Sp Crus3 and Sp Crus4 share high similarity in mud crab ( Scylla paramamosain ) exhibiting different antibacterial activities

Abstract Type I crustins are crucial effectors of crustacean immune system. Various type I crustins with high sequence diversity possess different antimicrobial activities. To date, the mechanism on how the sequence diversity of type I crustins affects their antimicrobial activities is largely unclear, and how different crustins function together against bacterial invasion still remains unknown. In this study, we identified two novel type I crustins, namely, Sp Crus3 and Sp Crus4, from an economically important crab, Scylla paramamosain . Either SpCrus3 or SpCrus4 was highly expressed in gill. After challenges with Vibrio parahemolyticus or Staphylococcus aureus , SpCrus4 was up-regulated, whereas SpCrus3 was down-regulated. No significant expression change of SpCrus3 and SpCrus4 was observed after white spot syndrome virus injection, suggesting that these two genes may not participate in the antiviral immune responses. SpCrus3 and SpCrus4 had the common 5′ terminus and high similarity of 66.06%, but Sp Crus4 exhibited stronger antimicrobial activity than that of Sp Crus3. Microorganism-binding assay results revealed that both Sp Crus3 and Sp Crus4 exhibited binding ability to all tested microorganisms. Furthermore, the polysaccharide-binding assay showed that these two proteins exhibited strong binding activity to bacterial polysaccharides, such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN). Sp Crus3 and Sp Crus4 exhibited stronger binding activity to LPS or LTA than to PGN. Moreover, Sp Crus4 showed stronger binding activity to LTA than that of Sp Crus3, which may be responsible for the significantly distinct antimicrobial activity between these two proteins. In addition, Sp Crus4 displayed stronger agglutination activity against several kinds of microorganisms than that of Sp Crus3. This increased agglutination activity may also contribute to the strong antibacterial activity of Sp Crus4. On the basis of all these results, a possible antibacterial mode exerted by Sp Crus3 and Sp Crus4 was proposed as follows. SpCrus3 was highly expressed in normal crabs to maintain low-level antibacterial activity without bacterial challenges. When crabs were challenged with bacteria, large amount of Sp Crus4 was generated to exhibit strong antibacterial activity against bacterial invasion. This study provides new insights to understand the antibacterial functions and mechanisms of type I crustins.