| Aeromonas hydrophila is an important bacterial pathogen of aquatic animals and human. The bacterium lives in biofilms in the natural environment, as another life way corresponding to planktonic cells, which plays a key role in the pathogenesis of many bacterial infections. Current research of biofilms is focused on the antimicrobial resistance and vaccine development, however, the relationship between virulence and biofilm formation ability and the differences in protein expression between A. hydrophila planktonic cells and biofilms have not been investigated. In this study, structure characteristics and forming progress of biofilm in vitro were observed, protein expression between biofilms and planktonic cells was compared, and the biological functions of proteins including OmpAII, FlaA, Omp38and LuxS related to biofilm formation were studied.1. Analysis of biofilm formation ability of A. hydrophila and its relationship with virulenceA. hydrophila can form biofilm, which causes the persistent infection of aquatic animals. In this study, using FITC-conA and FDA/PI staining techniques, the different components of biofilm were stained, and the characteristics of biofilm at different times were observed by fluorescence microscope, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and green fluorescent protein (GFP)-labled technology. The results showed that the bacteria began to cluster and form microcolony at8h, the biofilm was with certain thickness at24h, and the thickness and proportion of dead bacteria increased with the duration of culture time. In addition, the results of biofilm formation ability of Aeromonas strains showed that86.7%(100/113) could form biofilm. Additionally, the virulence of representative strains with different biofilm formation ability to zebrafish was investigated, and the positive correlation between biofilm formation ability and pathogenicity of strains was demonstrated. 2. Comparative proteomics analysis between biofilm and planktonic cells of A. hydrophilaThe differences in expression profiles of whole bacterial proteins and outer membrane proteins (OMPs) from A. hydrophila J-1between planktonic cells and biofilm were investigated by comparative proteomics analysis. The results showed that compared with the planktonic controls,17proteins were unregulated and other17proteins were downregulated in the whole bacterial proteins from A. hydrophila biofilms, and11proteins were unregulated and one protein were downregulated in the OMPs from A. hydrophila biofilms. In addition, an immunoproteomics assay was developed to identify candidate antigens in OMPs of planktonic cells and biofilm with convalescent serum from Crucian carp. Seven immunoreactive proteins were identified under both planktonic and biofilm growth conditions, among which six proteins were upregulated in biofilm, and one protein was immunoreactive only in the biofilm growth conditions.3. Biological functions of major outer membrane protein OmpAⅡ in A. hydrophilaTo explore the function of OmpAⅡ, the gene sequence of ompAII was analyzed and its distribution in252different Aeromonas strains was investigated, and the ompAⅡ mutant was constructed by homologous recombination method. The open reading frame (ORF) of ompAⅡ gene was1002bp at length, which encoded333aa major outer membrane protein OmpAⅡ. OmpAⅡ comprises the structural motifs characterized by members of the family of porin and OmpA. The distribution of ompAⅡ in A. hydrophila was83.3%(60/72). The hemolytic activity, protease activity and cytotoxicity of extracellular products in the ompAⅡ mutant were significantly decreased. As well, the biofilm formation ability of the mutant strain was decreased. Moreover, the adhesion capacity to HEp-2cell and antiphagocytosis capacity to RAW264.7of the mutant strain were decreased by25%and33%compared to wide type strain. It suggested that OmpAⅡ was an important virulence factor of A. hydrophila, which might be involved in the adhesion of A. hydrophila to host cells.4. Biological functions of FlaA in A. hydrophilaThe gene sequence of flaA was analyzed and its distribution in252different Aeromonas strains was investigated. The flaA mutant was constructed by homologous recombination method. The ORF of flaA gene was909bp, which encoded302aa FlaA. According to the evolutionary pathway of FlaA, the homolog in A. hydrophila J-1located in the second group of Aeromonas FlaA. The distribution of gene flaA in A. hydrophila was 55.6%(40/72), and the deduced amino acid was88%-99%identical to other Aeromonas strains. The expression of virulence genes aer and vgrG3in the flaA mutant was decreased to0.44and0.45, respectively, which correlated with the significant decrease of hemolytic activity. Moreover, the biofilm formation ability of the mutant strain was reduced to85%compared to wide type strain. Furthermore, the adhesion capacity to the HEp-2cell decreased by48%, and the cytotoxic activities of bacteria and supernantant to RAW264.7cell were reduced by35%and55%, respectively. It indicated that FlaA was related to the adhesion of bacterium and contributed to biofilm formation, and thus played an important role in pathogenesis of A. hydrophila.5. Biological functions of outer membrane protein Omp38in A. hydrophilaThe gene sequence of omp38was analyzed and its distribution in252different Aeromonas strains was investigated. The omp38mutant was constructed by homologous recombination method. The ORF of omp38gene was1074bp, which encoded357aa porin Omp38. Omp38belongs to the porin superfamily. PCR and Western blot analysis showed that the gene of Omp38was widespread in A. hydrophila, with the73.6%(53/72) distribution percentage, and this protein was the potential common protective antigen, which could be used as candidate antigen of genetic engineering subunit vaccine. The omp38mutant showed increased biofilm formation with crystal violet staining and CLSM, and increased polysaccharide production by fluorescence microscope with FITC-conA labled. The bacteriophage sensitivity results showed that the omp38mutant reduced the reproductive activity of bacteriophage, which revealed that Omp38maybe phage receptor. The expressions of virulence genes aer, ahe2and emp in the omp38mutant were decreased, which may associated with the reduce of hemolytic activity and protease activity. In addition, adhesion capacity to HEp-2cell, antiserum and antiphagocytosis capacity was significantly increased to11.45,10and1.73fold compared to wide type strain, which may associated with the protection of increased polysaccharide. As well, the cytotoxic activities of bacteria and supernantant to RAW264.7cell were notably increased by19%and10%, respectively. Furthermore, virulence of the mutant to zebrafish was increased, which associated with the increased expression of virulence gene vgrG2in T6SS of A. hydrophila. It is presumed that the increased polysaccharide resulted in other characteristic changes of the omp38mutant. It suggested that A. hydrophila virulence increased after omp38was deleted. 6. Construction and characterization of ompAⅡ and omp38double gene mutant strain in A. hydrophila J-1The ompAⅡ-omp38double gene mutant strain was constructed on the basis of the omp38mutant by homologous recombination method and biological characteristics were analyzed. The mutant strain showed well genetic stability and enhanced growth ability. Real time PCR analysis showed that expression of the virulence genes aer was decreased by0.83and the expressions of ahe2, emp, hcp2and vgrG2were increased by1.60,1.49,1.71and4.46compared to wide type strain, respectively. The decreased hemolytic activity and increased protease activity in the mutant strain, which corelated with the downregulation of aer and upregulation of ahe and emp. Moreover, antiserum ability and cytotoxicity to RAW264.7cell were increased, whereas, adhesion capacity to the HEp-2cell and antiphagocytosis to RAW264.7cell were decreased compared to wide type strain. It indicated that characteristics of the double gene mutant strain were not a simple adduct of characteristics of ompAⅡ and omp38single gene mutant strains.7. Characterization of luxS gene mutant strain in A. hydrophila J-1After the deletion of luxS gene in quorum sensing (QS), the biofilm formation capability of A. hydrophila J-1was significantly decreased. Real time PCR analysis showed that expressions of all virulence genes were decreased, among which expressions of aer, ahe2, emp and hcp2decreased by0.14,0.21,0.06and0.24, respectively, which might contrbute to the notably decreased hemolytic activity and protease activity.. Moreover, adhesion capacity to the HEp-2cell, cytotoxic activities of bacteria and supernantant to RAW264.7cell were significantly reduced to84%,85%and48%. It suggested that LuxS might be involved in biofilm formation and contribute to the virulence of the bacterium.8. Animal immune efficacy of recombinant protein Omp38of A. hydrophilaTo evaluate the immune efficacy of recombinant protein Omp38of A. hydrophila, mice and Parabramis pekinensis were immunized by injection, respectively. The results of vaccination in mice showed that IgG level of inactivation vaccine (group Ⅰ) and protein immunization (group Ⅱ) groups were significantly higher than that of control group (group Ⅲ), and reached the highest level at35d. Peritoneal macrophage phagocytosis activity and spleen lymphocyte proliferation activity were increased at the same trend as well as the production of antibody level. The percentages of CD3+and CD3+CD4+CD8-in spleen lymphocyte reached the highest level at42d after vaccination. They were65.17%and 52.08%in Group I, and53.22%and39.58%in group II. Real time PCR analysis showed that mRNA expression levels in IFN-y and IL-4began to increase at21d and reached the highest level at35d after vaccination in both immunization groups. The results of vaccination in Parabramis pekinensis showed that IgM level of inactivation vaccine (group I) and protein immunization (group Ⅱ) groups were significantly higher than that of control group (group Ⅲ), and reached the highest level at21d, at which time antibody titer of group Ⅱ was higher than that of group I, and maintained at a high level since this time. The activities of lysozyme (LSZ) and superoxide dismutases (SOD) in the sera of immunization groups had obvious changes at the early stage after immunization. The SOD activity of group II was higher than that of group Ⅲ at14d (P<0.05), and reached the highest level at21d. Likewise, the LSZ activity of immunization groups was at a high level at14d, and the activity of group I was higher than that of group II (P<0.05). Since this time the LSZ activity was fluctuated and higher than that of group III at the same day after immunization (P<0.05). The flow cytometer and plate count analysis showed that phagocytisis activity of lymphocytes in head kidney of immunization groups were significantly higher than that of group Ⅲ(P<0.05). The challenge test showed that the relative protection rates (RPS) of immunized mice in group I and group II were82.00%and58.82%, respectively, and the RPS of immunized Parabramis pekinensis in group I and group II was50.00%and57.14%, respectively. It suggested that the recombinant protein Omp38could stimulate the humoral and cellular immune response effectively, and provide better protection efficacy in mice and Parabramis pekinensis against infections caused by A. hydrophila. |
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