Mechanisms of recognition of non-self and initiation of defense reactions in insects

本课题的最终目的是研究入侵昆虫的细菌、真菌、寄生虫等外来物质被识别为“非我”的机制，以及“非我”的识别机制与昆虫防御反应的启动之间的联系。为了达到这一目的，本研究利用家蚕的两种昆虫组织——血淋巴和被皮进行了研究。血淋巴中存在酚氧化酶原级联反应（ProPO级联）。这种级联反应是由微量的-1,3-葡聚糖（β G）或肽聚糖（PG）触发的。我们之前已经证明PG和β ag分别与肽聚糖识别蛋白（PGRP）和β 1,3-葡聚糖识别蛋白（β GRP）特异性结合时触发级联反应。虽然人们推测这些识别蛋白可能在昆虫血液中将细菌或真菌识别为非自身的过程中起着核心作用，但我们对这些识别蛋白的化学结构和作用方式的了解非常有限。我们用免疫化学和免疫细胞化学的方法研究了这两种识别蛋白在血液液中的定位，并提出了这两种识别蛋白在血液液中将细菌或真菌识别为非自身过程中的作用的工作假设。proPO级联的组分包括两种以上的丝氨酸酶酶原。迄今为止获得的实验结果表明，活性形式的酶原或从酶原中提取的片段在其激活过程中调节血细胞的吞噬活性和血液动力学活性。为了明确证明这种可能性，我们目前正试图纯化所有proPO级联成分。我们最近成功地将proBAEEase及其活性形式纯化为同质性。该酶是否对血细胞的吞噬活性或血液动力学活性有影响，有待进一步研究。昆虫被皮的角质层通常被认为是一种保护性的盔甲和化学惰性（死亡）的身体的一部分。利用家蚕（Bombyx mori）体外培养的幼虫，我们可以证明，当角质层磨损部分含有细菌细胞壁成分（脂多糖或肽聚糖）时，角质层向被膜表皮细胞传递信号，指导其合成抗菌肽（抗菌肽）。抗菌肽分泌到角质层。细菌细胞壁成分在磨损角质层上的存在导致脂肪体中天蚕素mRNA的合成，但在脂肪体和血淋巴中未检测到天蚕素的活性。这种没有立即翻译的mRNA合成似乎表明，细菌细胞壁成分的角质层磨损使整个昆虫对细菌感染保持警惕。目前，我们正在研究细菌细胞壁成分的角质层磨损部分向表皮细胞和脂肪体发送信号的性质。少

The ultimate end of this research project is to study the mechanism by which foreign materials such as bacteria, fungi and parasites invading insects are recognized as non-self and how the recognition mechanism of non-self is linked to the initiation of defense reactions of insects. To reach the end the present research was carried out using two insect tissues, hemolymph and integument of the silkworm, Bombyx mori.The prophenoloxidase cascade (ProPO cascade) is present in hemolymph. The cascade is triggered by minute amounts of beta-1,3-glucan (beta G) or peptidoglycan (PG). We have shown previously that PG and betaG trigger the cascade when they bind specifically to peptidoglycan recognition protein (PGRP) and beta-1,3-glucan recognition protein (beta GRP), respectively. Although it is speculated that the recognition proteins may play a central role in the recognition of bacteria or fungi as non-self in insect hemocoel, our knowledge of the chemical structures and the mode of action o … More f the recognition proteins is very limited. We investigated the localization of the recognition proteins in hemocoel by immunochemical and immunocytochemical methods and put forward a working hypothesis on the role of the two recognition proteins in the process of recognition of bacteria or fungi as non-self in hemocoel.The components of proPO cascade include more than two serine enzyme zymogens. Experimental results so har obtained suggest that active forms of the zymogens or fragments derived from the zymogens during their activation modulate phagocytotic activity and hemokinetic activity of hemocytes. To prove this possibility unambiguously we are currently trying to purify all proPO cascade components. We recently have succeeded in purifying proBAEEase and its active form to homogeneity. It is expected that the active enzyme will be studied whether it has the effect on the phagocytotic or hemokinetic activity of hemocytes.Cuticle of insect integument is often considered to be a protective armor and chemically inert (dead) part of the body. Using axenically raised larvae of silkworm, Bombyx mori we could demonstrate that the cuticle transmit a signal to epidermal cells of integument to direct them to synthesize antibacterial peptide (cecropin) when the cuticle has bacterial cell wall component (lipopolysaccharide or peptidoglycan) on its abraded part. The antibacterial peptide is secreted to cuticle. The presence of bacterial cell wall component on abraded cuticle was shown to cause the synthesis of cecropin mRNA in fat body, but in this case no cecropin activity was detected in fat body and hemolymph. Seemingly this mRNA synthesis without immediate translation of the message indicate that cuticular abrasion with bacterial cell wall component make the the entire insect body alert to bacterial infection. Currently we are investigating the nature of signal sent to epidermal cells and fat body from the abraded part of cuticle with bacterial cell wall component. Less