A Preliminary Study Of Riemerella Anatipestifer Capsular Synthesis Gene Wza And Wzc

Riemerella anatipestifer?RA?is the most serious pathogen in duck industry.We found the gene encoded the B739-1124 and the gene encoded B739-1125 had the bioinformatics features of polysaccharide export protein gene?wza?and the tyrosine kinase gene?wzc?,when we carried out the functional annotation of RA CH-1 strain genome.To investigate the function of RA wza and RA wzc,some molecular biology methods were used,including the gene knock-out method.The main study and results were as followings:1 Construction and characterization of wza/wzc gene deletion strainRA CH-1?wza,RA CH-1?wzc,and the double deletion strains RA CH-1?wzac were constructed using homologous recombination method,the corresponding complementary strains were also made by transforming with the wild type corresponding genes?named cRA CH-1?wza,cRA CH-1?wzc?.The biological features of these strains were investigated.The results suggested that?1?The protuberant,moist surface,and shiny colonies changed into the flat and rough colony with shorter diameter and the colony liquefaction characteristics also disappear in the absence of wza or wzc gene;?2?India ink staining results showed that the capsule structure is incomplete or failed to form capsule;?3?Electron microscope results showed that the capsule structure became thinner or disappeared;?4?growth curves of these deletion strains were delayed for about 2 hours;?5?After proteinase K treatment,SDS-PAGE electrophoresis and silver staining,wza and wzc were involved in the synthesis of capsular polysaccharide?CPS?but not lipopolysaccharide?LPS?in RA;?6?Percoll density gradient centrifugation confirmed the increased buoyancy density;?7?Anti-drying and anti-oxidation?H₂O₂?ability were significantly reduced.Corresponding complementary strains can restore the above biological characteristics of the deleted strain to wild-type?RA CH-1?levels or close to wild-type strain.2 The effect of the deletion of wza or wzc on the bacterial surface propertyThe hydrophobicity and self-aggregating ability of bacteria surface are important factors affecting the adhesion of bacteria.Capsule is a substructure located on the surface of the bacteria,and it is one of the factors that determine the ability of the bacteria surface hydrophobic and automatic aggregation.To investigate the effect of the deletion of wza or wzc on the bacterial surface property,hydrophobic adhesion,the sedimentation method,and the glass tube and 96-well plate cultivation were used to determine the surface hydrophobicity,the ability of self-agglutination,the biofilm formation,respectively.The results showed that the surface hydrophobicity of RA CH-1?wza strain?26.10%?and RA CH-1?wzc strain?27.59%?was significantly higher than that of RA CH-1 strain?14.36%??p<0.001?.After 12 h sedimentation,the OD₆₀₀00 values of RA CH-1?wza strain and RA CH-1?wzc strain were 0.20 and 0.21,respectively.While the OD₆₀₀00 value of RA CH-1strain was 0.90,indicating that the self-aggregating ability was significantly increased after the deletion of wza and wzc genes?p<0.001?.In the culture of the gas-liquid interface in the glass tube and the 96-well plate,the biofilms of the wza,wzc,and wzac gene-deleted strains could be clearly observed after crystal violet staining,while the wild strain and the complemented strain showed no significant biofilm formation.The biofilm formation abilities of RA CH-1?wza strain and RA CH-1?wzc strain were both stronger than that of the wild strain by comparing the OD₅₉₅?p<0.001?.3 The effect of the deletion of wza or wzc on the virulence of RA CH-1The ability of cell adhesion and invasion,LD₅₀,and tissue damage and lesions are important indicators for determining the virulence of bacteria.The results showed:?1?The adherence and invasion capacities of these deletion strains to Vero cells were significantly increased?p<0.001?compared to the wild strain.?2?The virulences of the deletion strains were attenuated by comparative analysis of LD₅₀s of the wild and complemented strain by infecting 7-day-old ducklings.The LD₅₀0 of the CH-1?wza and CH-1?wzc were 1.99×10¹⁰CFU and 1.26×10¹⁰,respectively.The LD₅₀0 of the wild strain CH-1 strain was 7.94×10⁷CFU,indicating that the virulence of the CH-1?wza and CH-1?wzc was approximately250-fold and 159-fold attenuated,respectively.?3?No corresponding strains could be isolated from the blood of 1 day,3 days and 5 days after infection of 7-day-old ducklings,while the wild-type blood-borne bacteria reached 10⁶-10⁸CFU/mL.?4?In the infection assay,the ducklings infected with the wild type strain showed hepatic cord structure disorder,the necrosis,swelling,nuclei condensation and diffuse fatty degeneration of hepatocyte,the epicardial thickening and edema of the heart,the exudation of cellulose on the surface the heart,the expansion and congestion of blood vessels in the parenchyma of the brain,the diffuse infiltration of inflammatory cells,and the formation of visible lymphocytes and glial cells around the blood vessels.However,the liver,brain,and heart tissues of the ducklings infected with each deletion strain showed no obvious lesions,indicating that the pathogenicity abilities of the deletion were significantly reduced.?5?These deletion strains cannot survive in the 50%serum.When the deletion strains were complemented with the corresponding genes,the above biological features can be restored to the wild strain level.Therefore,after the deletion of wza or wzc gene,the adherence and invasion ability to Vero cells was significantly enhanced.However,it was more likely that these strains can be killed by the complement-mediated mechanism of the host after infection with 7-day-old ducklings,thus the pathogenic capacity of these deletion strainies were weakened.4.Tyrosine phosphorylation of Wzc and the interaction between Wzc and WzaThe truncated pET-28a?+?-Wzc E521-F765 and pET-28a?+?-Wzc E521-F790expression plasmids were constructed and transformed into the expression host strain E.coli BL21.The proteins were soluble existing in the expression strain.WzcE521-F765 is a fragment containing no C-terminal tyrosine-rich region and cannot be recognized by the specifically phosphorylated tyrosine monoclonal antibody 4G10;WzcE521-F790 contains a C-terminal tyrosine-rich region,which can be recognized by 4G10.Therefore,the Wzc phosphorylation site is located in the C-terminal tyrosine enrichment region.Chemical cross-linking confirmed that Wza and Wzc interaction.In summary,RA wza and wzc gene deletion influenced the export of the capsule,and enhanced surface hydrophobicity,the auto-aggregation and biofilm formation abilities.Meanwhile,RA wza and wzc gene deletion reduces the pathogenicity.Wzc protein C-terminal tyrosine region was the main phosphorylation site.The interaction of Wzc with Wza was involved in the synthesis and export of capsular polysaccharide.