Shuang Cheng

Characterization of DegQVh, 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 (degQVh), the gene encoding a DegQ homologue, was cloned from T4, a pathogenic V. harveyi strain isolated from diseased fish. DegQVh 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 degQVh in T4 was modulated by temperature, possibly through the σE-like factor. Enzymatic analyses demonstrated that the recombinant DegQVh 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 DegQVh protein were 50°C and pH 8.0. A vaccination study indicated that the purified recombinant DegQVh was a protective immunogen that could confer protection upon fish against infection by V. harveyi. In order to improve the efficiency of DegQVh as a vaccine, a genetic construct in the form of the plasmid pAQ1 was built, in which the DNA encoding the processed DegQVh protein was fused with the DNA encoding the secretion region of AgaV, an extracellular β-agarase. The E. coli strain harboring pAQ1 could express and secrete the chimeric DegQVh protein into the culture supernatant. Vaccination of fish with viable E. coli expressing chimeric degQVh 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.

Comparative study of the effects of aluminum adjuvants and Freund's incomplete adjuvant on the immune response to an Edwardsiella tarda major antigen.

Edwardsiella tarda is a severe aquaculture pathogen that can infect many different fish species cultured worldwide. Et49 is a major E. tarda antigen with weak immunoprotective potential. In this study, using Et49 as an example vaccine, the adjuvanticity of Freunds incomplete adjuvant (FIA), aluminum hydroxide, and aluminum phosphate adjuvant were evaluated in a Japanese flounder model. The results showed that the presence of FIA, aluminum hydroxide, and aluminum phosphate adjuvant increased the relative percent of survival of Et49-vaccinated fish by 47%, 19%, and 35%, respectively. Fish vaccinated with FIA-adjuvanted Et49 exhibited longer persistence of vaccine at the injection site and more severe intra-abdominal lesions than fish vaccinated with aluminum-adjuvanted Et49. Both aluminum adjuvants and, to a lesser degree, FIA augmented the production of specific serum antibodies, which reached the highest levels at 6 and 7 weeks post-vaccination. Passive immunization of Japanese flounder with sera from fish vaccinated with aluminum- and FIA-adjuvanted Et49 induced no protection against lethal E. tarda challenge. Examination of the transcription profile of immune-related genes showed that vaccination with aluminum-adjuvanted Et49 significantly enhanced the expression of the genes that are associated mainly with humoral immunity, whereas vaccination with FIA-adjuvanted Et49 induced the expression of a much broader spectrum of genes that are likely to be involved in humoral and innate cellular immunity. These results provide new insights to the action mechanisms of FIA and aluminum adjuvants in Japanese flounder and may be useful for the selection of adjuvant for vaccine formulations intended for Japanese flounder.

Identification and immunoprotective analysis of a Streptococcus iniae subunit vaccine candidate

Streptococcus iniae is a Gram-positive bacterium and a severe aquaculture pathogen that can infect a wide range of farmed fish species. In the summer of 2006, an epidemic broke out in a fish farm in north China, and examination of moribund fish (Japanese flounder) identified the possible etiological agent of the outbreak as a strain named SF1, which exhibited apparent virulence in a Japanese flounder infection model and conforms to the description of S. iniae by 16S rRNA sequence analysis and API 20 Strep test. Biochemical and random amplified polymorphic DNA analyses indicated that SF1 is of the serotype I. A putative iron-binding protein, Sip11, was identified from SF1 using a previously established molecular trap that selects exported proteins. Recombinant Sip11 was purified from Escherichia coli and found to be protective against SF1 infection when used as an injection vaccine administered intraperitoneally into Japanese flounder. To improve the vaccine potential of Sip11, an E. coli strain was constructed, which expresses and secrets recombinant Sip11 covalently linked to a carrier protein in the form of a chimera. Vaccination of Japanese flounder with live Sip11-secreting E. coli afforded complete protection upon the fish following lethal SF1 challenge. These results indicate that Sip11, especially when delivered by a live bacterial carrier, is an effective vaccine candidate against SF1 infection.

The iron-cofactored superoxide dismutase of Edwardsiella tarda inhibits macrophage-mediated innate immune response

Edwardsiella tarda is a Gram-negative bacterium that can infect a wide variety of marine and freshwater fish and cause severe economic losses worldwide. With an aim to elucidate the virulence mechanism of E. tarda, we in this study cloned and analyzed the function of an iron-cofactored superoxide dismutase (FeSOD) from a pathogenic E. tarda strain TX01 isolated from diseased fish. FeSOD is 192-residue in length and contains domain structures that are conserved among iron/manganese superoxide dismutases. Recombinant FeSOD purified from Escherichia coli exhibits apparent superoxide dismutase activity. Quantitative real-time RT-PCR analysis indicated that FeSOD expression is significantly upregulated immediately following TX01 infection of Japanese flounder (Paralichthys olivaceus) head kidney (HK) macrophages and cultured FG cells. Compared to the wild type strain TX01, the FeSOD mutant strain TXSod is (i) more sensitive to H(2)O(2)-induced oxidative damage, (ii) less resistant against serum- and macrophage-mediated bacterial killing, (iii) significantly weakened in the ability to invade into FG cells and to disseminate in fish blood and liver, and (iv) deficient in blocking macrophage respiratory burst activity and production of reactive oxygen species. Furthermore, HK macrophages infected by TXSod exhibits significantly increased expression of inflammatory cytokines compared to macrophages infected by TX01. Taken together, these results indicate that FeSOD is a virulence factor that plays an important role in the pathogenicity of E. tarda by inhibiting macrophage-mediated innate immune response.

Molecular analysis of the fur (ferric uptake regulator) gene of a pathogenic Edwardsiella tarda strain

The gene encoding the Edwardsiella tarda ferric uptake regulator (FurEt) was cloned from a pathogenic E. tarda strain isolated from diseased fish. FurEt shares 90% overall sequence identity with the Escherichia coli Fur (FurEc) and was able to complement the mutant phenotype of a furEc-defective E. coli strain. Mutational analysis indicated that C92S and C95S mutations inactivated FurEt whereas E112K mutation resulted in a superactive FurEt variant. FurEt negatively regulated its own expression; interruption of this regulation impaired bacterial growth, altered the production of certain outer membrane proteins, and attenuated bacterial virulence.

Analysis of the vaccine potential of a natural avirulent Edwardsiella tarda isolate

Edwardsiella tarda is a severe aquaculture pathogen that can infect many fish species and cause a systematic disease called edwardsiellosis, which can lead to high mortality under certain conditions. Currently, most vaccine candidates against edwardsiellosis are based on pathogenic E. tarda strains, which can be a concern in some cases. In this study, the vaccine potential of a natural E. tarda isolate, ATCC 15947, was examined in a Japanese flounder model. ATCC 15947 was found to be relatively avirulent to flounder but able to disseminate into and survive transiently in fish tissues following intraperitoneal (i.p.) injection. Fish vaccinated with ATCC 15947 via i.p. injection exhibited a high level of survival rate, which was increased to 100% by a booster injection. Fish vaccinated with ATCC 15947 orally in the form of alginate microspheres showed a moderate survival rate, while fish vaccinated with ATCC 15947 via the immersion route exhibited a low rate of survival. Following oral vaccination, ATCC 15947 could colonize transiently in the gut, liver, and spleen of the vaccinated fish. Both injection and oral vaccination with ATCC 15947 induced production of specific serum antibodies, the levels of which at different time points following vaccination were generally higher in fish vaccinated with ATCC 15947 via injection than in fish vaccinated with ATCC 15947 orally. Compared to control fish, fish vaccinated with ATCC 15947 showed enhanced serum bactericidal activity and significantly increased expression in genes encoding a number of immune-related factors. These results indicated that ATCC 15947 possesses good immunoprotective potential, which may be exploited in the development of E. tarda vaccines.

Analysis of Edwardsiella tarda DegP, a serine protease and a protective immunogen.

Edwardsiella tarda is a severe aquaculture pathogen with a broad host range that includes humans, animal, and fish. A gene (degP(Et)) encoding a DegP homologue was cloned from TX01, a pathogenic E. tarda strain isolated from diseased fish. DegP(Et) shares high sequence identities with the DegP proteins of several bacterial species. Functional analyses showed that degP(Et) could complement the temperature-sensitive phenotype of an Escherichia coli degP null mutant. Expression of degP(Et) in TX01 was modulated by growth phase and temperature, the latter possibly through the action of the sigma(E)-like factor. Overexpression of degP(Et) (i) enhanced the ability of TX01 to disseminate in fish blood at the advanced stage of infection, (ii) heightened the activity of type 2 autoinducer, and (iii) increased the expression of luxS and the genes encoding components of the virulence-associated type III secretion system. Recombinant DegP(Et) purified from E. coli was a serine protease that exhibited maximum activity at 40 degrees C and pH8.0. The proteolytic activity of recombinant DegP(Et) depended on the catalytic triad and the PDZ domains. Immunoprotective analyses showed that purified recombinant DegP(Et) was a protective immunogen that could induce the production of specific serum antibodies and elicit strong protective immunity in fish vaccinated with DegP(Et).

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

Vibrio harveyi is an important aquaculture pathogen that can infect a number of fish species and marine invertebrates. A putative protease, Vhp1, was identified from a pathogenic V. harveyi strain isolated from diseased fish as a protein with secretion capacity. Vhp1 is 530 amino acids in length and shares high sequence identities with several extracellular serine proteases of the Vibrio species. In silico analysis identified a protease domain in Vhp1, which is preceded by a subtilisin-N domain and followed by a bacterial pre-peptidase C-terminal domain. Purified recombinant protein corresponding to the protease domain of Vhp1 exhibited apparent proteolytic activity that was relatively heat-stable and reached maximum at pH 8.0 and 50 degrees C. The activity of purified recombinant Vhp1 protease was enhanced by Ca(2+) and inhibited by Mn(2+) and ethylenedinitrilotetraacetic acid. Cytotoxicity analyses indicated that recombinant Vhp1 protease was toxic to cultured Japanese flounder cells and could cause complete cell lysis. Immunoprotective analysis using Japanese flounder as an animal model showed that purified recombinant Vhp1 in the form of a denatured and proteolytically inactive protein was an effective subunit vaccine. To improve the vaccine potential of Vhp1, an Escherichia coli strain that expresses and secrets a cytotoxically impaired Vhp1 was constructed, which, when used as a live vaccine, afforded a high level of protection upon the vaccinated fish against lethal V. harveyi challenge. Taken together, these results demonstrate that Vhp1 is a cytotoxic protease and an effective vaccine candidate against V. harveyi infection.

Cys-92, Cys-95, and the C-terminal 12 residues of the Vibrio harveyi ferric uptake regulator (Fur) are functionally inessential

Ferric uptake regulator (Fur) is a global regulator involved in multiple aspects of bacterial life. The gene encoding the Vibrio harveyi Fur (Furvh) was cloned from a pathogenic V. harveyi strain isolated from diseased fish. Furvh shares 77% overall sequence identity with the Escherichia coli Fur (FurEc) and could complement a mutant of FurEc. Like FurEc, FurVh, possesses two cysteine residues at positions 92 and 95, yet unlike FurEc, in which these cysteine residues constitute part of the metal ion coordination site and hence are vital to the repressor activity, C92 and C95 of FurVh proved to be functionally inessential. Further study identified a Vibrio Fur signature sequence, which is preserved in all the ten Vibrio Fur proteins that have been discovered to date but in none of the non-vibrio Fur proteins. Site-directed and random mutation analyses of the signature residues, the cysteine residues, and seven highly charged amino acid residues indicated that D9, H32, C137, and K138 of Furvh are functionally important but D9, C137, and K138 can be replaced by more than one functional substitutes. Systematic deletion analysis demonstrated that the C-terminal 12 residues of FurVh are functionally inessential. These results (i) indicated that the activation mechanism, or certain aspects of which, of FurVh is possibly different from that of FurEc; and (ii) suggested that it is not very likely that the C-terminal 12 residues play any significant role in the activation or stability of FurVh; and (iii) provided insights into the potential function of the local structure involving C137 and K138.

Construction and evaluation of a live vaccine against Edwardsiella tarda and Vibrio harveyi: laboratory vs. mock field trial.

Edwardsiella tarda and Vibrio harveyi are Gram-negative bacterial pathogens that affect a wide range of cultured fish. In previous studies, we have reported an E. tarda live vaccine ATCC15947 and a V. harveyi subunit vaccine DegQ. On the basis of these studies, in the present study we developed a cross protective vaccine against both E. tarda and V. harveyi by constructing a recombinant ATCC15947, Et15VhD, that expresses and secrets V. harveyi DegQ as a soluble antigen. Laboratory studies in a turbot (Scophthalmus maximus) model showed that Et15VhD elicited significant protections against E. tarda and V. harveyi when administered via intraperitoneal injection, oral feeding, immersion, and oral plus immersion, respectively. Microbiological analysis indicated dissemination and transient colonization of Et15VhD in fish tissues following vaccination. Since, compared to injection, oral plus immersion is a practically more acceptable vaccination procedure in aquaculture, we conducted a mock field trial to further examine the potential of Et15VhD as an oral plus immersion vaccine. The results showed that during the period before artificial bacterial challenge, mortality was observed in both the vaccinated group and the control group; however, the mortality of Et15VhD-vaccinated fish was significantly lower than that of the control fish. Following experimental challenge with E. tarda and V. harveyi at one and two months post-vaccination, Et15VhD-vaccinated fish exhibited dramatically increased survival rates compared to control fish. Serum antibody analysis indicated specific antibody production in Et15VhD-vaccinated fish. Taken together, these results demonstrate that Et15VhD induces strong protective immunity against E. tarda and V. harveyi under both laboratory and mock field conditions, which suggests a potential for Et15VhD to be used in aquaculture.