Characterization of Inhibitor to Leptospiral Hemolysin Present in Bovine Serum

Abstract

Hemolysis by leptospiral hemolysin was strongly inhibited by bovine serum. The inhibitory activity was observed in the chloroform‐methanol‐soluble fraction of bovine serum. The inhibitor was eluted in a complex lipid fraction and was separated into two fractions (Fr. I and II) by silicic acid column chromatography. Fractions I and II inhibited approximately 75% and 95%, respectively, of hemolysis by leptospiral hemolysin. Fraction I was identified as phosphatidylethanolamine (PdE) by silica gel thin‐layer chromatography (TLC). Two kinds of phospholipids (PLs) were detected in Fr. II by TLC. One was resistant to alkaline treatment and was identified as sphingomyelin (Spm), and the other was sensitive to such treatment and was identified as phosphatidylcholine (PdC). PLs, such as Spm, PdC, phosphatidylglycerol, PdE, phosphatidylserine and cardiolipin, inhibited hemolysis by leptospiral hemolysin, but phosphatidylinositol did not show any inhibitory activity. PLs lacking the amino group in the polar backbone of the molecules were more effective. From experiments using erythrocytes of various kinds of animals, it was revealed that the hemolytic sensitivity of mammalian erythrocytes to leptospiral hemolysin depended on the Spm content in the erythrocyte membrane. On the other hand, phospholipase C (PLase C) activity with Spm and PdC as substrates was detected in the culture supernatant of Leptospira. Therefore, leptospiral hemolysin was presumed to be PLase C, perhaps sphingomyelinase. The inhibitors of leptospiral hemolysin present in bovine serum were identified as PLs. PLs in bovine serum were suggested to function as inhibitors of the interaction between leptospiral hemolysin and the surface of the erythrocyte membrane.

Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans

“…The ability to detect hemolytic activity in the presence of rat serum on erythrocyte agar plates suggests that the inhibitor is labile or highly diffusible in agar. The hemolysin inhibitor in bovine serum was shown to be phosphatidylethanolamine, phosphotidylcholine, and sphingomyelin [ 50 ]. In addition, because rats are a reservoir host for Leptospira , rat serum may contain anti-Sph2 antibodies that inhibit the function of Sph2.…”

Molecular analysis of a sphingomyelinase C gene from Leptospira interrogans serovar hardjo

“…Phospholipase assay and analysis by thin-layer chromatography. Sphingomyelinase activity was tested in a biphasic system, essentially as described previously (24), consisting of an ether-methanol (9:1, vol/vol) organic phase containing 2 mg of sphingomyelin isolated from bovine brain (Sigma Chemical Co., St. Louis, Mo.) per ml and a water phase containing 10 mM Tris hydrochloride (pH 7.4), 25 mM MgCl2, and (sonicated) bacteria and/or culture medium .…”

Copurification of Leptospira interrogans serovar pomona hemolysin and sphingomyelinase C

“…As long ago as 1959 it was suggested that the hemolysin might be a phospholipase (18). Much circumstantial evidence consistent with this suggestion has since accumulated, including repeated demonstration of phospholipase(s) in leptospiral culture supernatants (3,4,9,10,(14)(15)(16). The combined information, although strongly suggestive, does not provide proof that phospholipase is responsible for hemolysis.…”

“…Because it is firmly established that staphylococcal beta-hemolysin is a sphingomyelinase C (11,19), it seemed probable that the leptospiral hemolysin is a similar enzyme. Further evidence in support of this idea derives from the fact that phospholipase activity in pathogenic leptospiral culture filtrates or supernatants has been demonstrated repeatedly (4, 10, 14-16, 18,22) and also from the fact that hemolytic activity is inhibited by phospholipids (9,15,18).…”