Toll-like receptors (TLRs) represent a prominent category of pattern recognition receptors that have been extensively investigated for their pivotal role in combating pathogen incursions. Despite this, there has been a notable absence of comprehensive identification and exploration of the immune response associated with the TLR family genes in C. altivelis. This study successfully identified and named fourteen genes as Catlr1-1, Catlr1-2, Catlr2-1, Catlr2-2, Catlr3, Catlr5, Catlr7, Catlr8, Catlr9, Catlr13-1, Catlr13-2, Catlr18, Catlr21, and Catlr22. A series of bioinformatic analysis were performed, encompassing analysis of protein properties, examination of gene structures, evolutionary assessments, and prediction of protein tertiary structures. The expression patterns of Catlr genes were analyzed in five immune tissues: liver, spleen, kidney, gill, and intestine, in both healthy and bacterial stimulated-fish. The results showed that different tissue and different genes showed differed expression patterns after V. harveyi infection, indicating the involvement of all Catlr members in mounting immune responses following infection in various tissues. Additionally, histological evaluations of immune tissues unveiled varying levels of damage. In conclusion, this investigation into the TLR gene family offers novel information that contribute to a more profound comprehension of the immune response mechanisms in C. altivelis.

Chemokines are a family of small signaling proteins that are secreted by various cells. In addition to their roles in immune surveillance, localization of antigen, and lymphocyte trafficking for the maintenance of homeostasis, chemokines also function in induce immune cell migration under pathological conditions. In the present study, a novel CC chemokine gene (CaCC1) from humpback grouper (Cromileptes altivelis) was cloned and characterized. CaCC1 comprised a 435 bp open reading frame encoding 144 amino acid residues. The putative molecular weight of CaCC1 protein was 15 kDa CaCC1 contains four characteristic cysteines that are conserved in other known CC chemokines. CaCC1 also shares 11.64%-90.28% identity with other teleost and mammal CC chemokines. Phylogenetic analysis revealed that CaCC1 is most closely related to Epinephelus coioides EcCC1, both of which are in a fish-specific CC chemokine clade. CaCC1 was constitutively expressed in all examined C. altivelis tissues, with high expression levels in skin, heart, liver, and intestine. Vibrio harveyi stimulation up-regulated CaCC1 expression levels in liver, spleen, and head-kidney. Functional analyses revealed that the recombinant protein (rCaCC1) could induce the migration of head-kidney lymphocytes from C. altivelis. Moreover, rCaCC1 significantly enhanced phagocytosis in head-kidney macrophages from C. altivelis. In addition, rCaCC1 exhibited antimicrobial activities against Staphylococcus aureus, Edwardsiella tarda, and V. harveyi. In vivo, CaCC1 overexpression improved bacterial clearance in V. harveyi infected fish. Conversely, CaCC1 knockdown resulted in a significant decrease of bacterial clearance. These results demonstrate the important roles that CaCC1 plays in homeostasis and in inflammatory response to bacterial infection.