The Molecular Mechanism Of Lactobacillus Casei Resistance Against Aeromonas Infection In Zebrafish

Disease outbreaks,such as fish septicemia caused by Aeromonas,have become a major problem in limiting the healthy development of aquaculture.Probiotics plays important roles in animal health,showing a variety of beneficial effects,including promotion of gut health and homeostasis,growth,immune defenses and protection of host from pathogens infection.In this study,transposon library,target genes screening and gene knockout and other techniques were used to investigate the pathogenesis of Aeromonas spp.,and germ-free zebrafish model,gut microbiota transplant model,transcriptomics and other methods were used to investigate the mechanism of Lactobacillus on growth promotion and protecting against Aeromonas infection in zebrafish.The main contents and results are listed as follows:1.The mechanism of Aeromonas infection in zebrafishDuring the period of 2009-2014,there were five Aeromonas species detected in cyprinid fish septicemia in southern China,inculding A.veronii,A.hydrophila,A.jandaei,A.schubertii and A.aquariorum.Among the isolates from fish with clinical symptoms of motile Aeromonad septicemia(MAS),A.veronii and A.hydrophila infections were dominant.In addition,the mixed infection of A.veronii and A.hydrophila was the main way that caused septicemia in fish in south China,and A.veronii was the prerequisite for infection.According to results of the oro-intestinal infection model and the intraperitoneal injection model,it was found that A.hydrophila infection caused 100%of zebrafish mortality after 10-18h injection,while A.veronii caused only 10-40%zebrafish mortality after 96h injection.These indicated that the pathogenicity of A.veronii and A.hydrophila were quite different.A.veronii strains showed higher virulence than A.hydrophila strains in an oro-intestinal infection model,while the virulence of A.hydrophila was much higher than A.veronii strains in the intraperitoneal injection model.During the mixed infection process,the toxin aerolysin secreted by A.veronii species via type II secreted system caused serious peri-oral and intestinal lesions in fish,which made bacterial pathogen of A.hydrophila species or other species easy to invade into the body through the lesion sites,and finally led to septicemia and an enhanced mortality in fish.We found that A.veronii aerolysin(Aer)knockout strain lost the ability to cause peri-oral and intestinal damage in zebrafish,and also lost the ability to cause mortality in zebrafish.A.hydrophila RtxA(ACD)knockout strain showed lower virulence compared with the wild type strain(P<0.01).These data indicate that Aer and RtxA play a crucial role in the pathogenesis of A.veronii and A.hydrophila respectively.2.The mechanism of probiotics on growth promotion in zebrafishWe investigated the effects of six lactobacillus strains(L.rhamnosus LGG,L.rhamnosus 20300,L.casei BL23,L.plantarum JCM 1149 and L.acidophilus JCM 1132)on growth performance of zebrafish,respectively.The results showed that L.casei BL23 and L.rhamnosus 20300 could significantly increase the body weight of zebrafish larvae after continuous administrated with a dose of 106 CFU/mL for 21 days or 35 days(P<0.05).Furthermore,only viable cells of BL23(106 CFU/mL)could significantly increase the body weight of zebrafish larvae after 14 days or 28 days treatment,while the heat inactivated bacteria cells did not improve the body weight of the fish.Digital gene expression profiling(DGE)showed that BL23 viable bacteria cells could participate in the regulation of PPAR signal pathway,amino acid metabolism,fatty acid metabolism and steroid hormone synthesis in zebrafish,which are involved to promoting the growth and development in zebrafish.3.The mechanism of probiotics on promoting zebrafish resistance against Aeromonas infectionFirstly,zebrafish larvae(just hatching)pre-colonized with the potential probiotic bacterial strains and then the strains possessing anti-infective effect against A.veronii were screened.It showed that L.casei BL23 significantly increased the survival rate of zebrafish larvae upon A.veronii infection(P<0.01).The results of germ-free(GF)zebrafish model and gut microbiota transplant experiment showed that L.casei BL23 per se has anti-infective effects in zebrafish larvae,which was not involved in gut microbiota.Furthermore,an exopolysaccharide-protein complex(EPSP)extracted from L.casei BL23 cells was identified,which consisted of a 40-45 KD size protein and an exopolysaccharide composed of a-Rha,a-Glc,?-GlcNAc and ?-GalNAc.EPSP significantly increased the survival rate of GF zebrafish at a dose of 10-20?g/ml after A.veronii infection(P<0.01).In addition,the EPSP induced a higher gene expression of tlrl and tlr2(P<0.05),and modulated the expression profile of pro-inflammatory and anti-inflammatory cytokines in zebrafish liver(ZFL)cells(P<0.05).All these data indicated that the anti-infective effect of EPSP from L.casei BL23 was mediated by enhancement of immune responses in zebrafish,which might be involved in TLR1/TLR2 signal pathway.