Immunogenicity And Secretion Regulation Mechanism Of Fructose 1,6-diphosphate Aldolase In Edwardsiella Piscicida

Intensive fish farming results in frequent disease occurrence. Edwardsiella piscicida is one kind of important pathogens which can infect kinds of fish, and leads to systematic hemorrhagic septicaemia, ulcer and necrosis of the muscular tissue or the organs such as liver, kidney and spleen. It is imperative to develop safe and effective vaccines with clear immune mechanism in controlling disease outbreaks and stopping antibiotics abuse in aquaculture.In our previous antigen screening experiment based on the genome sequence of E. piscicida with reverse vaccinology, fructose 1,6-diphosphate aldolase (FBA) showed excellent relative percent survival (RPS) in zebrafish against E. piscicida. FBA is an enzyme in glycolytic pathway, responsible for energy metabolism. In recent years, some glycolytic enzymes in pathogens were identified as moonlighting proteins which exerted metabolism functions of energy production in cytoplasm and virulence functions in extracellular matrix. Though a few studies reported that FBA in some pathogens had immunogenicity and could be secreted to the extracellular environment or transported to the surface of the pathogens to participate in pathogen adhesion to cell, there were few researches on immunogenicity and secretion regulation of FBA in E. piscicida.Hence, this study focuses mainly on immunogenicity, secretion pathway and regulation, and virulence related functions of FBA in E. piscicida. The results are as follows.Firstly, based on sequence alignment analysis, FBAs in many pathogens were found to be highly conserved under their sub-classification. In the analysis of non-cytoplasm localization, FBAs in five common aquaculture pathogens E. piscicida, Aeromonas hydrophila, Vibrio anguillarum, V. harveyi and V. alginolyticus could be secreted to extracellular environment. In subsequent vaccine evaluation in zebrafish model, FBA showed excellent cross protection against different pathogens with 45～80% of RPS. In turbot model (economic fish species), FBA showed 68% of RPS against E. piscicida. The sera sampled from turbot vaccinated with rFBA showed significant specific reaction against rFBA, and revealed cross-reaction and dramatic bactericidal activity against the five aquaculture pathogens. The up-regulated expression of immune related genes in vaccinated zebrafish further demonstrated that specific immune response of vaccinated fish could be effectively aroused. Therefore the housekeeping enzyme FBA in the glycolytic pathway in E. piscicida had excellent immune protective efficacy, and could be applied to broad spectrum vaccine development.Secondly, the FBA secretion levels in different E. piscicida secretion pathway mutants were analyzed while the FBA amount in the outer membrane vesicles (OMV) of E. piscicida was determined. These results indicated that the inactivation of single T3SS, T4SS, T6SS and Tat pathway could not block FBA secretion, and that OMV was not the main secretion pathway of FBA. In subsequent Pull-down assay, FBA was proved to interact with SecB, a chaperone in Sec pathway, and inferred probably to be transported by Sec machine. Based on the ELISA method, in the inserted inactivation mutant library (75.6% of genes coverage), no FBA secretion deficient strain was screened. It was inferred that FBA secretion might be closely related to bacterial growth and metabolism. Thus FBA might be not dependent on one single secretion pathway but be secreted by complex multiple secretion pathway, and its secretion was indispensable during the growth of bacteria.Moreover, in the research on secretion regulation, FBA could be secreted in E. piscicida without signal peptide. FBA secretion level was regulated by bacteria itself, and in the late secretion phage, its secretion level was in the dynamic equilibrium of FBA secretion and degradation. By screening of inserted inactivation mutant library and subsequent verification, the gene esrC was proved to play an important role in FBA secretion regulation. Its inactivation resulted in significant up-regulation of FBA secretion level. With culture time extension, the transcriptional level of gene fbaA in the AesrC mutant showed an insignificant difference compared to that in the wild type strain, but the transcriptional level of gene secB in the AesrC mutant indicated down-regulation tendency compared to that in the wild type strain. In addition, the transcriptional level of gene fbaA in the wild type strain was negatively correlated with FBA secretion level. During the growth of bacteria, the secretion level of FBA increased while on the contrary the transcriptional level of gene fbaA decreased. These results demonstrated that FBA secretion level was not controlled by its transcriptional level and also its transcriptional level was not regulated directly by transcription factor EsrC. EsrC indirectly regulated FBA secretion by directly regulating the gene secB (whose expression product SecB was proved to interact with FBA) or other secretion related genes, which indicated the complexity of FBA secretion regulation.Finally, it was found in the research on extracellular functions of FBA that the FBA in the extracellular medium or on the surface of bacteria still possessed the glycolytic enzyme activity. The structure of FBA in cytoplasm, periplasm and extracellular medium was consistent, and was not largely modified for secretion or exerting virulence associated functions. In the cell infection assay, FBA contributed to adhesion of E. piscicida strain to HeLa cells. By analyzing the FBA secretion level in 48 different E. piscicida strains, all the strains could secreted FBA, and their virulence was not positively correlated with their FBA secretion level.This study will provide theoretical foundations not only for vaccine development but also for the research on secretion regulation mechanism and virulence functions of glycolytic enzymes.