Characterization of DegQ _Vh , a Serine Protease and a Protective Immunogen from a Pathogenic Vibrio harveyi Strain

Abstract

Vibrio harveyi is an important marine pathogen that can infect a number of aquaculture species. V. harveyi degQ (degQ Vh ), the gene encoding a DegQ homologue, was cloned from T4, a pathogenic V. harveyi strain isolated from diseased fish. DegQ Vh was closely related to the HtrA family members identified in other Vibrio species and could complement the temperature-sensitive phenotype of an Escherichia coli strain defective in degP. Expression of degQ Vh in T4 was modulated by temperature, possibly through the E -like factor. Enzymatic analyses demonstrated that the recombinant DegQ Vh protein expressed in and purified from E. coli was an active serine protease whose activity required the integrity of the catalytic site and the PDZ domains. The optimal temperature and pH of the recombinant DegQ Vh protein were 50°C and pH 8.0. A vaccination study indicated that the purified recombinant DegQ Vh was a protective immunogen that could confer protection upon fish against infection by V. harveyi. In order to improve the efficiency of DegQ Vh as a vaccine, a genetic construct in the form of the plasmid pAQ1 was built, in which the DNA encoding the processed DegQ Vh protein was fused with the DNA encoding the secretion region of AgaV, an extracellular ␤-agarase. The E. coli strain harboring pAQ1 could express and secrete the chimeric DegQ Vh protein into the culture supernatant. Vaccination of fish with viable E. coli expressing chimeric degQ Vh significantly (P < 0.001) enhanced the survival of fish against V. harveyi challenge, which was possibly due to the relatively prolonged exposure of the immune system to the recombinant antigen produced constitutively, albeit at a gradually decreasing level, by the carrier strain.Under stress conditions, such as those induced by elevated temperatures, extreme pH, osmolarity shock, and host immune responses, damaged and misfolded proteins are accumulated in bacteria; the removal and correction of these otherwise harmful proteins depend on the operation of a number of protease/chaperon systems, among which are the HtrA/DegP proteins (5, 6, 28). Initially discovered in Escherichia coli as an essential protein required for growth at high temperatures (22,23,41), HtrA (high temperature requirement A) homologues have been identified in diverse prokaryotic and eukaryotic organisms, including humans (10). These proteins possess the dual function of protease and chaperon and can switch roles according to the input of environmental stimuli (39). Members of the HtrA family, which include DegP (also known as DO), DegQ, and DegS, share a relatively high level of sequence identity and the common feature of an N-terminal protease domain followed by one or two PDZ domains. In E. coli, degQ and degS are located next to each other on the chromosome and are transcribed independently (44); their respective encoded proteins, DegQ and DegS, are ϳ36% identical to DegP, whose coding sequence is physically separated from the degQdegS cluster. DegP is involved in the refolding/degradation of abnormal prot...

Evaluation of the vaccine potential of a cytotoxic protease and a protective immunogen from a pathogenic Vibrio harveyi strain

“…V. harveyi T4D1 is genetically isogenic to V. harveyi T4, which was isolated from diseased Japanese flounder using TSAYE medium and demonstrated to be pathogenic to flounder in live animal infection studies [31,32]. T4D1 was obtained as described previously [33].…”

“…The protease activity of purified recombinant Vhp1 was determined in the standard assay buffer as described previously using azocasein (0.5%; Sigma, USA) as a substrate [31]. One unit (U) of enzyme activity was defined as the amount of enzyme that caused an increase of 0.001 at 350 nm absorbance in 1 min.…”

“…One unit (U) of enzyme activity was defined as the amount of enzyme that caused an increase of 0.001 at 350 nm absorbance in 1 min. The effects of temperature, pH, metals ions, and ethylenedinitrilotetraacetic acid (EDTA) were determined as described previously [31]. …”

“…For vaccination with purified recombinant Vhp1, Bacillus sp. B187, an avirulent strain isolated from the gut of healthy Japanese flounder [31], was used as an adjuvant as described previously [31]. Briefly, B187 was grown in LB medium to an OD 600 of 0.6; the cells were harvested by centrifugation, washed with PBS, and resuspended in PBS to 1 × 10 8 CFU/ml (named B187-PBS).…”

“…These protein-based vaccines include the outer membrane protein OmpK, glyceraldehyde-3-phosphate dehydrogenase, and haemolysin [28][29][30]. In addition, our recent study has identified a chaperon protease from V. harveyi and found it highly immunoprotective when used as a secreted vaccine [31].…”