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.昆虫免疫的核心环节之一，是对微生物实施黑色素包囊（melanin encapsulation）。黑化反应（melanization）同时也是结瘤（nodulation）免疫过程的组成部分——当昆虫血细胞（hemocytes）包裹微生物并合成黑色素时，便会触发结瘤过程。昆虫结节与哺乳动物的免疫肉芽肿（immune granulomas）功能相似。学界普遍认为，黑色素可通过生成毒性中间产物及引发氧化损伤来杀灭微生物。然而，目前尚不清楚在昆虫宿主感染过程中，免疫源性黑色素在多大程度上可直接发挥杀真菌效应。我们此前的研究表明，新型隐球菌（C. neoformans）的菌体可在血细胞结节中被宿主来源的黑色素包囊。本文中，我们报道了大蜡螟（Galleria mellonella）幼虫的黑色素依赖性免疫反应，与感染过程中新型隐球菌的细胞死亡之间存在关联。为原位监测黑化反应，我们对大蜡螟幼虫应用了组织透明化技术，结果显示结瘤过程遍布虫体全身。此外，我们开发了一套针对离体血淋巴（hemolymph）暴露于真菌菌体后的延时显微成像（time-lapse microscopy）方案，借此可可视化并量化黑色素依赖性免疫反应的动力学特征。利用该技术，我们发现隐球菌自身的黑色素与漆酶（laccase）可增强血淋巴中的免疫黑化反应。我们还借助上述技术，研究了白色念珠菌（Candida albicans）对大蜡螟的感染过程。结果显示，白色念珠菌的酵母态（yeast form）菌体是宿主黑色素包囊的主要靶标，而丝状菌体结构（filamentous structures）则可逃逸黑色素包囊。约23%被黑色素包囊的白色念珠菌酵母菌体可存活并突破包囊。总体而言，本研究结果提供了直接证据，证明黑化反应可在昆虫宿主中作为直接的抗真菌免疫机制发挥作用。