Identification and classification of all potential hemolysin encoding genes and their products from Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai1

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Abstract
To identify and classify all potential hemolysin candidates of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai. All of the potential hemolysin encoding genes were characterized in silico. These genes were cloned and expressed in Escherichia coli. The hemolytic activities of the expressed proteins were assayed observing the hemolysis on sheep blood agar plates. 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. The hemolytic activities of hemolysin candidates (LA0327, LA0378, LA1027, LA1029, LA1650, LA3050, LA3 937, LA4004) from L interrogans strain Lai were confirmed. They were further divided into two groups, sphingomyelinase hemolysins and non-sphingomyelinase hemolysins, based on their ability to hydrolyze sphingomyelin. Most of these hemolysins were actually expressed in living L interrogans and some of them were secreted into the environment. This study establishes an essential and complete basis for further studying the contribution of hemolysins to the patho-genesis of L interrogans.