Nosema bombycis suppresses host hemolymph melanization through secreted serpin 6 inhibiting the prophenoloxidase activation cascade
Nosema bombycis suppresses host hemolymph melanization through secreted serpin 6 inhibiting the prophenoloxidase activation cascade
复制标题
家蚕微孢子虫通过分泌丝氨酸蛋白酶抑制剂 6 抑制原酚氧化酶激活级联来抑制宿主血淋巴黑化
DOI：
10.1016/j.jip.2019.107260
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发表时间：
2019-11-01
影响因子：
3.4
通讯作者：
Zhou, Zeyang
中科院分区：
文献类型：
--
作者：
Bao, Jialing;Liu, Lulu;Zhou, Zeyang
家蚕微孢子虫是家蚕的一种病原体，属于微孢子虫，是一组与真菌有关的专性细胞内寄生虫。家蚕乳杆菌感染可引起该病。昆虫利用血淋巴黑色素化作为对抗病原体的先天免疫反应的一部分，黑色素化依赖于丝氨酸蛋白酶介导的酚氧化酶（PPO）激活级联反应，该反应受到丝氨酸蛋白酶抑制剂（蛇肽）的严格调节。已有研究表明，家蚕乳螨感染可抑制家蚕血淋巴黑色素化，但其作用机制尚未阐明。我们假设瓢虫可以分泌蛇形蛋白（NbSPNs）来抑制宿主丝氨酸蛋白酶在PPO激活级联中，从而抑制酚氧化酶（PO）活性和随之而来的黑化。在本研究中，我们证实了蚕蛾感染抑制了家蚕PO活性和黑素化，并在感染寄主的血淋巴中鉴定了蚕蛾丝蛋白6 （NbSPN6）的表达。重组NbSPN6加入正常血淋巴后，PO活性呈剂量依赖性抑制。此外，通过RNA干扰技术进行体内分析发现，阻断NbSPN6表达可逆转其对PO活性的抑制作用，抑制家蚕在宿主体内的增殖。这些结果表明NbSPN6在病原菌成功感染中起着不可或缺的作用。为了进一步阐明NbSPN6抑制宿主防御的分子基础，我们确定宿主丝氨酸蛋白酶酚氧化酶活化酶（PPAE）是NbSPN6抑制的直接靶点。综上所述，我们的新研究首次阐明了病原体来源的蛇形蛋白抑制血淋巴黑色素化的分子机制，从而调节宿主的先天免疫反应。本研究也可能为预防微孢子虫感染提供新的策略。
Nosema bombycis is a pathogen of the silkworm that belongs to the microsporidia, a group of obligate intracellular parasites related to fungi. N. bombycis infection causes the disease pebrine in silkworms. Insects utilize hemolymph melanization as part of the innate immune response to fight against pathogens, and melanization relies on a serine protease-mediated prophenoloxidase (PPO) activation cascade that is tightly regulated by serine protease inhibitors (serpins). Previous studies showed that N. bombycis infection suppressed silkworm hemolymph melanization, however the mechanism has not been elucidated. We hypothesize that N. bombycis can secret serpins (NbSPNs) to inhibit host serine proteases in the PPO activation cascade, thus suppressing phenoloxidase (PO) activity and the consequent melanization. We demonstrated in this study that N. bombycis infection suppressed silkworm PO activity and melanization and we identified the expression of N. bombycis serpin 6 (NbSPN6) in the hemolymph of the infected host. When recombinant NbSPN6 was added to normal hemolymph, PO activity was inhibited in a dose-dependent manner. Moreover, in vivo analysis by RNA interference technology showed that when NbSPN6 expression is blocked, the inhibitory effects on PO activity can be reversed and the proliferation of N. bombycis within host can be suppressed. These results demonstrated the indispensable role of NbSPN6 in successful pathogen infection. To further elucidate the molecular basis of NbSPN6 suppressing host defense, we determined that the host serine protease prophenoloxidase-activating enzyme (PPAE) is the direct target of NbSPN6 inhibition. Taken together, our novel study is the first to elucidate the molecular mechanism of pathogen-derived serpin inhibiting hemolymph melanization and, thus, regulating host innate immune responses. This study may also provide novel strategies for preventing microsporidia infection.