# Acid-treated Staphylococcus aureus induces acute silkworm hemolymph melanization

## TL;DR

This study shows that acid-treated Staphylococcus aureus activates the innate immune response in silkworms, as indicated by increased hemolymph melanization.

## Contribution

The novel finding is that acid treatment of S. aureus enhances its ability to induce innate immunity in silkworms, possibly through bacterial proteins.

## Key findings

- Acidification of Staphylococcus aureus increases silkworm hemolymph melanization.
- Protease treatment inhibits the melanization activity of acid-treated S. aureus.
- C. acnes culture supernatant lowers pH and enhances S. aureus-induced immune activation.

## Abstract

The skin microbiome maintains healthy human skin, and disruption of the microbiome balance leads to inflammatory skin diseases such as folliculitis and atopic dermatitis. Staphylococcus aureus and Cutibacterium acnes are pathogenic bacteria that simultaneously inhabit the skin and cause inflammatory diseases of the skin through the activation of innate immune responses. Silkworms are useful invertebrate animal models for evaluating innate immune responses. In silkworms, phenoloxidase generates melanin as an indicator of innate immune activation upon the recognition of bacterial or fungal components. We hypothesized that S. aureus and C. acnes interact to increase the innate immunity-activating properties of S. aureus. In the present study, we showed that acidification is involved in the activation of silkworm hemolymph melanization by S. aureus. Autoclaved-killed S. aureus (S. aureus [AC]) alone does not greatly activate silkworm hemolymph melanization. On the other hand, applying S. aureus [AC] treated with C. acnes culture supernatant increased the silkworm hemolymph melanization. Adding C. acnes culture supernatant to the medium decreased the pH. S. aureus [AC] treated with propionic acid, acetic acid, or lactic acid induced higher silkworm hemolymph melanization activity than untreated S. aureus [AC]. S. aureus [AC] treated with hydrochloric acid also induced silkworm hemolymph melanization. The silkworm hemolymph melanization activity of S. aureus [AC] treated with hydrochloric acid was inhibited by protease treatment of S. aureus [AC]. These results suggest that acid treatment of S. aureus induces innate immune activation in silkworms and that S. aureus proteins are involved in the induction of innate immunity in silkworms.