Galleria mellonella immune response to infection with Streptococcus pyogenes

The larvae of Galleria mellonella are increasingly popular as infection model organism to study virulence mechanisms and pathogenicity of bacteria and fungi. In this study, the Streptococcus pyogenes-specific immune response of the larvae was investigated using a proteomics approach. Larvae of the greater wax moth G. mellonella were obtained from Bugs-International GmbH (Irsingen, Germany). The larvae were stored and fed in a wheat bran mixture at 37°C. Larvae with a weight of 0.15 – 0.2 g were used in the experiments. S. pyogenes M49 strain 591 was grown overnight in THY, washed twice in a 0.9% NaCl solution, and suspended in 0.9% NaCl to a final concentration of 2.4 – 4 x 108 CFU/mL. Larvae were infected with 2.4 – 4 x 106 CFU/larva in 10 µL 0.9% NaCl. In each experiment, 45 larvae were infected with S. pyogenes and 30 larvae were injected with 0.9 % NaCl (mock control). Hemolymph was extracted after 4 h, 24 h, and 72 h from 15 infected larvae and 10 non-infected larvae per time point. The hemolymph was separated by centrifugation into cell-free hemolymph and hemocytes. Each experiment was performed in three biological replicates. The protein abundance was regulated in the course of the infection. In the early infection phase, four hours post infection (p.i.), proteins responsible for recognition of foreign surfaces were upregulated, e.g. peptidoglycan recognition proteins. After 24 h, sensory proteins, receptors involved in recognition and opsonisation, and antimicrobial peptides were detected with increased amounts compared to the control group. 72 h p.i., proteins involved in development and metabolism were present at reduced levels. Overall, the immune response of larvae showed relative specificity to S. pyogenes infection, high similarity to the mammalian innate immune response, and a greater complexity than appreciated today.