Identification and partial characterization of a novel hemolysin from Leptospira interrogans serovar lai

Abstract

It has been suggested that leptospiral hemolysins are important in the virulence and pathogenesis of leptospirosis. We have isolated an Escherichia coli clone carrying the 7.8 kb DNA insert from a genomic library of Leptospira interrogans serovar lai by plaque hybridization using a sequence derived from the sphingomyelinase C gene (sphA) of L. borgpetersenii. The clone showed a clear β-hemolytic zone on sheep blood agar and high hemolytic activities on both human and sheep erythrocytes in liquid assays. The clone carried at least two genes responsible for the hemolytic activities, encoded by two open reading frames of 1662 and 816 nucleotides, which are named sphH and hap-1 (hemolysis associated protein-1), respectively. The SphH showed 75% homology to the SphA at the amino acid level, and the Hap-1 showed no significant homology in major databases. Interestingly, however, E. coli cells harboring sphH did not show sphingomyelinase or phospholipase activities. Moreover, SphH-mediated hemolysis was osmotically protected by polyethylene glycol 5000, suggesting that the hemolysis is likely to be caused by pore formation on the membrane. The SphH was successfully expressed in E. coli as a histidine (His)–SphH fusion protein. Both sphH and hap-1 were highly conserved among the Leptospira species, except for the absence of sphH in non-pathogenic L. biflexa serovar patoc. We concluded that the SphH is a novel hemolysin of a pathogenic Leptospira species, which may be a putative pore-forming protein.

Introduction

Leptospirosis is a worldwide zoonosis caused by bacteria belonging to the genus Leptospira and its severity ranges from mild to rapidly fatal (Faine, 1994). All pathogenic leptospires were formerly classified as Leptospira interrogans (Kmety and Dikken, 1993). Recently, DNA homology studies have led to the reclassification of L. interrogans into seven pathogenic Leptospira species: L. borgpeterseni, L. inadai, L. interrogans, L. kirschneri, L. noguchii, L. santarosai, and L. weilii (Ramadass et al., 1992). Various serovar strains show overall differences in the severity of the disease produced in a particular host. The most severe human diseases are caused by L. interrogans serovar icterohemorrhagiae (Faine, 1994). Human leptospirosis in Korea has shown prominent respiratory symptoms with pulmonary hemorrhage in half of the cases, resulting in several deaths (Kim et al., 1991). The severity of the illness may be associated with the virulence level of the most prevalent serovar lai.

Vascular endothelial lesions are characterized in the early stage of leptospirosis. The widespread petechial hemorrhages are apparent in various organs and tissues in man and animals. In the late stage, renal failure, liver cell necrosis, and jaundice have been seen. As virulent factors, lipopolysaccharide (Isogai et al., 1986), glycolipoprotein (Alves et al., 1992), peptidoglycan (Dobrina et al., 1995), heat shock proteins (Stamm et al., 1991), flagellin (Goldstein and Charon, 1990) and others can contribute to the pathogenesis. It has been claimed that leptospiral hemolysins, known as phospholipases, are one of the potential virulent factors which act on erythrocytes and possibly other cell membranes containing substrate phospholipids, leading to cytolysis (Faine, 1994, Thompson, 1986). Sphingomyelinase C has been purified previously (Bernheimer and Bey, 1986), and the corresponding genes have been sequenced (Segers et al., 1990), but its role in the pathogenesis of leptospirosis still remains to be determined. Multiple copies of the sph homologs (sphB to sphG) were found among pathogenic leptospires (Segers et al., 1992).

In this study we identified and characterized a novel hemolysin (SphH) from L. interrogans serovar lai, which is homologous to sphingomyelinase C of L. borgpetersenii reported previously (Segers et al., 1990). The SphH conferred hemolytic activity but no sphingomyelinase or phospholipase activities. SphH-mediated hemolysis seems to be caused by pore formation on target cell membranes. In addition, we found a novel hemolysis-associated protein (Hap-1) which seems to act additively with SphH. The sphH was highly conserved in only pathogenic leptospires, whereas the hap-1 was conserved in both pathogenic and saprophytic strains.