Functional characterization of complement factor D on the defense against Gyrodactylus kobayashii (Monogenea) infection in goldfish (Carassius auratus)

Abstract

Complement factor D (CfD), a member of serine peptidases superfamily, acts as the rate-limiting enzyme of alternative complement pathway (AP) and is important for the AP-initiated C3 activation and subsequent amplification of complement cascade. The functions of the CfD in antibacterial and antiviral have been demonstrated; however, much less is known about the anti-parasite effect of CfD in teleost fish. In this study, we firstly demonstrated that the complements from goldfish serum showed high lethal effect on Gyrodactylus kobayashii acting as the AP. The serum activity against G. kobayashii was abolished by addition of EDTA, but not by EGTA + Mg2+. Subsequently, we cloned and characterized the goldfish (Carassius auratus) CfD (CaCfD), whose open reading frame consists of 753 bp, encoding 250 amino acids for a 48 kDa polypeptide. Bioinformatics analysis results showed that CfD had only one serine protease trypsin domain, and the serine active sites were completely conserved in teleost fish during evolution. The ubiquitous expression of CaCfD in all tested tissues was displayed, with the highest expression in the liver. After G. kobayashii infection, the mRNA levels of CaCfD were significantly up-regulated in blood, liver, spleen and kidney. The hemolysis percentage of the rabbit red blood cells (RRBCs) was enhanced by serum from G. kobayashii-infected goldfish, indicating that more complement components in serum were stimulated. Besides, we confirmed that recombinant CaCfD protein ((r)CaCfD) significantly improved the RRBCs lysis mediated by the complements, probably the increase of CaCfD level in complement system. (r)CaCfD also showed high anti-parasitic effect against G. kobayashii in vitro. Taken together, this study enriches the function of CfD participating in host defense against fish parasites, and the role of the complements in the protection of fishes against gyrodactylids infection deserves further investigation.