Studies On Action Mode Of Vibrio Harveyi Hemolyisin (VHH)

With the rapid development of aquaculture, bacterial pathogens for marineanimals have caused severe economic loss. Vibriosis is a common and widespreadepidemic disease caused by Vibrios which are normal inhabitants in aquaticenvirments. Among these bacteria, Vibrio harveyi has become recognized as a seriouspathogen for marine animals. We know little about the mechanism for thepathogenesis of Vibrio harveyi.Among the potential virulent determinants, Vibrio harveyi hemolysin, i.e. VHH,a TLH (thermolabile hemolysin from Vibrio. Parahaemolyticus) -like protein wascertainly determined to involve in fish pathogenesis. In this study, gaschromatographic (GC) analysis was used to identify hydrolytic type of VHH. And theresult indicated that VHH has either phospholipase A1 activity or A2 activity. Hence itcould be considered as a phospholipase B. The result of chemical modificationshowed recombinant VHH was sensitive to the reagents for histidine and disulfidebond modification, revealing that there might be a histidine residue within the activesite of the phopholipase domain of VHH and one or more disulfide bonds werenecessary for the stability of VHH conformation. Ser153 was putatively identified asa very important residue of active site for VHH by bioinformatic method.Site-directed mutagenesis was performed to substitute this residue for anon-functional glycine. The mutant protein was overexpressed in Escherichia coli andpurified by metal chelating affinity chromatography. Enzymatic activity assays andpathogenicity test indicated that the mutant protein lost phospholipase and hemolyticactivities and pathogenicity to fish, which indicated that both the hemolytic activityand pathogenicity to fish of VHH were due to its phospholiapse activity. 3 keyresidues of a possible lipocalin-like domain localized at N terminal of VHH weresubstituted with alanine. And all the resulting mutant proteins lost phospholiapseactivties, suggesting the potential N terminal domain was necessary element for thephospholiapse activity of VHH. A 3-D model obtained from homology modelling of VHH phospholipase domain was employed to facilitate an in silico docking analysiswith phosphatidylcholine. And the result showed the potential binding sites of VHHto the substrate in catalysis process. Among these residues, tryptophan 174, theresidue binding to the choline part of phosphatidylcholine, might play a pivotal role insubstrate-enzyme binding because its substitution with alanine resulted in VHH lostthe phospholipase activity at all. At last, we examined the distribution of vhh genesamong the genome of V. harveyi strain VIB645. And the result indicated that one inchromosome and another in plasmid. This study facilitated the understanding ofpathogenesis of Vibrio harveyi and the mode of action of VHH/TLH-like hemolysin,and would be helpful to the development of vaccine against Vibrio harveyi.