| Vibrio alginolyticus is a genus of Vibrio.It is a gram-negative short bacilli,with end flagellum,no spores and capable of movement.It is widely distributed in the sea water.Vibrio alginolyticus can cause Vibrio disease and cause incalculable economic losses to aquaculture industry.Therefore,the pathogenicity of Vibrio alginolyticus has been widely studied by scientists.Type II secretory pathway is widespread in gram negative bacteria,including human,animal and plant pathogenic bacteria.Type II secretion system can secrete various proteins to host cells,including extracellular enzymes,proteases,toxins and toxic factors.These extracellular protein toxic factors usually destroy host cells,leading to tissue necrosis and diseaseAs an important regulator of the general stress response of bacteria,rpoS expression can be induced when the external environment changes,such as carbon source or nitrogen source starvation,high osmotic pressure,low pH,and higher temperature stress.In bacteria,a large number of environmental and intracellular signals are involved in the regulation of rpoS.These regulatory processes can occur both at the level of transcription and translation,as well as in various aspects of degradation and activity regulation,and eventually form a complex and delicate regulatory network.The rpoS gene,as a global regulatory factor,also plays an important role in the anti-inverse effect of pathogenic bacteria.In this research,the relationship between the adhesion of Vibrio alginolyticus and the gene(secA,secD,secF,yajC and yidC)in the Type II secretory system was investigated by gene silencing,qRT-PCR and in vitro adhesion test.The results showed that the expression and adhesion of target genes were significantly down regulated after transient silencing and stable silencing.After transient silencing,at 3h,secA decreased by 2.69-times,secD decreased by 8.61-times,secF decreased by 3.13-times,yajC decreased by 2.16-times,and yidC decreased by 4.51-times.In the control group,each field of vision consisted about 1349 ±76 cell.However,secA,secD,secF,yajC,and yidC RNAi group consisted about 424 ± 41525 ± 34,312 ± 44,and 338 ± 32,283 ±50 cells/vision,respectively.After stable silencing,the expression levels of secA,secD,secF,yajC and yidC genes were down regulated by 2.04-,1.46-,1.14-,2.78-and 5.95-times respectively.Compared with the control group,there were 1078 + 58 cells in each field of vision in the control group,while the secA,secD,secF,yajC,and the yidC RNAi group were about 214 ± 44,302 ± 48932 ± 144808 ± 48,and 234 ± 22 cells/vision,and.It shows that secA,secD,secF,yajC and yidC genes are closely related to the adhesion of Vibrio alginolyticus.The expression levels of secA,secD,secF,yajC and yidC genes were significantly affected by temperature,salinity,pH and starvation.At the temperature of 44℃,the expression of the 5 genes reached the peak.When the salinity was 3.5%,the expression of the 5 genes was the highest.When treated with PH,the expression of the five target genes presented the trend of the inverted U type.In addition to the yidC,the other 4 genes reached the highest level when the gene was at pH 7.With the prolongation of starvation time,the expression levels of 5 genes were significantly decreased.This indicates that secA,secD,secF,yajC and yidC genes are sensitive to different environmental factors,and the same environmental stress can lead to different gene expression changes.Therefore,environmental factors can influence the adhesion of Vibrio alginolyticus through the expression of secA,secD,secF,yajC and yidC.At the same time,we also carry on the gene silencing of rpoS gene,qRT-PCR,in vitro adhesion test,detection of growth curve,hemolytic experiment,soft agar plate motility assay,and biofilm assay.The results showed that the expression level of target gene after stable silencing was 1.90-times lower than that of wild type.There were 1078 ± 58 adhered cells in each field of vision in the control group,while the rpoS silence strain was 514 ± 37 adhered cells/vision in the control group,and the expression of rpoS gene was significantly influenced by temperature,salinity,pH and the last time of hunger.At 44℃,the expression level of rpoS was significantly induced.When the salinity was 4.5%,the expression of rpoS was the highest.The expression of rpoS showed the trend of inverted U,and the gene expression level reached the highest at pH 7.The expression level of rpoS decreased significantly with the time of starvation.These proves that rpoS has different responses to different environmental stresses,which indicates that the rpoS gene is sensitive to environmental factors.Compared with the wild type,the growth curve of the silenced strain was lower,while the growth rate of the silent strain was faster before 6.5h,and the growth rate of the wild type from 6.5h to the stable period was faster.Hemolytic activity of the silenced strain is also lower than the wild strain.The hemolytic rate of wild type is 4.23-times of that of the silenced strain.In the biofilm formation experiment,the silenced strain biofilm productionis generally lower,and the wild strain is 1.32-times of the silenced strain.The Vibrio alginolyticus motility is stronger than that of s rpoS silenced strain.This indicates that rpoS gene is closely related to hemolysis,biofilm production,growth and movement of Vibrio alginolyticus.In conclusion,the results of this study showed that the type II secretory system and rpoS gene had an important regulatory effect on the pathogenectiy of Vibrio alginolyticus.The loss of the expression of the above genes would decrease the virulence and it also showed that the type II secretory system and the rpoS gene were sensitive to different environmental stresses. |
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