Galleria mellonella immune melanization is fungicidal during infection

A key component of the insect immune system is the ability to induce melanization during microbial infection, including during nodulation – a mechanism in which insect immune cells (hemocytes) surround microbes. Melaninization causes oxidative damage and releases toxic intermediates that control microbial infections. However, a direct fungicidal role of melanin during infection has not been previously demonstrated. We previously reported that the fungus Cryptococcus neoformans is encapsulated with insect-derived melanin within nodules. Here we developed and applied several techniques to Galleria mellonella wax moth larvae infected with C. neoformans to better study melanin’s role during infection. We provided evidence that in vivo melanin encapsulation within nodules was fungicidal. To monitor melanization that occurred during infection, we applied a tissue-clearing technique to G. mellonella larvae to visualize melanized nodules in situ throughout the organism following infection. Further, we developed a protocol using time-lapse microscopy to visualize and quantify the kinetics of the melanization response in extracted hemolymph following exposure to C. neoformans. Using this technique, we found evidence that cryptococcal melanin and laccase enhance immune melanization. We extended this in vitro approach to study the fungal pathogens Candida albicans and Candida auris. We find that the yeast forms of C. albicans and C. auris were the primary targets of host melanization, while filamentous and pseudohyphal structures were melanin evasive. Approximately 23% of melanin-encapsulated C. albicans yeast survive, replicate, and breakthrough the encapsulation. Overall, our results provide direct evidence that the melanization reaction functions as a direct antifungal mechanism in G. mellonella hosts.