Studies On The Function Of Genes Involved In Invasion And Adhesion Of Edwardsiella Tarda

Edwardsiella tarda is a Gram-negative bacterial pathogen with a wide host range,including fish, amphibians, reptiles and mammals, and is responsible for tremendouseconomical losses in aquaculture. Meanwhile, E. tarda is the only species of thegenus Edwardsiella that can cause infection in humans. Several virulence-associatedsystems, such as adhesins, the resistance to the immune system of host, siderophores,hemolysin, T3SS and T6SS have been implicated in the pathogenesis of E. tarda, butthe pathogenic mechanism of E. tarda have not been clarified. In order to prevent andtreat edwardsiellosis, and facilitate the sustainable development of aquaculture, it isnecessary to fully understand the pathogenesis of E. tarda.Invasion of epithelial cells is associated with the initiation of infection for manybacteria. This process involves many virulence factors, such as invasin, adhesin andhemagglutinin. Invasin mainly exists in Gram-negative pathogen, consisting of an N-terminal β domain located in outer membrane and a C-terminal structure combinedwith the host cell, which play an important role in the invasion of host cells. Adhesin,including pilus adhesin and non-pilus adhesin, functions in bacteria adherence tosurface structures of host cells. Temperature sensitive hemagglutinins, discovered inavian pathogenic Escherichia coli (APEC), is also related to the pathogenicity ofbacteria.In order to investigate the function of inv, adh and tsh in E. tarda, in-frame deletionmutants of each gene of E. tarda H1was constructed by overlap PCR and doublecrossover allelic exchange using suicide vector pRE112. Briefly, the upstreamfragment and downstream part of inv, adh or tsh were amplified by PCR, respectively.Fusion of the upstream and downstream fragments was performed by overlap PCR.The fused segment was then cloned into suicide vector pRE112to produce theinsertional construct. The resulting construct was transformed into Esc. coli SY327and then introduced into Esc. coli S17-1for mating into E. tarda H1by conjugation. Allelic exchange between the chromosomal gene and the in-frame deleted plasmidcopy was achieved in a second crossover event which was counter-selected on TSAcontaining10%sucrose to determine the excision of pRE112from the chromosome.In addition, an inv overexpression E. tarda strain (inv+) was constructed byelectrotransforming Δinv with a plasmid carrying an intact inv gene with the putativepromoter region. Series of mutational properties of in-frame deletion mutantscompared with the wild type strain E. tarda H1were investigated，including thegrowth, biofilm formation, haemolytic activity, invasion to EPC cells, lethality in fishmodel, LPS synthesis and the secretion of ECP.Compared to the wild type H1, the haemolytic activity, biofilm formation andinternalization into EPC cells were decreased in Δinv while increased significantly ininv+. In addition, Δinv showed decreased virulence compared to H1, while inv+restored the wild type virulence in zebrafish model. Moreover, Δinv and inv+displayed the same growth rates as the wild-type H1, and showed no change in LPSsynthesis and the secretion of ECP.The adhesin in-frame deletion mutant Δadh represented the same growth rate as thewild type strain and increased resistance to aminoglycoside antibiotics (Kanamycin,Neomycin) and tetracycline antibiotics (Tetracycline, Doxycycline). This might berelated to the changes in the structures of the outer membrane proteins andlipopolysaccharide. Meanwhile, Δadh displayed decrease in biofilm formation,haemolytic activity, adherence to EPC cells and the lethality in fish.Compared to the wild type H1, the temperature sensitive hemagglutin in-framedeletion mutant Δtsh showed the same growrh rate and increased resistance toaminoglycoside antibiotics (Amikacin, Gentamicin, Kanamycin and Neomycin),while the hemolytic activity and biofilm formation was decreased. Moreover, Δtshexhibited attenuated virulence to zebrafish.The results suggest that inv, adh and tsh play an important role in virulence of E.tarda. As important virulence factors, they effect on the biofilm formation, haemolyticactivity, adherence and internalization to epithelial cells and the lethality of fish hosts,and thus contribute to the pathogenicity of E. tarda.