Construction and characterization of different hemolysin gene deletion strains in Vibrio parahaemolyticus (ΔhlyA, ΔhlyIII) and evaluation of their virulence.

Vibrio parahaemolyticus, a halophilic food-borne pathogen, possesses an arsenal of virulence factors. The pathogenicity of V. parahaemolyticus results from a combination of various virulence factors. HlyA and hlyIII genes are presumed to function in hemolysis, in addition to tdh and trh in V. parahaemolyticus. To confirm the hemolytic function of genes hlyA and hlyIII, ΔhlyA and ΔhlyIII strains of V. parahaemolyticus were separately constructed via homologous recombination. The cytotoxicity and pathogenicity of the ΔhlyA and ΔhlyIII strains were evaluated using a Tetrahymena-Vibrio co-culture model and an immersion challenge in Litopenaeus vannamei. Results indicated that the hemolytic activity of the ΔhlyA and ΔhlyIII strains decreased by approximately 31.4 % and 24.9 % respectively, compared to the WT strain. Both ΔhlyA and ΔhlyIII exhibited reduced cytotoxicity towards Tetrahymena. Then shrimp infection experiments showed LD ₅₀ values for ΔhlyA and ΔhlyIII of 3.06 × 10 ⁸  CFU/mL and 1.23 × 10 ⁸  CFU/mL, respectively, both higher than the WT strain's value of 2.57 × 10 ⁷  CFU/mL. Histopathological observations revealed that hepatopancreas from shrimps challenged with ΔhlyA and ΔhlyIII exhibited mild symptoms, whereas those challenged with the WT strain displayed severe AHPND. These findings indicate that the ΔhlyA and ΔhlyIII strains are significantly less virulent than the WT strain. In conclusion, both hlyA and hlyIII are vital virulence genes involved in hemolytic and cytotoxic of V. parahaemolyticus.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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