To confirm that all changes in phenotypes were caused by the deletion of fliC, the Δ fliC mutant was provided with the intact fliC gene in plasmid pACYC184 for complementation analysis. The correct deletion was verified by DNA sequencing of the resulting PCR product. The Δ fliC mutant was confirmed by the ability to grow on TSA supplemented with ColB and inability to grow with Cm. The nonpolar mutant was an in-frame deletion within the open reading frame of the fliC gene, and disruption of fliC was confirmed by the mutant’s inability to transcript mRNA, which was verified by RT-PCR (data not shown). ![]() tarda H1, thus obtaining the Δ fliC mutant with loss of an internal region of the FliC from 56 to 323th amino acid residues. Using the double selection strategy of allelic exchange mutagenesis by means of suicide vector pRE112, we deleted 804-bp (residues 166–969) of the fliC gene in E. tarda H1 and the corresponding complemented strain ii) compared the fliC in-frame deletion mutant and the wild-type in terms of virulence-associated phenotypes and pathogenicity.Ĭonstruction of the Δ fliC Mutant and the Complementary Strain fliC + In an attempt to explore the role of FliC as a virulence associated protein, we i) constructed a fliC in-frame deletion mutant of E. Knock-out of virulence related genes can be used as a strategy to produce attenuated bacterial vaccines. Īlthough FliC has been considered associated with virulent strains, no information is available about its exact involvements in the pathogenesis of E. Purified recombinant FliC showed no apparent immunoprotectivity in a Japanese flounder model when used as a subunit vaccine, yet elicited significantly stronger protective immunity when FliC was fused to other DNA vaccines. tarda, FliC was revealed to be a virulent-strain-specific protein, and was further identified as an antigenic protein through the use of rabbit polyclonal antiserum. įliC as a flagellar filament structural protein was identified in a variety of organisms. In addition, flagellin was proposed to be a potent activator of innate immune response and thus plays a role either in stimulating host defense or in disease causation. Exhibiting regions of highly homologous amino acid sequences to several proteins of type III secretion system (TTSS), the bacterial flagellum has been considered to serve as a secretory system that might transport various virulence factors. The flagellum is an ultrastructure which mediates a number of functions in addition to motility, attachment and chemotaxis. ![]() ![]() The phenotypic characteristics of the fliC deletion mutant uncovered in this investigation suggest that fliC plays an essential role in normal flagellum function, bacterial growth, protein secretion by TTSS and bacterial virulence.Įdwardsiella tarda is an enteric pathogen responsible for significant economic loss in aquaculture with a wide host range including humans, and is usually flagellated and motile. In addition, the mutant showed reduced pathogenicity to fish by increasing the LD 50 value for 100-fold compared to the wild-type strain, as well as showed impaired bacterial growth, reduced motility, decreased biofilm formation and reduced levels of virulence-associated protein secretion involved in the type III secretion system (TTSS). It was found that the deletion of fliC significantly decreased the diameter of flagella filaments. tarda was constructed through double crossover allelic exchange by means of the suicide vector pRE112, and its virulence-associated phenotypes and pathogenicity were tested. In this study, a fliC in-frame deletion mutant of a virulent isolate of E. FliC, as a flagellar filament structural protein, is hypothesized to be involved in the pathogenesis of infection. Edwardsiella tarda is a flagellated Gram-negative bacterium which causes edwardsiellosis in fish.
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