Functional Deficiencies Of Hemolysin A Cause Nonhemolysis Of Epidemic Strains Of Vibrio Cholerae Serogroup O1 Biotype El Tor

Objective:By analyzing the relationship between the hlyA gene and the hemolytic phenotype of the serogroup O1 El Tor strain of Vibrio cholerae during the 7th cholera pandemic.The mechanism leading to hemolytic phenotypic differences in Vibrio cholerae serogroup O1 El Tor strains during the seventh cholera pandemic has been explained.Methods:The hemolytic phenotype of 294 strains of Vibrio cholerae serogroup O1 El Tor toxigenic strain isolated from 1961 to 2011 were verified.hlyA gene was amplified and sequenced or obtained hlyA gene form genome-wide information for sequence analysis.294 strains of serogroup O1 El Tor toxigenic cholerae strain which were isolated from different regions and sources in different years were selected in the background of the seventh cholera pandemic.The sequence variation in hlyA gene was illustrated.ML tree was constructed based on the SNPs in the non-repetitive and non-recombinant genome region of 230 01 El Tor epidemic V.cholerae isolated in China since 1961.The distribution of the non-hemolytic mutant was mapped on the ML tree.By analyzing the hemolytic phenotype and hlyA gene sequence type,the epidemiological characteristics of the hemolytic strain of cholera in the context of the seventh cholera pandemic,were summarized.The relationship between hlyA gene and hemolytic phenotype has been further discussed.85 strains were selected for further analysis.The effect of hlyA gene on hemolytic phenotype was studied by knockout,complement or high expression of hlyA gene in some strains.The relative transcription level of hlyA was detected by qRT-PCR after reverse transcription of cDNA.The hemolysin secretion of the experimental strain was examined by ultrasonic crushing method.The relationship between hlyA gene mutation and the hemolytic phenotype of serogroup O1 El Tor vibrio cholerae strains was comprehensively analyzed and discussed and the cause of the negative hemolytic phenotype of some strains was explained.Results:1.Among 294 strains of toxigenic Vibrio cholerae serogroup O1 El Tor strain,the carrying rate of hlyA gene was 100%.Under the background of the seventh cholera pandemic,all the 60 strains isolated at the first cholera epidemic peak in China were positive in hemolytic phenotype.In the second epidemic peak of cholera,75 strains were isolated,45 were hemolytic positive and 30 were hemolytic negative.During the third cholera epidemic peak,64 of the 115 strains were hemolytic positive and 50 were hemolytic negative.In the subsequent low epidemic period,a total of 45 strains of vibrio cholerae Ol serogroup El Tor were collected,which 26 were hemolytic positive and 19 were hemolytic negative.2.By comparing with the hlyA gene of the standard strain C6706 of serogroup O1 El Tor Vibrio cholerae,the 85 strains selected for further functional studies were divided into 5 genotypes of ST.1-5.hlyA gene was the same as the standard hlyA gene,with a total of 37 strains There were 37 strains of ST.1 hlyA gene.Compared with the ST.1 hlyA gene,ST.2 type hlyA gene has a point mutations at 1358(thymine mutated into cytosine).Almost all strains isolated at the beginning of the seventh pandemic were ST.2 strains.ST.3 is identical to ST.2,except that there is a synonymy mutation in 1979.The ST.4 and ST.5 hlyA genes were missing a total of four bases in 1088-1091.In addition,the point mutation site of ST.2 type was also found in ST.5 type.3.The isolation time of ST.2 strain was at the beginning of the seventh pandemic,and all hemolytic phenotypes were positive.The isolation time of ST.1,ST.3,ST.4 and ST.5 strains was relatively late,and there were hemolytic negative phenotypic strains.4.Vibrio cholerae serogroup O1 El Tor sreference strain C6706 was hemolytic positive strain.After hlyA gene was knocked out,the hemolytic activity was lost,hlyA deletion strain obtained hemolytic activity when pBAD33 expression plasmid carrying hlyA gene was complemented.5.The non-hemolytic mutants emerged after the first high epidemic phase and became a common type in the second epidemic.The non-hemolytic mutant dispersed in the 2nd,3rd epidemic and the inter-epidemic periods.However,certain clones of non-hemolytic mutants spread for several years.6.After cloning type ST.2-ST.5 hlyA gene and complementing with the deletion strain C6706,CΔhlyA-C-ST.2 and CΔhlyA-C-ST.3 were hemolytic positive while CΔhlyA-C-ST.4 and CΔhlyA-C-ST.5 were hemolytic negative.7.Among the 12 strains of ST.1 hemolytic negative strain(ST.1H-),the cytoplasm of 7 strains were hemolytic positive and 5 were hemolytic negative.pBAD33-hlyA was transferred to these 12 strains of ST.1H-,so that hlyA was highly expressed in the strain.The hemolytic activity was obtained in 8 strains of ST.1H-after high expression,and the hemolytic activity was still negative in 4 strains.Four strains with high expression of hlyA were still hemolytic negative after ultrasonic crushing,but the cell content solution was hemolytic positive.Conclusion:1.In the seventh global cholera pandemic,,The epidemic strain of the non-hemolytic El Tor Vibrio cholerae appeared in China shortly after the cholera epidemic began in 1961,and the proportion of the hemolytic negative strain in the subsequent epidemic gradually increased.This result is similar to the previously reported hemolytic phenotype changes of Vibrio cholerae in Asia and America,indicating that China,as a part of Asiatic cholera transmission,participated in the transmission of strains between continents and countries.The evolution of hlyA gene is independent of that of th e whole genome in the 7th cholera pandemic.It is probably a random mutation.The results showed the role of hemolytic phenotype in the biotyping of epidemic strains.2.Through sequencing and hemolytic activity analysis of the hlyA gene of Vibrio cholerae serogroup O1 El Tor strain,it was determined that ST.2 type hlyA gene was the most common wild type hlyA gene of serogroup O1 El Tor strain,and the other four hlyA genotypes were formed by gene mutation during the epidemic.Various factors influenced the hemolytic phenotype of the epidemic strain of Vibrio cholerae.One of the reasons was that the deletion mutation of 4 bases in the hlyA gene resulted in the hemolytic negative of some strains.In addition,the reduced transcription level of hlyA and the blocked secretion of hemolysin hlyA are also the reasons for the hemolytic negative phenotype of the strain.Reduced hlyA transcription levels are relatively common in non-hemolytic strains.This study also provides a direction for further research on the non-hemolytic mutations of these strains.The transcriptional regulation of hlyA gene and secretion deficiency of HlyA should be the focus of future research.