Molecular characterization, functional analysis, and defense mechanisms of two CC chemokines in Nile tilapia (Oreochromis niloticus) in response to severely pathogenic bacteria

• Two cDNAs encoded for CC chemokine genes in Nile tilapia (On-CC1 and On-CC2) were firstly cloned and characterized.
• The expression levels of On-CC1 and On-CC2 in response to S. agalactiae and F. columnare were intensively investigated.
• Functional analysis of rOn-CC1 and rOn-CC2 proteins at 10–100 µg/ml efficiently enhanced phagocytic activity of phagocytes.
• Southern assay and searching in Ensembl database indicated two functional CC chemokine genes exist in Nile tilapia genome.

Two full-length cDNAs encoding CC chemokine genes in Nile tilapia (Oreochromis niloticus) (On-CC1 and On-CC2) were cloned and characterized. On-CC1 and On-CC2 showed signature cysteine motifs consisting of four cysteines. The expression levels of On-CC1 and On-CC2 were analyzed by RT-PCR, which showed that low expression of these two genes was only observed in the peripheral blood leukocytes (PBLs) and spleen of normal fish. Expression levels of these two molecules were quantified in 13 tissues of fish infected with virulent strains of Streptococcus agalactiae and Flavobacterium columnare. Most tissues, especially PBLs, the spleen and the liver, expressed significantly higher mRNA levels than the controls, particularly at 12 and 24 h after infection (P < 0.05). The current study strongly indicates that CC chemokine genes in Nile tilapia are crucially involved in the early immune responses to pathogens. Functional analyses clearly demonstrated that 10 and 100 μg/ml of recombinant rOn-CC1 and rOn-CC2 proteins efficiently enhanced the phagocytic activity (in vitro) of Nile tilapia phagocytes. Finally, Southern blot analysis and searching in Ensembl databases demonstrated that two different functional CC chemokine genes and other pseudogene fragments were discovered in the Nile tilapia genome.