CC25-like from Cromileptes altivelis (CaCC25-like) promotes antimicrobial immunity defense through chemotactic and direct antibacterial activity.

The humpback grouper (Cromileptes altivelis), a high-value marine species, encounters substantial challenges in aquaculture due to frequent disease outbreaks and elevated mortality rates. Chemokines, which serve as pivotal mediators of both innate and adaptive immunity, play an essential role in orchestrating leukocyte migration and pathogen clearance. However, their functional roles remain poorly characterized compared to those in mammals. In this study, we identified a CC25-like gene in C. altivelis (CaCC25-like) and investigated its immune-related functions. The CaCC25-like encodes a 96-amino-acid protein that contains a conserved SCY domain and NCCL motif, showing high homology to teleost CC25 orthologs (42.45 %-97.92 %) but low similarity to mammalian CC25 (<25 %). Under normal physiological conditions, CaCC25-like was ubiquitously expressed, with the highest expression levels in the liver. Following challenge with Vibrio harveyi, its expression was significantly upregulated in both the spleen and head kidney. Recombinant CaCC25-like protein (rCaCC25-like) induced dose-dependent chemotactic activity and proliferation of head kidney lymphocytes. Furthermore, rCaCC25-like exhibited direct antibacterial activity against V. harveyi, Streptococcus agalactiae, and Escherichia coli, reducing their survival rates by 19.83 %-79.57 %. Overexpression of CaCC25-like in vivo decreased bacterial colonization in immune tissues by 16.07 %-78.11 %, whereas siRNA-mediated knockdown of CaCC25-like increased pathogen loads by up to 6.59-fold. These findings demonstrate CaCC25-like as a multifunctional immune effector that enhances leukocyte recruitment, proliferation, and microbial clearance in humpback grouper. This study not only deepens the understanding of teleost chemokine biology, but also provides a solid foundation for developing immunomodulatory strategies aimed at improving disease resistance in aquaculture.