Physiological Function Of The Zebrafish Pore-forming Protein Aep1

Pore forming proteins(PFPs)have been identified in all kingdoms of life,which are featured with the existence of both water-soluble monomers and transmembrane assembly.After the soluble-form PFPs specifically recognize the receptor in the target cell membrane,they oligomerize into a ring-like complex that penetrates the cell membrane,leading to the permeability change and eventually the death of the target cell.This characteristic of PFPs makes they also exhibit various functions,such as participating in host defense,bacterial attack,apoptosis,programmed cell death and cell signal transduction and so on.As the second largest family of ?-PFPs,the aerolysin family is first identified from Aeromonas hydrophila.Currently,the research of this family is mainly focused on various types of bacterial PFPs,whereas aerolysin-like proteins from eukaryotes have not yet been clearly studied.Recently,we reported the receptor recognition and in vitro perforation mechanism of the aerolysin-like protein Dln1/Aep1 from zebrafish.However,its physiological function remains unknow.Here we detected that aepl gene is constitutively expressed in various immune-related tissues of adult zebrafish;and moreover,its expression is significantly upregulated upon bacterial challenge,indicating its involvement in antimicrobial infection.Pre-injection of recombinant Aep1 into the infected zebrafish greatly accelerated the clearance of bacteria,resulting in significantly increased survival rate.Meanwhile,the induced expression of cytokines such as interleukin IL-1? and tumor necrosis factor TNF-a in zebrafish upon injection of recombinant Aepl suggested that Aepl may be a pro-inflammatory protein that triggers the antimicrobial immune responses.However,compared to the overproduction of these cytokines in the infected zebrafish,pre-injection of Aepl could significantly reduce the expression level of these cytokines,accompanying with a reduced bacterial load.Moreover,the expression profiles through the developmental stages of zebrafish demonstrated that aepl is activated at the very early stage prior to the maturation of adaptive immune system.Based on the above data and analysis,we proposed that Aepl is an innate immune molecule that prevents the bacterial infection.This study fills the gap in the study of the physiological function of zebrafish aerolysin-like protein,and moreover,it provides hints for the research on the function of other aerolysin proteins.