Cytotoxic activities of Leptospira interrogans hemolysin SphH as a pore-forming protein on mammalian cells

Abstract

Leptospirosis is a spirochetal zoonosis that causes an acute febrile systemic illness in humans. Leptospira sp. hemolysins have been shown to be virulence factors for the pathogenesis of leptospirosis. Previously, we cloned a hemolysin SphH of Leptospira interrogans serovar lai, a homologue of L. borgpetersenii sphingomyelinase (SphA), from a genomic library (S. H. Lee, K. A. Kim, Y. K. Kim, I. W. Seong, M. J. Kim, and Y. J. Lee, Gene 254:19-28, 2000). Escherichia coli lysate harboring the sphH showed high hemolytic activities on sheep erythrocytes. However, it neither showed sphingomyelinase nor phospholipase activities, in contrast to SphA which was known to have sphingomyelinase activity. Interestingly, the SphH-mediated hemolysis on erythrocytes was osmotically protected by PEG 5000, suggesting that the SphH might have caused pore formation on the erythrocyte membrane. In the present study, we have prepared the Leptospira hemolysin SphH and investigated its hemolytic and cytotoxic activities on mammalian cells. SphH was shown to be a pore-forming protein on several mammalian cells: When treated with the SphH, the sheep erythrocyte membranes formed pores, which were morphologically confirmed by transmission electron microscopy. Furthermore, the SphH-mediated cytotoxicities on mammalian cells were demonstrated by the release of LDH and by inverted microscopic examinations. Finally, the immune serum against the full-length hemolysin could effectively neutralize the SphH-mediated hemolytic and cytotoxic activities. In conclusion, these results suggest that the virulence of Leptospira SphH was due to the pore formation on mammalian cell membranes.

FIG. 1.

Hemolytic activities of cell-free lysate and 0.1 M NaCl SphH eluate. Assays were performed on 1% sheep erythrocytes at 37°C for 1 h with 50 μl of cell-free lysate from E. coli harboring pHLK-2(4d) (○), 0.1 M NaCl SphH eluate (•), and cell-free lysate from E. coli harboring pBS vector (▴) as a negative control. The percent hemolysis was expressed in relation to 100% hemolysis of erythrocytes in distilled water. The data are the means ± standard deviations (error bars) of triplicates.

FIG. 2.

Detection of Leptospira hemolysin SphH expressed in E. coli. (A) SDS-PAGE was performed in SDS-8% polyacrylamide gel and stained with Coomassie brilliant blue. (B) Immunoblot analysis was performed with rabbit immune serum against His-SphH fusion protein. E. coli harboring pET-sphH which contained full-length hemolysin was induced by IPTG for 3 h at 37°C, and sonic lysates were separated into soluble and insoluble fractions by centrifugation. Lanes: 1 to 2, whole-cell E. coli lysate harboring pET-sphH without IPTG induction (lane 1) and with IPTG induction (lane 2); 3 to 4, soluble fraction (lane 3) and insoluble fraction (lane 4) from E. coli lysate harboring pET-sphH induced by IPTG; M, molecular mass marker. The arrow indicates the approximately 64-kDa His-SphH fusion protein.

FIG. 3.

Transmission electron micrographs of sheep erythrocyte membranes treated with the SphH. Sheep erythrocytes were mixed with 0.1 M NaCl SphH eluate and reacted at 37°C for 20 min. (A) Control sheep erythrocyte mixed with PBS (pH 7.2) or lysate of E. coli harboring pBS vector. (B and C) Sheep erythrocytes treated with SphH eluate. Arrowheads indicate the membrane pores induced by SphH, and arrows indicate the burst membrane regions caused by water influx through the SphH-induced pores by osmotic pressure. Magnification, ×16,000 (A and B) and ×40,000 (C).

FIG. 4.

Kinetics of the SphH-mediated cytotoxic effect on mammalian cells. Kinetics of cytotoxicity induced by the 0.1 M NaCl SphH eluate on Vero, A549, H1299, and L132 cells were determined. Cells began to die 2 h after addition of the SphH, and total cell lysis occurred after 6 to 8 h. Cytotoxicity was not induced by E. coli lysate harboring pBS vector which was used as a negative control (data not shown). Cells were cultured as described in Materials and Methods. Cytotoxicity of mammalian cell lines was assayed by measuring the LDH released from the lysed cells and is expressed as a percentage of cell lysis, as indicated in Materials and Methods. Results represent the means ± standard deviations (error bars) of triplicates.

FIG. 5.

Dose-dependent cytotoxicity of SphH on mammalian cells. Vero cells (A), A549 cells (B), H1299 cells (C), and L132 cells (D) were incubated with twofold serially diluted 0.1 M NaCl SphH eluate under 5% CO₂ at 37°C for 8 h. Increasing concentrations of the SphH (•) proportionally induced a rapid injury of cells. E. coli lysate harboring pBS vector (○) was used as a negative control. Membrane damage of mammalian cells was evaluated by the measuring LDH release, as indicated in Material and Methods. Results represent the means ± standard deviations (error bars) of triplicates.

FIG. 6.

The inverted photomicrographs of SphH-induced morphological changes on Vero cells. Vero cells were cultured in a 96-well plate until they reached 90 to ∼100% confluence. A 20-μl aliquot of 0.1 M NaCl SphH eluate was added to each well. After incubation under 5% CO₂ at 37°C for 4 or 8 h, the cells were examined with an inverted microscope. (A) Vero cells treated with lysate of E. coli harboring pBS vector as a negative control. (B and C) Vero cells treated with the eluate for 4 or 8 h, respectively. Magnification, ×140.