Effects Of Antibiotics On T6SS Expression And Screening Of Genes Affecting Virulence Of Vibrio Anguillarum

V.anguillarum is one of the common pathogenic pathogens in aquaculture and marine environment,which can cause fish,shrimp,shellfish and other aquatic animals hemorrhagic sepsis,leading to significant economic losses.The VI secretion system?T6SS?is widely distributed in Gram-negative bacteria and has the functions of improving the virulence,inter-or intra-species competition,reciprocal symbiosis with host,and coping with changes in the external environment.In the previous study,we found that the T6 SS of M3 strain was not expressed,and even changing various culture conditions and environmental factors failed to activate T6 SS.In order to find the factors affecting the expression of T6 SS,this study analyzed the effects of several antibiotics on the expression of M3 T6 SS,and constructed a M3 mutant library to screen the factors that affect T6 SS expression.However,neither of these methods successfully screened apposite factor to activate M3 T6 SS.Based on the mutant library,the genes affecting several virulence phenotypes of V.anguillarum were screened.The specific research contents are as follows:1.Effects of antibiotics on the expression of V.anguillarum T6 SS.The activation of V.anguillarum MHK3 T6 SS is characterized by expressing and secreting hemolysin co-regulating protein?Hemolysin co-regulated Protein,Hcp?.In this study,we constructed a fusion strain of V.anguillarum,M3 lacZ::T6SS and MHK3lacZ::T6SS,to detect the expression changes of T6 SS under antibiotic stress.The results showed that the growth of V.anguillarum MHK3 was inhibited in the logarithmic growth period with different concentrations of tetracycline,streptomycin,roxithromycin and polymycin?p<0.05?,but growth was restored in the platform growth period?p<0.05?.At the same time,the expression of V.anguillarum T6 SS was significantly inhibited throughout the growth period?p<0.05?.These results showed that the effects of antibiotics such as tetracycline,streptomycin,roxithromycin and polymycin can reduce the expression of V.anguillarum MHK3 T6 SS rather thaninduce the expression of M3 T6 SS.2.The construction of V.anguillarum M3 transposon mutant library and screening the of Hcp.Using transposon mini-Tn10?pLOF/Kana?,the transposon mutant library of V.anguillarum M3 strain was constructed by electric excitation conversion and bacterial binding.We obtained 1152 strains of mutant strain.The transcription and expression of hcp gene of mutant library strain were detected by RT-PCR and Westernblot.The results showed that the transcription level of hcp gene of the mutant strain 2B₅ was 40 times higher than wild-type M3 strain,but we failed to detect the expression of Hcp protein in the mutant strain.3.Screening of mutants and genes with phenotypic changes in virulence related to V.anguillarum.The 1152 strains of the M3 mutant library were screened,and 1strain?6G₁?,3 strains?5A₁1,7B₁2,7E₁2,?,1 strains?7H₁?and 3 strains?5E₂?6A₂?6E₁2?of the mutant strain changed significantly in casein enzyme activity,gelatin enzyme activity and bacterial membrane formation ability respectively.Further analysis showed that mutations in a phosphodiesterase related gene caused increased swimming capacity?P < 0.05?,mutations in leuD,rseB and thiQ led to significant weakening of casein enzyme activity?p<0.05?,mutations in potD led to a significant weakening of gelatin protease activity?p<0.05?,and mutations in leuO,ilvH and grpB caused a significant decrease in the formation capacity of the bacteria?p<0.05?.To sum up,this study failed to find the factors that activate V.anguillarum M3T6 SS from the environment and gene level,and found 8 genes related to virulence such as mobility,protease activity and bacterial membrane formation of V.anguillarum M3,which laid a foundation for further study of the pathogenic mechanism of V.anguillarum.