Secretion Regulation Of GAPDH In Edwardsiella Tarda

Edwardsiella tarda is a critical pathogenic bacterium in aquaculture industry,especially for some economically important fish species.Its infection could potentially lead to huge losses resulted from the outbreaks of severe diseases,such as sepsis.In the early stage of the experiment,a strong virulent strain,E.tarda EIB202,was isolated from disease turbot.Subsequently,huge amount of experiment was carried out to explore potential vaccine.Glyceraldehyde-3-phosphate dehydrogenase（GAPDH）,an important housekeeping enzyme in glycolytic pathway,could be used as protective antigen protein which offers an optimizing target in subunit vaccine of aquaculture.In this paper,the transcription and extracellular secretion of GAPDH will be analyzed in E.tarda EIB202 to illuminate the mechanism of GAPDH secretion,and the main results in this study were as follows.Firstly,secretion of GAPDH in E.tarda EIB202 and its insertional inactivated strains of secretory pathway were analyzed by Western-blot and ELISA.It is shown that EIB202 does not secrete GAPDH via any of these secretory pathway.Secretion of GAPDH in different condition of culture was analyzed in addition.It was found that 30℃、DMEM medium and static culture were the best conditions for GAPDH secreting in E.tarda EIB202.According to the result,western-blot is better in qualitative detection whereas ELISA is better in quantitative determination.Through high-throughput screening of mutant library,three strains,ETAE₀885、ETAE₀861 and ETAE₀986,were found out to have higher secretion of GAPDH.Their genes were named esrA,esrC and major facilitator superfamily gene respectively,of which esrA and esrC belong to a two-component regulatory system.Lacking of the genes lead to significant up-regulation of secretion of GAPDH.Quantitative real-time PCR was used to compare the mRNA expression of these genes.It is shown that all of their products played a negative regulation role in gap expression..These findings laid a solid foundation to further explore the mechanism of secretion of GAPDH in E.tarda and to exploit subunit vaccine.