Identification and classification of all potential hemolysin encoding genes and their products from Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai
Abstract
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 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. Results and Conclusion: The hemolytic activities of
hemolysin candidates (LA0327, LA0378, LA1027, LA1029, LA1650, LA3050,
LA3937, 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 pathogenesis
of L interrogans.
Keywords:
interrogans serogroup Icterohaemorrhagiae serovar Lai. Methods: 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. Results and Conclusion: The hemolytic activities of
hemolysin candidates (LA0327, LA0378, LA1027, LA1029, LA1650, LA3050,
LA3937, 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 pathogenesis
of L interrogans.