| Vibrio parahaemolyticus, an emerging pathogen of animal and human, is a leading cause of seafood poisoning worldwide. The global dissemination of this pathogen underscores the importance of understanding its numerous virulence factors and their effects on the human host. V. parahaemolyticus produces an array of factors associated with bacterial virulence, including thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), and type III secretion system (T3SS). The recently identified type VI secretion system (T6SS) has been reported to be a key player in regulation of virulence, biofilm formation and resistance to environmental stress in bacteria. Microbial adhesion to host tissues is the initial event of most infectious process. Surface proteins can directly participate in microbial virulence by facilitating pathogen dissemination via interactions with host factors. Enolase has been found playing critical roles in bacterial pathogenicity. However, there are fewer report related to the T6SS and enolase of V. parahaemolyticus, and their roles in the pathogenesis of V. parahaemolyticus are yet to be established. Therefore, it is important to examine them from the pathogenic points of view.1. Distribution of the major virulence factors of Vibrio parahaemolyticusThe genes of hemolysin (tdh and trh), adhesins (MAM7 and end), Type III secretion system (T3SS-1 and T3SS-2) and Type VI secretion system (T6SS-land T6SS-2) from 155 environmental isolates and 30 clinical isolates were detected by PCR. The positive rate of MAM7, eno, T3SS-1 and T6SS-2 genes in all clinical and environmental isolates were up to 100%, suggesting that these factors may be important for bacterial survival, but cannot be used as the virulence markers of V. parahaemolyticus. In clinical isolates, the positive rates of hemolysin genes tdh and trh, T3SS-2 and T6SS-1 were 93.3%,6.7%,83.3% and 86.7%, respectively; whereas, the positive rates from the environmental isolates were 2.5%, 0.6%,1.3% and 21.9%, respectively. The results showed that tdh, trh, T3SS-2 and T6SS-1 genes were present in majority of clinical isolates, suggesting that they play important roles in the pathogenesis of V. parahaemolyticus.2. Construction of the vipAl, hcpl and dotUl mtutants in Vibrio parahaemolyticus T6SS-1The vipAl, hcp1 and dotUl mutants were constructed by homologous recombination method, and the genetic complementation strains were constructed on the basis of the single gene mutant. The genes vipAl, hcpl, hcp2 and dotUl were cloned, expressed, and the antisera to recombinant proteins were also prepared. The result of PCR and sequence analysis showed that the mutant and complementary strains were successfully constructed, and had good genetic stability. The results of real-time RT PCR and Western blot revealed that the levels of transcription and expression of the each target gene in the mutant were at a lower level. Both Hcp1 and Hcp2 pro tens could be detected in the supernatant and pellet of wild type strains, indicating V. parahaemolyticus strain SH112 both has functional T6SS-1 and T6SS-2. Only Hcp2 but not Hcpl was detectable in the supernatant of vipAl, hcpl and dotUl mutant strains, suggesting that the deletion of T6SS-1 gene does not affect the function of T6SS-2, and the three genes all play an important functional role in the Hcpl secretion of T6SS-1.3. Characterization of the vipAl, hcpl and dotUl mutant strains in Vibrio parahaemolyticus T6SS-1Biological characteristics of the vipAl, hcpl and dotUl mutants were systematically analyzed, including anti-acid stress, motility, biofilm forming ability, cell adhesion ability, cytotoxicity, apoptosis and drug resistance. We also analyzed the effects of three major genes on the environmental adaptation and pathogenicity of V. parahaemolyticus. The results showed that there were no significant difference in drug resistance and motility in the three mutants relative to wild type strain. Growth experiments showed that the hcpl mutant displayed a fast growth phenotype. The anti-acid stress ability showed ascending in Ahcpl strain and descending in ΔvipAl, but no obvious difference in ΔdotUl, relative to the wild type strain. The anti-NaCl stress ability showed ascending in Δhcpl strain and no obvious difference in ΔvipA1 and ΔdotUl, relative to the wild type strain. The biofilm forming ability showed obvious descending in hcpl, vipAl and dotUl gene deletion mutant. Cell adhesion experiments showed that all the three mutants could significantly decrease the adhesion ability of V. parahaemolyticus onto HeLa cells. Cytotoxicity of bacteria and cell-free culture supernatant of vipAl, hcpl and dotUl mutants indicated the most significant decline. Compared to wild type strains, the vipAl, hcpl and dotUl mutants can cause cell apoptosis declined. Furthermore, vipAl, hcpl and dotUl are required for induction of pro-inflammatory cytokines, such as IL-1β and IL-6 in macrophages.These results suggest that the vipAl, hcpl and dotUl genes in the T6SS-1 cluster play an important role on environmental adaptation and pathogenicity of V. parahaemolyticus.4. Construction and characterization of the vipAl-hcpl mutant in Vibrio parahaemolyticusWe further constructed a vipAl-hcpl dual mutant, and analyzed biological characteristics, including anti-acid stress, motility, biofilm forming ability, cell adhesion ability, cytotoxicity, apoptosis and drug resistance. The results suggested that there was no difference in the motility and drug resistance between the vipAl-hcpl mutant and wild type strain. The anti-acid stress ability showed ascending in Ahcpl strain and descending in AvipAl, but no obvious difference in vipAl-hcpl mutant, relative to the wild type strain. The biofilm forming ability showed obvious descending in dual gene deletion mutant. The abilities of cell adhesion, cytotoxicity, apoptosis and induction of pro-inflammatory cytokines in vipAl-hcpl mutant descended significantly, and they were lower than those in the single gene mutant. The results suggest that vipAl and hcpl may have synergistic effects on environmental adaptability and pathogenicity of V.parahaemolyticus.5. Cloning, expression and activity of Vibrio parahaemolyticus enolaseSequence analysis showed that the open reading frame of eno gene is 1,302 bp in length encoding an enzyme of 433 amino acids with a calculated molecular mass of 48 kDa, having a functional region on the surface of the menbrane, a transmembrane domain and being absent for an N-terminal signal sequence. The nucleotide homology is 85%-98% same from other parahaemolyticus. The recombinant enolase protein was expressed in a prokaryotic system and purified.52-kDa protein was observed as a band in SDS-PAGE analysis, and the recombinant protein enolase reacted strongly with convalescent phase serum from mice clinically infected with V. parahaemolyticus, showing that enolase triggered an antibody response during the course of infection. In addition, immunoassay results showed that V. parahaemolyticus enolase is immunogenic in rabbits, and an ELISA titer of 1:32,000 could be achieved. Our enzymatic test results revealed that in vitro overexpressed V. parahaemolyticus enolase successfully catalyzes the conversion of 2-PGE to PEP, indicating that recombinant enolase retains enzymatic activity.6. Biological functions and immune efficacy of enolase in Vibrio parahaemolyticusConsiderable evidence has shown that enolase present on the bacterial surface mainly acts as a receptor for plasminogen and functions as a mediator of microbial virulence. This paper analyzed the role played by enolase in the process of pathogenesis and immune protection of V. parahaemolyticusn. Western blot analysis showed that VP-enolase was detectable in the extracellular, outer membrane (OM), and cytoplasmic protein fractions using antibodies against the recombinant enolase. Surface expression of enolase was further confirmed by immunogold staining and mass spectrometry (LC-MS/MS) analysis of OM protein profiles. Notably, VP-enolase was identified as a human plasminogen-binding protein with the enzyme-linked immunosorbent assay. The values obtained for adherence and inhibition suggest a role of surface-exposed enolase in epithelial adherence of V. parahaemolyticus. We further showed that enolase confers efficient immunity against challenge with a lethal dose of V. parahaemolyticus in a mouse model. The protection rate was up to 86.7%. These results suggest that enolase might play a potential role in the pathogenesis of V. parahaemolyticus, and can be used as a new vaccine candidate against V. parahaemolyticus infection. |
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