Diversity Of Edwardsiella Tarda And Functional Studies On Its Flagellin Genes

Edwardsiella tarda, a Gram-negative bacterium, is the causative agent of edwardsiellosis with a wide host range including fish, amphibians, reptiles and mammalia, which is responsible for major economic losses in aquaculture industry worldwide. More importantly, Edw. tarda is the solely species of Edwardsiella genus that can cause infections in human, while relatively infrequent. Although several virulence-associated systems have been implicated in the pathogenesis of Edw. tarda, the major virulence factors and the pathogenic mechanism of Edw. tarda have not been clarified. The development of an effective strategy to control Edw. tarda infections calls for intensive researches on this microorganism, and its interactions with host, which are crucial to understand the pathogenic mechanisms of the pathogen for the continuous development of the aquaculture industry.Flagella formation as a potential virulence factor has been demonstrated in many bacteria for mediating a number of processes during the pathogenesis of the bacteria. FliC, as a flagella structural protein, is hypothesized to be involved in the development of infection. However, the role of FliC in regards to the pathogenesis has not been clearly defined. In this study, the flagella formation, the flagellin gene (fliC), the bacteria motility and so on were investigated among 10 Edw. tarda strains with different origins. In an attempt to explore the role of FliC as virulence associated protein and its contribution during the infection process, we focused our studies on the functional analysis of the flagellin of Edw. tarda H1 by constructing two flagellin mutation strains (ΔfliC1 andΔfliC2, respectively) through double crossover allelic exchange by means of a suicide vector. We further compared the series of mutational characteristics of fliC in-frame deletion mutants with the wild type strain Edw. tarda H1 in terms of virulence-associated phenotypes and pathogenicity. The main objectives of this study are to investigate the function and the involvement of fliCs during the pathogenesis of Edw. tarda for detailed interpretation of the molecular mechanism exploited by Edw. tarda during infection, which could provide crucial insights into the virulence mechanisms used by Edw. tarda and the development of useful vaccine against Edw. tarda infection.The main results of this study are as follow:1. In this research, the flagella formation, flagellin gene (fliC), bacteria motility (swimming motility and swarming motility), biofilm formation, extracellular protein, plasmid profiles and antibiotic susceptibilities were investigated in 10 Edw. tarda strains from different geographic origins and hosts. Edw. tarda strains exhibited diversities in phenotypic characteristics which were dependent on the geographic areas and origins. It was found that Edw. tarda strains varied greatly in the number of flagella which were considered positively related to the ability of motile and biofilm formation. Strains with quantitatively more flagella production or more stable flagella formation were more efficient in motility (both swimming and swarming motility) and biofilm formation, and vice versa; There existed two flagellin genes in Edw. tarda through checking the fliC sequences in 10 different strains and blasting them with the genomic sequence from EIB202. The two flagellin genes fliC1 and fliC2 had 86% identities in nucleotide sequences and 83% identities in predicted amino acid sequences. Alignment of FliCs amino acids sequence indicated that FliC from different strains were conserved in the N- and C- terminal sequences which constitute the backbone of flagella, while sequences in the middle which was exposed to the flagella surface were low in similarity which indicated the fact of the diversities in flagella surface antigens. In addition, extracellular products were correlated with the pathogenecity of Edw. tarda and the 55kD, 21kD, 18kD proteins were thought to be virulence proteins; Moreover, the plasmid profiles and antibiotic susceptibilities were dependent on their geographic areas. All these distinctive characteristics of different strains implied the existence of diverse pathogenic mechanisms of Edw. tarda.2. In this research, an in frame deletion mutant of fliC1 (ΔfliC1) of a virulent isolate of Edw. tarda H1 and its complemented strain (fliC1⁺) were constructed based on suicide plasmid pRE112 and the complemented plasmid pACYC184, and its virulence-associated phenotypes were tested including flagella formation, motility, growth condition, autoaggregation, biofilm formation, extracellular protein secretion, drug resistance and pathogenecity to zebra fish. It was found that the deletion of fliC significantly decreased the diameter of flagella filaments. In addition, the mutant caused a number of other pathogenicity-impairing phenotypic effects including impaired bacterial growth, reduced motility, decreased biofilm formation, reduced levels of virulence-associated protein secretion involved in the typeâ…¢and typeâ…¥secretion system, and most importantly, attenuated virulence to fish by increasing the LD₅₀ value for 100-fold compared to the wild-type strain. Almost all the observed phenotypes were restored by the complemented strain fliC1⁺. Therefore, it was assumed that FliC1 as the flagellin domain protein is directly involved in the flagella formation of Edw. tarda, as it has been proved to have positive effects on bacteria growth, motility, auto-aggregation, biofilm formation and virulent protein secretion through the typeâ…¢and typeâ…¥secretion system, making great contribution to the pathogenic process and the virulence network of Edw. tarda. In addition, changes in the ability of drug resistance ofΔfliC1 might relate with changes in the structures of outer membrane proteins and lipopolysaccharide.3. Similarly, an in frame deletion mutant of fliC2 (ΔfliC2) and its complemented strain (fliC2⁺) were constructed, and its virulence-associated phenotypes were tested accordingly. It was found that theΔfliC2 mutant can still produce flagella and there is no difference with the wild type H1; in addition, the deletion of fliC2 resulted in reduced motility, arrested bacterial growth, decreased biofilm formation, changed drug resistance ability; however, secretion of extracellular proteins and autoaggregation remained the same with the wild type H1, and the LD₅₀ value ofΔfliC2 as well. Almost all the observed phenotypes were restored by the complemented strain fliC2⁺. Therefore it was assumed that FliC2 as the flagellin associated protein might not be involved in flagella formation. It played a limited role in the process of bacterial growth, motility and biofilm formation, but had no detectable effects on auto-aggregation, extracellular protein secretion and the pathogenicity of Edw. tarda.