Supplementary Material for: CLIPA7 Exhibits Pleiotropic Roles in the <b><i>Anopheles gambiae</i></b> Immune Response

Clip domain serine proteases and clip domain serine protease homologs (cSPHs) are key components of serine protease cascades that drive the melanization response. Despite lacking catalytic activity, cSPHs play essential roles in regulating melanization, but the spectrum of functions they catalyze within and outside these cascades is not fully understood. Aside from their classical role as cofactors for PPO activation, we have previously revealed an unprecedented complexity in the function and molecular organization of these cSPHs in the immune response of the malaria vector <i>Anopheles gambiae</i>. Here, we add yet another dimension to the complex roles underpinning the contributions of cSPHs to mosquito immunity by showing that CLIPA7, a member of the expanded cSPH family, defines a novel branch within the cSPH network that is essential for the melanization of <i>Escherichia coli</i> but not <i>Plasmodium</i> ookinetes or Gram-positive bacteria. Despite its dispensability for the melanization of Gram-positive bacteria, we show that CLIPA7 is required for the clearance of systemic infections with <i>Staphylococcus aureus</i>. CLIPA7 is produced by hemocytes and associates with the surfaces of live <i>E. coli</i> and <i>S. aureus</i> cells in vivo as well as with those of melanized cells. Based on its RNAi phenotypes and its unique domain architecture among <i>A. gambiae</i> cSPHs including the presence of an RGD motif, we propose that CLIPA7 exhibits pleiotropic roles in mosquito immunity that extend beyond the regulation of melanization to microbial clearance.含clip结构域的丝氨酸蛋白酶（clip domain serine proteases）与clip结构域丝氨酸蛋白酶同源物（cSPHs）是驱动黑化反应的丝氨酸蛋白酶级联反应的关键组成成分。尽管缺乏催化活性，cSPHs在调控黑化反应中发挥着不可或缺的作用，但目前对于它们在这些级联反应内外所催化的功能谱仍未完全明晰。除了作为酚氧化酶原（PPO）激活的辅因子这一经典功能外，我们此前在疟疾媒介冈比亚按蚊（Anopheles gambiae）的免疫反应中，揭示了这类cSPHs在功能与分子组织层面前所未有的复杂性。本研究为阐明cSPHs在蚊虫免疫中所发挥的复杂作用增添了新的维度：研究发现，扩增的cSPH家族成员CLIPA7构成了cSPH网络中的一个全新分支，该分支对于大肠杆菌（Escherichia coli）的黑化过程是必需的，但对疟原虫（Plasmodium）动合子或革兰氏阳性细菌的黑化则并非必需。尽管CLIPA7对于革兰氏阳性细菌的黑化并非必需，但我们的实验显示其对于清除金黄色葡萄球菌（Staphylococcus aureus）引发的系统性感染是必需的。CLIPA7由血细胞产生，并可在体内结合活的大肠杆菌与金黄色葡萄球菌细胞表面，同时也能结合黑化细胞的表面。基于其RNA干扰（RNAi）表型，以及在冈比亚按蚊cSPHs中独有的域结构（包括存在RGD基序），我们提出CLIPA7在蚊虫免疫中发挥多效性作用，其功能不仅局限于黑化反应的调控，还延伸至微生物清除过程。