Graduate School of Bio-Application and Systems Engineering, Tokyo University of Agriculture and Technology, Naka-cho 2-24-16, Koganei, Tokyo 184-8588, Japan.
Graduate School of Bio-Application and Systems Engineering, Tokyo University of Agriculture and Technology, Naka-cho 2-24-16, Koganei, Tokyo 184-8588, Japan.
J Insect Physiol. 2021 Jul;132:104252. doi: 10.1016/j.jinsphys.2021.104252. Epub 2021 May 20.
Previously, we found that nodule formation, a cellular defense response in insects, is regulated by humoral factors called C-type lectins in the hemolymph. To elucidate the factors that elicit nodule formation following the recognition of microorganisms by C-type lectins, a reproducible quantitative in vitro assay system was constructed. Then, using this system, the inhibitory activities of antisera raised against hemolymph proteases (HPs), serine protease homologues (SPHs), and pathogen-associated molecular pattern (PAMP)-recognition proteins were assessed. Among the antisera raised against HP and SPH, only that against HP8, a terminal proteinase that activates Spätzle, consistently inhibited in-vitro nodule-like aggregate formation in all three tested microorganisms, Micrococcus luteus, Escherichia coli, and Saccharomyces cerevisiae. Antisera raised against C-type lectins, BmLBP, and BmMBP also inhibited nodule-like aggregate formation, while those against β-glucan recognition proteins and peptidoglycan recognition protein-S1 did not. Microorganisms pretreated with hemolymph, which contains HP8 and C-type lectins, also induced nodule-like aggregate formation, indicating that nodulation factors are present on microbial cells. Furthermore, antisera raised against HP8, BmLBP, and BmMBP showed inhibitory activities in the in vivo nodule formation system using Bombyx mori larvae. Thus, two humoral factors in the hemolymph of B. mori larvae, BmHP8 and C-type lectins, were found to play significant roles in eliciting the cellular defense response of nodule formation.
先前，我们发现昆虫细胞防御反应的结节形成受血淋巴中称为 C 型凝集素的体液因子调节。为了阐明 C 型凝集素识别微生物后引发结节形成的因素，构建了一个可重复的体外定量分析系统。然后，使用该系统评估了针对血淋巴蛋白酶 (HP)、丝氨酸蛋白酶同源物 (SPH) 和病原体相关分子模式 (PAMP) 识别蛋白的抗血清的抑制活性。在针对 HP 和 SPH 的抗血清中，只有针对 HP8 的抗血清能够一致地抑制三种测试微生物（藤黄微球菌、大肠杆菌和酿酒酵母）中体外结节样聚集物的形成，HP8 是一种激活 Spätzle 的末端蛋白酶。针对 C 型凝集素、BmLBP 和 BmMBP 的抗血清也抑制结节样聚集物的形成，而针对β-葡聚糖识别蛋白和肽聚糖识别蛋白-S1 的抗血清则没有。用含有 HP8 和 C 型凝集素的血淋巴预处理微生物也诱导了结节样聚集物的形成，表明结节形成因子存在于微生物细胞上。此外，针对 HP8、BmLBP 和 BmMBP 的抗血清在使用家蚕幼虫的体内结节形成系统中表现出抑制活性。因此，家蚕幼虫血淋巴中的两种体液因子，BmHP8 和 C 型凝集素，被发现在家蚕细胞防御反应的结节形成中发挥重要作用。
