Acute melanization of silkworm hemolymph by peptidoglycans of the human commensal bacterium Cutibacterium acnes

Abstract

Cutibacterium acnes is a pathogenic bacterium that cause inflammatory diseases of the skin and intervertebral discs. The immune activation induced by C. acnes requires multiple cellular responses in the host. Silkworm, an invertebrate, generates melanin by phenoloxidase upon recognizing bacterial or fungal components. Therefore, the melanization reaction can be used as an indicator of innate immune activation. A silkworm infection model was developed for evaluating the virulence of C. acnes, but a system for evaluating the induction of innate immunity by C. acnes using melanization as an indicator has not yet been established. Here we demonstrated that C. acnes rapidly causes melanization of the silkworm hemolymph. On the other hand, Staphylococcus aureus, a gram-positive bacterium identical to C. acnes, does not cause immediate melanization. Even injection of heat-killed C. acnes cells caused melanization of the silkworm hemolymph. DNase, RNase, and protease treatment of the heat-treated C. acnes cells did not decrease the silkworm hemolymph melanization. Treatment with peptidoglycan-degrading enzymes, such as lysostaphin and lysozyme, however, decreased the induction of melanization by the heat-treated C. acnes cells. These findings suggest that silkworm hemolymph melanization may be a useful indicator to evaluate innate immune activation by C. acnes and that C. acnes peptidoglycans are involved in the induction of innate immunity in silkworms.

Effects of insecticidal proteins of Enterobacter cloacae NK on cellular immunity of Galleria mellonella larvae

“…Most insects can trigger a cascade reaction of polyphenol oxidase after being invaded by pathogens, which results in the melanization reaction within a few minutes ( Matsumoto et al, 2022 ). This melanization reaction produces some toxic small molecular substances that can help kill most pathogens ( Ashida et al, 1990 ).…”

“…Although insecticidal proteins can reduce the number of hemocytes in G. mellonella larvae, they have different mechanisms, implying the diversity of the toxic mechanism of the insecticidal proteins. Most insects can trigger a cascade reaction of polyphenol oxidase after being invaded by pathogens, which results in the melanization reaction within a few minutes (Matsumoto et al, 2022). This melanization reaction produces some toxic small molecular substances that can help kill most pathogens (Ashida et al, 1990).…”

Acid-treatedStaphylococcus aureusinduces acute silkworm hemolymph melanization

“…C. acnes ATCC6919 and S. aureus Newman strains were used in this study. The C. acnes ATCC6919 strain was spread on Gifu anaerobic medium agar and incubated under anaerobic conditions at 37°C for 3 days [16]. The S. aureus Newman strain was spread on tryptic soy broth agar and incubated under aerobic conditions at 37°C for 1 day [16].…”

“…The C. acnes ATCC6919 strain was spread on Gifu anaerobic medium agar and incubated under anaerobic conditions at 37°C for 3 days [16]. The S. aureus Newman strain was spread on tryptic soy broth agar and incubated under aerobic conditions at 37°C for 1 day [16]. To prepare the C. acnes culture supernatant (CS), C. acnes ATCC6919 strain (8 x 10 9 cells) was added to 10 mL of tryptic soy broth + 2% glucose medium and incubated at 37°C for 6 days under anaerobic conditions.…”

“…The silkworm, an invertebrate, has benefits as a model animal to overcome these ethical issues [10,12,13]. Moreover, experimental systems using silkworms have been established to evaluate the innate immune activation by pathogenic microorganisms [14][15][16]. The melanization response is an innate immune mechanism of insects, including silkworms [17][18][19].…”