh2a pepC变体、PfRh5变体1以及EBA-181 RVNKN变体的存在具有相关性。在呈现NrTrCr表型的野外分离株中，ebl与Pfrh的表达水平同样提示PfRh2a、PfRh5与EBA-181可能是该入侵通路中的关键介导因子。值得注意的是，本研究证实了秘鲁恶性疟原虫野外分离株在入侵表型与配体多态性方面的独特性，同时为研究恶性疟原虫重新传入人群后拓展其入侵谱的能力提供了独特的研究契机。本研究与以入侵通路蛋白为靶点的无性血液阶段疫苗研发直接相关，研究结果提示，区域性入侵变异与全球地理差异可能会让一刀切式的疫苗研发方案难以奏效。