Study On The Immunomodulatory Activity Of Phosvitin-derived Antibacterial Peptide Pt5in Zebrafish

Innate immunity plays an important role in the anti-infectious response of fish.Antimicrobial peptides (AMPs) is an important innate immune effector moleculeinvolved in this process. When fish encounters invading pathogens, they cansynthesize and release AMPs by immune-rellevant cells to fight the microbes. Inaddition to direct antibacterial activity, AMPs also play other biological functions likeimmunomodulatory activity, helping the fish to keep physiological homoestasis.This research was conducted on the basis of our previous study, whichdemonstrated that phosvitin (Pv) is associated with the immune defense of developingembryos in zebrafish and is a multivalent pattern recognition receptor as well as anantimicrobial effector capable of damaging Gram-negative and-positive microbes.Our previous studies also revealed that Pv-derived peptide Pt5(consisting of theC-terminal55residues) showd an antibacterial activity similar to Pv, indicating theprotective effects of Pv-derived peptide against pathogen infection in fish innateimmunity. A growing number of studies has been shown that in addition tofunctioning as antibacterial agents, AMPs also have immunomodulatory activity.There have been a large amount of reports showing the the antibactyerial activities ofAMPs in fish, but research on their immune modulatory activity and relativemechanism is rather limited to date. The aim of this study was therefore to examinethe immune and immunomodulatory activities of Pt5in zebrafish in more detail.Initially, we used the technique of self-induced expression of exogenous genesreported by Studier and synthesized enough quantity of high quality of recombinantPt5, laying a foundation for the subsequent experiments. The recombinant proteinsynthysized thus retained full antibacterial activity as observed previously. We thenestablished an acute sepsis model by injection of pathogenic A. hydrophila into the zebrafish Danio rerio to examine the antibacterial activity of Pt5. The experimentalresults showed that after acute bacterial infection, the survival percentage of thezebrafish injected with Pt5was significantly higher than that of the fish injected withbuffer alone or with the inactive Pt5mutant, M13. Moreover, the number of A.hydrophilia detected in the infected tisssues was significantly reduced in Pt5-injectedfish compared with control fish. Peripheral blood cell composition analysis showedthat Pt5was able to induce a marked reduction in the leukocyte number in sepsismodel fish. It was clear that Pt5play an immunomodulatory activity, capable ofreducing inflammatory responses in fish, protecting them from acute septicemia.To understant the mechanism Pt5plays in the acute infection of zebrafish,semi-quantitative real-time PCR was used to analyze the effects of Pt5on theexpression profiles of proinflammatory cytokines as well as inflammatory cytokinesin the spleen and head kidney of zebrafish following injection with A. hydrophilia orbuffer alone. The results showed that in the zebrafish which had not been challengedwith A. hydrophilia, injection with Pt5could not induce up-regulation ofproinflammatory cytokines as well as inflammatory cytokines, whereas in thezebrafish which had been challenged with A. hydrophilia, injection with Pt5couldcause a significant decrease in the expression levels of proinflammatory cytokines aswell as inflammatory cytokines at6and12h. By contrast, injection of M13or bufferalone into zebrafish (which had been challenged with A. hydrophilia) resulted in aconsiderable elevation of proinflammatory cytokines as well as inflammatorycytokines.It is apparent from the above experimental results that the antimicrobial peptidePt5plays an immunomodulatory role capable of maintaining the homeostasis, therebyprotecting the infected fish.