Study On The Regulatory Effect Of Ⅵ Secretory System-related FHA Domain Protein TagH On Vibrio Cholerae Hemolysin HlyA

Objective:The previous study of our research group found that the knockout of the FHA domain protein Tag H related to the type Ⅵ secretion system can significantly enhance the hemolytic activity of Vibrio cholerae.Our project intends to further study the molecular mechanism of Tag H protein regulating the hemolytic activity and cytotoxicity of Vibrio cholerae Hly A,in order to enrich the molecular regulation mechanism of Hly A and provide experimental basis for in-depth understanding of the biological function of Tag H protein.Methods:Prokaryotic expression and purification of Hly A protein and preparation of polyclonal antiserum for analysis of changes in Hly A levels in the strain to be tested.On the basis of the previous gene knockout,theΔtag H::tag H complementary strain was further constructed to clarify the effect of tag H gene on the hemolytic activity and cytotoxicity of Vibrio cholerae.TheΔtag HΔhly A double-knockout strain was constructed to explore whether Tag H mediated pathological changes such as hemolysis and cytotoxicity by regulating the expression of Hly A.T6SS mutant strainsΔtss B andΔtss M were constructed to explore whether the effect of Tag H on Hly A expression is a downstream effect of T6SS impairment.The expression of hly A transcriptional regulator before and after tag H gene knockout was analyzed by fluorescence quantitative PCR,and the promoter activity was analyzed by luciferase reporter assay.TheΔtag H+p BAD24-fur overexpression strain and theΔtag HΔhly U double knockout strain were further constructed to explore the possible molecular mechanism of Tag H regulating hly A transcription at the transcriptional level.By constructingΔtag HΔprt V double-knockout strains and complementary strains,and performing related phenotype verification analysis,we explored whether Tag H protein regulates Hly A degradation by Prt V protease at the post-translational level.The hemolytic activity and Hly A protein expression of the mutant strains were analyzed by point mutation of the predicted conserved residues related to Tag H binding to phosphopeptide at serine and lysine sites to verify whether these conserved phosphopeptide binding residues also interact with the regulation of hemolytic activity by Tag H in Vibrio cholerae.Results:Hly A protein was successfully expressed and purified,and polyclonal antiserum with high efficiency could be obtained after immunizing mice.TheΔtag H::tag H complementary strain and theΔtag HΔhly A double-knockout strain were successfully constructed.The results of hemolysis and cytotoxicity experiments showed that the hemolytic activity and cytotoxicity of theΔtag H strain were significantly enhanced compared with the wild strain,and the hemolytic activity and cytotoxicity of theΔtag H::tag H complementary strain were similar to those of the wild strain.It is suggested that Tag H negatively affects the hemolytic activity and cytotoxicity of Vibrio cholerae.The hemolytic activity and cytotoxicity of theΔtag HΔhly A culture supernatant were almost completely lost.Western blot results showed that the expression of Hly A in the supernatant and whole bacterial lysate of theΔtag H mutant strain was significantly higher than that of the wild strain,and the expression of Hly A in theΔtag H::tag H complementary strain was similar to that of the wild strain.The above results suggest that Tag H regulates the hemolytic activity and cytotoxicity of Vibrio cholerae mainly depending on the expression of Hly A.T6SS mutant strainsΔtss B andΔtss M were successfully constructed.The results of blood plate hemolytic zone assay and western blot showed that both tss B and tss M knockout inhibited Hcp secretion,but had no effect on hemolytic activity and Hly A expression.These results suggest that the regulation of Tag H on hemolytic activity is independent of the integrity of T6SS.The results of fluorescence quantification and promoter luciferase reporter experiments showed that compared with wild type,the transcription levels and promoter activities of hly A and hly U were significantly up-regulated,and the transcription levels and promoter activities of fur were significantly down-regulated inΔtag H mutants,while the transcription levels and promoter activities of hly A,hly U and fur inΔtag H::tag H mutants recovered to the levels close to those of the wild type.Δtag H+p BAD24-fur overexpression strain andΔtag HΔhly U double knockout strain were successfully constructed.Fluorescence quantification and hemolysis experiments showed that the hemolytic activity and hly A transcription ofΔtag HΔhly U were significantly lower than those of theΔtag H mutant and wild strain,while the hemolytic activity and hly A transcription ofΔtag H+p BAD24-fur were partially restored compared with those of theΔtag H strain.The above results suggest that Tag H regulates the transcription of hly A at the transcriptional level and depends on the co-regulation of Hly U and Fur.Real-time quantitative PCR results showed that Tag H negatively regulates the expression of Prt V protease,and further successfully constructedΔtag HΔprt V double-knockout strain andΔtag HΔprt V::prt V complementary strain.Hemolysis and cytotoxicity experiments were performed.The results showed that the hemolytic activity of the supernatant of theΔtag HΔprt V mutant was slightly higher than that of theΔtag H mutant,and the hemolytic activity and cytotoxicity of theΔtag HΔprt V::prt V complementary were significantly lower than those of theΔtag H andΔtag HΔprt V strains.Western blot results showed that Hly A was expressed at similar levels in the supernatants ofΔtag H andΔtag HΔprt V strains.InΔtag HΔprt V::prt V strain,the expression of Hly A was significantly reduced.The above results suggest that Tag H regulates the degradation of Hly A at the post-translational level may depend on expression of Prt V protease.Tag H^S38A,Tag H^K54Aand Tag H^S38AK54Amutant strains were successfully constructed and the hemolytic activity was carried out.The results showed that compared with the hemolytic activity of theΔtag H::tag H strain,the hemolytic activity of theΔtag H::tag H^S38AΔtag H::tag H^K54AandΔtag H::tag H^S38AK54Amutant strains increased sequentially.Western blot results showed that the secretion amount of Hly A in supernatant ofΔtag H::tag H^S38A,Δtag H::tag H^K54AandΔtag H::tag H^S38AK54Aincreased successively.These results suggest that the phosphopeptide binding site of Tag H plays a vital role in the regulation of hemolysin Hly A expression.Conclusions:In this study,we identified for the first time that Tag H plays an important role in controlling V.cholerae hemolytic activity,in addition to regulating the T6SS.Tag H negatively regulates the expression of Hly A at the transcriptional and post-translational levels,and the phosphopeptide binding site of the FHA domain of Tag H plays a key role in regulating hemolytic activity.This study provides evidence to help unravel the regulatory role of Tag H in Hly A and the T6SS,expands our understanding of the function of Tag H and Hly A,and provides critical insights for the development of strategies to manage Hly A,which plays a key role in the pathogenesis of V.cholerae.