Studies On The Resuscitation-promoting Factor Like Glycoprotease Of Vibrio Parahaemolyticus And V. Harveyi

Vibrio parahaemolyticus and V. harveyi are opportunistic pathogens of marine animals. V. parahaemolyticu is also an important food-poisoning pathogen in coastal countries. The bacteria are able to adapt to environmental stresses and enter into the viable but nonculturable (VBNC) state. The VBNC cells could recover to culturable state under suitable conditions. However, the resuscitation mechanism remains unknown. Resuscitation-promoting factor is a novel bacterial cytokine family, which was first discovered in Micrococcus luteus. Purified Rpf increased the viable cell count of dormant M. luteus cultures at 100 pM concentrations and stimulated the growth of viable M. luteus cells and some mycobacteria. There have little reports about Rpf of marine vibrios. The aim of this study was to clone and express the resuscitation-promoting factors like glycoprotease (Gcp) gene from V. parahaemolyticus 8621.4 and V. harveyi SF-1, construct the gcp mutant strain, and evaluate the roles of gcp in entering into the VBNC state under starvation stresses and recovering from the VBNC state.The gcp genes were cloned by PCR amplification from the chromosomal DNA of V. parahaemolyticus 8621.4 and V. harveyi SF-1. The nucleotide sequences were determined and both of the gcp genes consist of 702 bp, which encoded a polypeptide of 233 amino acids. Neither have a clear secretion signal and transmembrane domain. The similarities of the polypeptide sequence from V. parahaemolyticus 8621.4 with those of other V. parahaemolyticus strain, V. harvey, Vibrio sp. Ex25, Vibrio coralliilyticus, Vibrio mimicus and Vibrio salmonicida were from 67% to 100%. It also showed 55% of similarities with the Rpf (Gcp) of Proteus mirabilis HI4320 and the M22 peptidase YeaZ of Yersinia, and 57% similarities with the Rpf of Salmonella and Escherichia coli. The amino acids sequence from V. harveyi SF-1 showed 69% to 98% of identity to the corresponding amino acids sequence of other V. harveyi strains, V. alginolyticus, V. parahaemolyticus, V. vulnificus, V. coralliilyticus, V. cholerae, V. metschnikovii and V. splendidus.The genes were subcloned to pET-28a (+) and expressed in BL21 (DE3). The expressed protein were purified by Ni2+-affinity chromatography. The polyclonal antibody was prepared by immunization of rabbits with the purified Gcp protein of V. parahaemolyticus 8621.4. A specific protein band of 27 kDa was detected in the whole-cell lysate preparation of normal cultural cells and 45 days culture cells of V. parahaemolyticus 8621.4 and V. harveyi SF-1 at low temperature conditions by western-blotting analysis, one additional band could be detected in the VBNC cells of V. parahaemolyticus 8621.4. The Gcp promoted the growth of viable V. harveyi SF-1 significantly (OD590, p<0.01). A gcp mutant was constructed by integration of the suicide plasmid containing the 425 bp fragment of gcp gene into the chromosomal gcp gene of V. harveyi SF-1 by homologous recombination. Both wild-type and the mutant strains of V. harveyi SF-1 grew well in 2216E broth at 28℃, the mutant growed faster than the wild type (OD590, p<0.01), and there were no difference between the mutant supplemented Gcp and the wild type (OD590, p>0.05).Virulence of the gcp mutant strain was also decreased when they were intraperitoneally inoculated into zebra fish, the LD50 of the wild-type and the mutant were 2.66×104 CFU/ mL and 3.13×105 CFU/ mL respectively.