Structural Analysis of the Leptospiral Sphingomyelinases: in silico and Experimental Evaluation of Sph2 as an Mg⁺⁺-Dependent Sphingomyelinase

Abstract

<i>Background:</i> Leptospiral sphingomyelinases are candidate virulence factors present only in pathogenic <i>Leptospira</i> spp. <i>Leptospira interrogans</i> serovar Lai encodes Sph1, Sph2, Sph3, Sph4 and SphH. Except for Sph4, they all possess the exo-endo-phosphatase domain that groups them under the DNase I superfamily. <i>Methods, Results and Conclusions:</i> Modeling of exo-endo-phosphatase domains reveals high-level structural similarity of Sph2 with the crystal structure of SmcL and BC SMase sphingomyelinases from <i>Listeria ivanovii</i> and <i>Bacillus cereus, </i>respectively. A β-hairpin loop, essential for host cell membrane interaction, is absent in leptospiral sphingomyelinases. Instead, several aromatic amino acids were oriented outward from the surface of these molecules and formed clusters of hydrophobic regions that possibly enables the anchoring of these molecules into the host cell membrane, as demonstrated in Sph2 and Sph3. Sph2 is unique and possesses the Mg<sup>++</sup>-binding Glu53 residue in the metal-binding site and two His residues (His151 and His286) in the catalytic site. We demonstrate experimentally the Mg<sup>++</sup>-dependent hemolysis of erythrocytes by rSph2 and its ability to cleave sphingomyelin to ceramide. Anti-Sph2 antibodies neutralized the hemolytic activity of Sph2. In conclusion, we provide evidence showing that Sph2 is a Mg<sup>++</sup>-dependent hemolysin with both sphingomyelinase and hemolytic activities.

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

“…The molecular masses of bacterial SMase Cs vary from 27 to 39 kDa for most of the enzymes, except for those of Pseudomonas and Leptospira, which have a molecular mass of 58 and 63 kDa, respectively, due to the presence of additional sequences at their C termini (19,20). The three-dimensional structures of enzymes in this group (Bacillus cereus PDB ID 2DDT, Listeria ivanovii PDB ID 1ZWX, Staphylococcus aureus ␤-toxin PDB ID 3I5V, and Streptomyces griseocarneus PDB ID 3WCX) (21)(22)(23) reveal that, although sharing less than 17% sequence identity with mammalian DNase I, they adopt the DNase I fold, as do the mammalian neutral SMases (23).…”

“…Leptospires migrate through skin abrasions or mucosal surfaces into the circulation and can cause severe systemic infections, leading in the severest forms to acute vasculitis, renal tubular interstitial necrosis, hepatic dysfunction, diffuse pulmonary hemorrhage, and respiratory failure (20). There are up to five SMases encoded in the genomes of pathogenic Leptospira strains which are absent in the genomes of nonpathogenic strains (20,51). Leptospiral SMases, known to be cytotoxic to endothelial cells, lymphocytes, and macrophages (20,29,30), are likely involved in several aspects of leptospirosis pathogenesis (20).…”

“…There are up to five SMases encoded in the genomes of pathogenic Leptospira strains which are absent in the genomes of nonpathogenic strains (20,51). Leptospiral SMases, known to be cytotoxic to endothelial cells, lymphocytes, and macrophages (20,29,30), are likely involved in several aspects of leptospirosis pathogenesis (20). Among Leptospira interrogans SMases, sph2, which is likely secreted by a TolC-based type I secretion system, has the most prominent SMase C and hemolytic activities (51).…”

Membrane-damaging and cytotoxic sphingomyelinases and phospholipases

“…The hydrophobic β-hairpin is not found in other structurally characterized members of the DNase I-like fold superfamily, and it constitutes an excellent example of the evolutionary adaptation of a protein scaffold to enable substrate selectivity [29]. However, the absence of the β-hairpin structure in the Pseudomonas and the L. interrogans SMases C suggests the presence of other membrane association mechanisms [36,37]. Accordingly, deletion of the C-terminus in a Pseudomonas SMase mutant abolishes its capacity to bind erythrocyte membranes but not its enzymatic activity against SM in detergent micelles.…”

“…Leptospires migrate from the skin through small cuts or abrasions into the systemic circulation and could cause severe and even fatal infections characterized by hemorrhagic diathesis, jaundice, acute vasculitis, renal tubular interstitial necrosis, and renal failure [37]. Genome sequencing has revealed multiple SMases C encoded in several pathogenic Leptospira, which are absent in the genomes of nonpathogenic strains [37]. Leptospiral SMases C are cytotoxic to endothelial cells, lymphocytes, and macrophages [37,42,43].…”