Molecular characterization, expression analysis, and functional identification of Lccystatin M from Large Yellow Croaker (Larimichthys crocea) in immune defense against Pseudomonas plecoglossicida infection.

Cystatins are key molecules widely involved in the regulation of cellular proteases and innate immune responses. In recent years, research on this family in the innate immunity of teleosts has deepened, gradually revealing their crucial roles in immune defense. However, the immune functions and mechanisms of this family in the economically important marine aquaculture species, large yellow croaker (Larimichthys crocea), have yet to be elucidated. Visceral white nodule disease (VWND), caused by Pseudomonas plecoglossicida, poses a serious threat to the healthy development of the large yellow croaker aquaculture industry, making the identification of disease resistance immune targets at the molecular level of great theoretical and practical value. In this study, a type II cystatin, named Lccystatin M, was successfully identified in large yellow croaker. Expression profile analysis showed that this gene was constitutively expressed at high levels in the gill tissue of healthy fish. Further functional experiments demonstrated that recombinant LcCystatin M not only exhibited bacterial agglutination activity and significant in vitro antibacterial effects against Gram-negative bacteria (P. plecoglossicida), but also showed the same effects against Gram-positive bacteria (Staphylococcus aureus), suggesting it might play a vital barrier defense role in the gills, which are direct contact with the external environment. In addition, following infection with P. plecoglossicida, the expression of Lccystatin M in the spleen, the main target tissue of the pathogen, was significantly upregulated, and the recombinant protein exhibited typical papain inhibitory activity. These results indicated that Lccystatin M not only participated in the immune response of large yellow croaker against P. plecoglossicida, but might also exert its immune defense function by inhibiting pathogen-associated protease activity. This study provided a new perspective for understanding the functions of cystatins in the innate immunity of teleosts and laid a theoretical foundation for the development of molecular targets for disease prevention and control of P. plecoglossicida infection in large yellow croaker.