A Study On The Intracellular Trafficking Pathways Of Edwardsiella Tarda And The Interaction Between E.Tarda And Host Complement Regulatory Factor CD59

Edwardsiella tarda is a Gram-negative bacterial pathogen that can infect a variety of hosts,especially fish,and has been one of the most serious fish pathogens.Previous studies have shown that E.tarda can reproduce in host phagocytic cells such as macrophages and can also resist immune killing of host serum.However,the mechanism of these phenomena is unclear.The purpose of this paper is to study the infection pathway of E.tarda in macrophages and to explore the interaction between E.tarda and complement regulatory factor CD59(CsCD59)of Cynoglossus semilaevis,which will promotes us to comprehend the mechanism of immune evasion of E.tarda.To explore the intracellular trafficking pathway of E.tarda,we found that E.tarda TX1 entered RAW264.7 and multiplied intracellularly in a robust manner.Cellular invasion of E.tarda was significantly impaired by inhibition of clathrin-and caveolin-mediated endocytic pathways and by inhibition of endosome acidification,but not by inhibition of macropinocytosis.Consistently,RAW264.7-infecting E.tarda was co-localized with clathrin,caveolin,and hallmarks of early and late endosomes,and intracellular E.tarda was found to exist in acid organelles.In addition,E.tarda in RAW264.7 was associated with microfilament and microtubule,and blocking of the functions of these cytoskeletons by inhibitors significantly decreased E.tarda infection.Furthermore,formaldehyde-killed E.tarda exhibited routes of cellular uptake and intracellular trafficking similar to that of live E.tarda.Together these results provide the first evidence that entry of live E.tarda into macrophages is probably a passive,virulence-independent process of phagocytosis effected by clathrin-and caveolin-mediated endocytosis and cytoskeletons,and that the intracellular traffic of E.tarda involves endosomes and endolysosomes.The complement system is an integral part of immunity system and consists of multiple proteins.CD59 is a regulator of the complement system that inhibits the formation of membrane attack complexes in the complement system and protects endogenous cells from lysis.To investigate whether E.tarda can utilize CD59 to escape the killing of the fish complement system,we firstly performed molecular cloning,protein expression and purification of CsCD59.CsCD59 possesses the conserved structural features of CD59 and shares 33%-46% sequence identities with other fish CD59.Expression of CsCD59 was high in liver,spleen,and muscle,and was stimulated by infection of bacterial pathogens.Recombinant CsCD59(rCs CD59)exhibited an apparent inhibition effect on the activation of tongue sole serum complement.ELISA and microscopy detected binding of rCsCD59 to a number of Gram-negative and Gram-positive bacteria.Interaction with rCsCD59 did not affect bacterial viability but significantly enhanced bacterial resistance against the killing effect of fish serum.Together these results indicate that fish CD59 may to some degrees facilitate a general escape of bacteria from complement-mediated immunity.