Haemolysin Sph2 of Leptospira interrogans induces cell apoptosis via intracellular reactive oxygen species elevation and mitochondrial membrane injury

Abstract

Leptospira interrogans causes widespread leptospirosis in humans and animals, with major symptoms of jaundice and haemorrhage. Sph2, a member of the sphingomyelinase haemolysins, is an important virulence factor for leptospire. In this study, the function and mechanism of Sph2 in the pathogenesis of leptospirosis were investigated to further understand the pathogenesis of leptospire. Real-time PCR analysis of expression levels during cell invasion showed that sph2 gene expression was transiently induced in human umbilical vein endothelial cells (HUVECs), human embryo liver cells (L02), and human epithelial lung cells (L132), with expression levels reaching a peak after 45 min of infection. Further functional analysis of recombinant Sph2 (rSph2) by LDH assays and confocal microscopy showed that rSph2 can be internalised by cells both by causing cell membrane damage and by a damage-independent clathrin-mediated endocytosis pathway. Subsequently, rSph2 is able to translocate to mitochondria, which led to an increase in the levels of reactive oxygen species (ROS) and a decrease of the mitochondrial membrane potential (ΔΨ m ). Further flowcytometry analyses after rSph2 exposure showed that 28.7%, 31%, and 27.3% of the HUVEC, L02, and L132 cells, respectively, became apoptotic. Because apoptosis could be decreased with the ROS inhibitor N-acetyl cysteine, these experiments suggested that rSph2 triggers apoptosis through mitochondrial membrane damage and ROS elevation. The ability of leptospiral haemolysin rSph2 to cause apoptosis likely contributes to the pathogenesis of leptospirosis.

Characterization of a Novel Peptide from Pathogenic Leptospira and Its Cytotoxic Effect

“…The apoptosis inductivity of pathogenic Leptospira has been found to be associated with virulence proteins. Recently, several studies reported the role of the Fas-binding outer membrane protein and recombinant hemolysin (Sph2) of L. interrogans in the induction of apoptosis in macrophage, umbilical vein endothelium, embryo liver, and human lung epithelial cells [29][30][31]. Here, we demonstrated that Vero cells exposed to 100 µM peptide for 24 h developed late apoptosis.…”

Use of an Integrated Multi-Omics Approach To Identify Molecular Mechanisms and Critical Factors Involved in the Pathogenesis of Leptospira

“…Mce binds to the α5β1 and αVβ3 integrins and mediates the Leptospira internalization into macrophages ( 81 ). Pathogenic Leptospira (LIC, LA, and LIHH) genome consists of five sphingomyelinase genes ( sph-1 to sph-4 , as well as sphH ), out of which sph-2 is reported to be most secreted during infection and is involved in mediating the invasion of bacteria in lung and liver cells through clathrin-mediated endocytosis, thereby inducing apoptosis ( 33 , 35 , 87 , 88 ). Next, in our conjoint analysis, we found that 19 proteins predicted to be secretory are upregulated ( Table 1 ; see Fig.…”

Next Generation Sequencing for Diagnosis of Leptospirosis Combined With Multiple Organ Failure: A Case Report and Literature Review

“…Most deaths from leptospirosis are attributed to renal failure and/or gastrointestinal, pulmonary, or cerebral hemorrhage ( 24 – 26 ) caused by extensive capillary damage including extensive renal necrosis and glomerular atrophy. Some bacterial pathogens produce hemolysins including Sph2, and the expression of Sph2 is highest among epithelial cells ( 27 ). L. interrogans Sph2 was recently found to trigger inflammatory reaction, cytomembranous injury, and apoptosis by damaging the membranes of blood vessel endothelial cells through increasing intracellular reactive oxygen species (ROS) and decreasing the mitochondrial membrane potential (MMP).…”