State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, 430071, China; Savaid Medical School, University of Chinese Academy of Sciences, Beijing, 100049, China.
State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, 430071, China.
Insect Biochem Mol Biol. 2022 Aug;147:103775. doi: 10.1016/j.ibmb.2022.103775. Epub 2022 Jun 17.
Melanization is a key immune response mediated by serine protease (SP) cascade in insects. Multiple SP pathways exist in different species and it is unclear how conserved these cascades are. The cotton bollworm Helicoverpa armigera is a major worldwide agricultural pest. We reported a conserved melanization pathway in this species, which consists of SP41, cSP1, and cSP6. In this study, we attempted to identify an insect pathogen that elicits the cascade and test whether or not there are other SP cascades in H. armigera. After Micrococcus luteus, Enterobacter cloacae, Beauveria bassiana, or Helicoverpa armigera nucleopolyhedrovirus were injected into larvae, pathogen-induced hemolymph samples were collected for in vitro biochemical assays, which failed to detect proSP41 or procSP1 activation. In contrast, we found that procSP4, a protein proposed to participate in H. armigera melanization, was activated in M. luteus infected hemolymph. We further revealed that cSP8 was a prophenoloxidase (PPO) activating protease downstream of cSP4, and cSP4 was activated by cSP10. The pathway of cSP10-cSP4-cSP8 activated PPO in vitro. Efficiently cleaved procSPH11 and procSPH50 by cSP8 substantially enhanced phenoloxidase activity, suggesting they work together as a cofactor for cSP8 mediated PPO activation. Hemolymph from larvae challenged with M. luteus or its peptidoglycan effectively activated procSP10. Collectively, these results revealed a new PPO activation cascade specifically triggered by the bacterium. In addition, we found that the PPO activation cascades in H. armigera and Manduca sexta are conserved.
黑化是昆虫中丝氨酸蛋白酶（SP）级联反应介导的关键免疫反应。不同物种中存在多种 SP 途径，这些级联反应的保守程度尚不清楚。棉铃虫 Helicoverpa armigera 是一种世界性的农业害虫。我们在该物种中报道了一个保守的黑化途径，该途径由 SP41、cSP1 和 cSP6 组成。在本研究中，我们试图鉴定一种引发级联反应的昆虫病原体，并测试 H. armigera 是否存在其他 SP 级联反应。在向幼虫注射微球菌（Micrococcus luteus）、阴沟肠杆菌（Enterobacter cloacae）、球孢白僵菌（Beauveria bassiana）或棉铃虫核多角体病毒（Helicoverpa armigera nucleopolyhedrovirus）后，收集病原体诱导的血淋巴样本进行体外生化分析，但未能检测到 proSP41 或 procSP1 的激活。相比之下，我们发现一种拟参与 H. armigera 黑化的蛋白 procSP4 在感染微球菌的血淋巴中被激活。我们进一步揭示 cSP8 是 cSP4 下游的酚氧化酶（PPO）激活蛋白酶，cSP10 激活 cSP4。该途径在体外激活了 cSP10-cSP4-cSP8 的 PPO。cSP8 有效地切割 procSPH11 和 procSPH50，显著增强了酚氧化酶活性，表明它们作为 cSP8 介导的 PPO 激活的辅助因子共同作用。幼虫受到微球菌或其肽聚糖挑战后产生的血淋巴有效地激活了 procSP10。总的来说，这些结果揭示了一种由细菌特异性触发的新的 PPO 激活级联反应。此外，我们发现 H. armigera 和曼陀罗夜蛾（Manduca sexta）的 PPO 激活级联反应是保守的。
