| The gene locus of capsular polysaccharide synthesis was predicted, and the wza gene involved in capsular polysaccharide export across outer membrane was identified by bioinformatics analysis. Then the wza gene-deleted strain CH-1 Δwza of RA CH-1 was constructed by allele replacement. We found that this gene was involved in biofilm formation, antibiotic resistance, cell adhesion and invasion, serum killing resistance, capsule formation, virulence, and other biological characteristics. The main results of this paper are as follows: 1. Bioinformatics analysis of RA wza geneRA wza gene is a 726bp open reading frame, with a typical TATA promoter in the 11bp site of upstream of the open reading frame. In addition, in the downstream of wza gene, there are wzc gene, glycosyltransferase genes, and lipid biosynthesis genes. Wzc protein is a casein kinase that can regulate the capsular polysaccharide biosynthesis and export. The wza gene encodes a 241 amino-acid protein with molecular weight 26.6 KD. After Sequence alignment analysis, we found Wza protein have high identity with polysaccharide transporters of other Flavobacterium bacteria. Besides, the Wza protein has Poly_export family domain, which is responsible for the export of polysaccharides. Subcellular localization prediction of this protein showed that it was an outer membrane protein, which confirmed the protein was likely to participate in the transport of RA capsular polysaccharide. The secondary structure of Wza protein contained the main components:alpha helix (23.24%), extended strand (30.29%), beta turn (14.11%) and random coil (32.37%). In addition, the homology modeling structure of RA Wza was constructed using the SWISS-MODEL2. Construction of RA CH-1 AwzaIn this study, overlapping PCR was used to construct the integration PCR fragment Awza-spc, which contained the left arm fragment, right arm fragment, spc fragment. Due to T-end feature of pYA4278 suicide plasmid after digested by AdhI, we could directly clone the PCR integration fragment Awza-spc into the pYA4278 to construct the recombination pYA4218-Awza-spc suicide plasmid. The conjugation of pYA4278-Awza-Δwza-spc from Escherichia coli X7213 into RA CH-1 would be done. RA CH-1 Δwza was constructed by allelic exchange that involved a 704-bp deletion of wza gene ORF (726bp).The wza gene deletion mutant was confirmed by PCR identification.3. The function of RA wza geneRA wza gene deletion would lead to slower growth of RA, the colony characteristics almost unchanged. Observed using a transmission electron microscope, the capsular of the deletion strain apparently disappeared of just left a thin layer. RA CH-1 is a typical biofilm strain deficient strain, which only had an average OD595=0.049 by crystal violet staining. However, the deletion of wza gene had significantly increased biofilm formation capacity with average OD595=0.269. One of main ingredients of capsular is capsular polysaccharide, which has strong hydrophilic and antioxidant capacity. Through desiccation condition experiment and antioxidant experiments, we found that RA CH-1 Awza had increased sensitivity to desiccation condition and H2O2. In addition, surface hydrophobicity results show a significant increase in CH-1Δwza, which indicated the loss of capsular polysaccharide of CH-1Δwza again. By selecting the commonly used antibiotics, we detected the susceptibility of parent strain and mutant strain to antibiotics, the data showed that CH-1Δwza had larger sensitivity to most antibiotics, but there was still little significant difference to a few antibiotics. The data suggest that RA capsule was related with antibiotic resistance. In terms of capsules of many bacteria, they were natural barriers to antibiotics. The results of complement-mediated killing experiment showed that the survival rate of the parent strain in 90% normal duck serum concentration was about 85%; while CH-1Δwza cannot survive in 90% normal duck serum concentration, which indicated that wza gene played an important role in the sepsis infection of RA.4. Wza is a virulence factor of RACell adhesion and invasion is a prerequisite for a bacterial infection. The bacterial adherence and invasion abilities of CH-1Δwza were lower than those of RA CH-1. Adhesion colonies of CH-lAwza and the parent strain in adhesion experiment were 5.8×104 CFU/well and 3.6×104 CFU/well. LD50s of RA CH-1 and CH-1Δwza were determined according to the method of Reed and Muench. The LD50 of CH-1 was 5.62x107, while the LD50 of CH-1Δwza was 2.37x1010. The virulence of RA CH-lAwza was attenuated approximately 421-fold by comparative analysis of LD50S of CH-1 and CH-lAwza. This result indicated that the deletion of wza gene could attenuate the pathogenicity of RA to ducklings. |
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