Ookinete-Interacting Proteins on the Microvillar Surface are Partitioned into Detergent Resistant Membranes of <i>Anopheles gambiae</i> Midguts

Lipid raft microdomains, a component of detergent resistant membranes (DRMs), are routinely exploited by pathogens during host-cell entry. Multiple membrane-surface proteins mediate Plasmodium ookinete invasion of the Anopheles midgut, a critical step in the parasite life cycle that is successfully targeted by transmission-blocking vaccines (TBV). Given that lipid rafts are a common feature of host-pathogen interactions, we hypothesized that they promote the partitioning of midgut surface proteins and thus facilitate ookinete invasion. In support of this hypothesis, we found that five of the characterized Anopheles TBV candidates, including the leading Anopheles TBV candidate, AgAPN1, are present in Anopheles gambiae DRMs. Therefore, to extend the repertoire of putative midgut ligands that can be targeted by TBVs, we analyzed midgut DRMs by tandem mass spectrometry. We identified 1452 proteins including several markers of DRMs. Since glycosylphosphotidyl inositol (GPI)-anchored proteins partition to DRMs, we characterized the GPI subproteome of An. gambiae midgut brush-border microvilli and found that 96.9% of the proteins identified in the GPI-anchored fractions were also present in DRMs. Our study vastly expands the number of candidate malarial TBV targets for subsequent analysis by the broader community and provides an inferred role for midgut plasmalemma microdomains in ookinete cell invasion.脂筏微结构域（lipid raft microdomains）作为去垢剂抗性膜（DRMs）的组成部分，常被病原体用于宿主细胞入侵过程。多种膜表面蛋白介导疟原虫（Plasmodium）动合子入侵按蚊（Anopheles）中肠，这是寄生虫生命周期中的关键步骤，且已被传播阻断疫苗（TBV）成功作为靶向靶点。鉴于脂筏是宿主-病原体相互作用的普遍特征，我们提出假说：脂筏可促进中肠表面蛋白的分区富集，进而助力动合子的入侵过程。为验证该假说，我们经检测发现，5种已被鉴定的按蚊传播阻断疫苗候选蛋白（包括主流候选蛋白AgAPN1）均存在于冈比亚按蚊（Anopheles gambiae）的DRM组分中。因此，为扩充可被传播阻断疫苗靶向的潜在中肠配体库，我们通过串联质谱对中肠DRM组分展开了分析，共鉴定出1452种蛋白，其中包含多种DRM标志物。由于糖基磷脂酰肌醇（GPI）锚定蛋白可富集至DRM组分中，我们进一步对冈比亚按蚊中肠刷状缘微绒毛的GPI亚蛋白质组进行了表征，结果显示，在GPI锚定蛋白组分中鉴定出的蛋白有96.9%同时存在于DRM组分中。本研究极大拓展了可供学界后续研究的疟疾传播阻断疫苗候选靶点数量，并为中肠质膜微结构域在动合子入侵过程中的作用提供了推断性依据。