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Aim:To identify and classify all potential hemolysin candidates of Leptospirainterrogans serogroup Icterohaemorrhagiae serovar Lai.Methods:All of thepotential hemolysin encoding genes were characterized in silico.These geneswere cloned and expressed in Escherichia coli.The hemolytic activities of theexpressed proteins were assayed observing the hemolysis on sheep blood agarplates.Sphingomyelinase activities of the hemolysin candidates were measuredby thin-layer chromatography(TLC)and HPLC for sphingomyelin-hydrolysis.Expression and secretion of the hemolysins in L interrogans were studied byreverse transcription polymerase chain reaction,Western blot,and enzyme-linkedimmunosorbent assays.Results and Conclusion:The hemolytic activities ofhemolysin candidates(LA0327,LA0378,LA1027,LA1029,LAI650,LA3050,LA3937,LA4004)from L interrogans strain Lai were confirmed.They were furtherdivided into two groups,sphingomyelinase hemolysins and non-sphingomyelinasehemolysins,based on their ability to hydrolyze sphingomyelin.Most of thesehemolysins were actually expressed in living L interrogans and some of themwere secreted into the environment.This study establishes an essential findcomplete basis for further studying the contribution of hemolysins to the patho-genesis of L interrogans.
Aim: To identify and classify all potential hemolysin candidates of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai. Methods: All of the potential hemolysin encoding genes were in in silico. The gene genes cloned and expressed in Escherichia coli. The hemolytic activities of the expressed proteins were assayed for the hemolysis on sheep blood agarplates. Sphingomyelinase activities of the hemolysin candidates were measured by thin-layer chromatography (TLC) and HPLC for sphingomyelin-hydrolysis. Expression and secretion of the hemolysins in L interrogans were studied by reverse transcription polymerase chain reaction, Western blot, and enzyme- linked immunosorbent assays. Results and Conclusion: The hemolytic activities of hemolysin candidates (LA0327, LA0378, LA1027, LA1029, LAI650, LA3050, LA3937, LA4004) from L interrogans strain were isolated and the even weredivided into two groups, sphingomyelinase hemolysins and non-sphingomyelinasehemolysins based on their ability to h ydrolyze sphingomyelin. Host of these hemolysins were actually expressed in living L interrogans and some of themwere secreted into the environment. This study establishes an essential findcomplete basis for further studying the contribution of hemolysins to the patho-genesis of L interrogans.