The Insect Melanization Response in Galleria mellonella is Directly Fungicidal

A key component of insect immunity is melanin encapsulation of microbes. Melanization is also a part of an immune process known as nodulation, which occurs when insect hemocytes surround microbes and produce melanin. Insect nodules are analogous to mammalian immune granulomas. Melanin is believed to kill microbes through the production of toxic intermediates and oxidative damage. However, it is unclear to what extent immune melanin is directly fungicidal during infections of insect hosts. We reported previously that C. neoformans cells are encapsulated with host-derived melanin within hemocyte nodules. Here we report an association between melanin-based immune responses by Galleria mellonella wax moth larvae and fungal cell death of C. neoformans during infection. To monitor melanization in situ, we applied a tissue-clearing technique to G. mellonella larvae, revealing that nodulation occurs throughout the organism. Further, we developed a protocol for time-lapse microscopy of extracted hemolymph following exposure to fungal cells, which allowed us to visualize and quantify the kinetics of the melanin-based immune response. Using this technique, we found evidence that cryptococcal melanins and laccase enhance immune melanization in hemolymph. We used these techniques to also study the fungal pathogen Candida albicans infections of G. mellonella. We find that the yeast form of C. albicans was the primary targets of host melanization, while filamentous structures were melanin-evasive. Approximately 23% of melanin-encapsulated C. albicans yeast survive and break through the encapsulation. Overall, our results provide direct evidence that the melanization reaction functions as a direct antifungal mechanism in insect hosts.

昆虫免疫机制中的一个关键组成部分为微生物的黑色素包裹。黑色素化亦为一种被称为结节的免疫过程的一部分，该过程发生时，昆虫的血细胞环绕微生物并产生黑色素。昆虫结节与哺乳动物的免疫肉芽肿相类似。据信，黑色素通过产生有毒中间体和氧化损伤来杀死微生物。然而，关于免疫黑色素在昆虫宿主感染期间直接具有抗真菌作用程度的问题尚不明确。我们先前报道，新型隐球菌细胞在血细胞结节中被宿主来源的黑色素所包裹。在本研究中，我们报告了家蚕蜡虫幼虫基于黑色素产生的免疫反应与新型隐球菌在感染期间的真菌细胞死亡之间的关联。为了在原位监测黑色素化过程，我们应用了一种组织清亮技术于家蚕蜡虫幼虫，揭示了结节在整个机体中发生。此外，我们开发了一种在暴露于真菌细胞后对提取的血液淋巴进行时间推移显微摄影的方案，该方案使我们能够可视化并量化基于黑色素的免疫反应的动力学。利用这一技术，我们发现隐球菌黑色素和漆酶增强了血液淋巴中的免疫黑色素化。我们利用这些技术还研究了家蚕蜡虫感染的白假丝酵母。我们发现，白假丝酵母的酵母形态是宿主黑色素化的主要靶标，而丝状结构则逃避黑色素。大约23%的黑色素包裹的白假丝酵母酵母能够存活并突破包裹。总体而言，我们的研究结果直接证明了黑色素化反应在昆虫宿主体内作为一种直接的抗菌机制发挥作用。