Staphylococcal β-Toxin Modulates Human Aortic Endothelial Cell and Platelet Function through Sphingomyelinase and Biofilm Ligase Activities

Abstract

Staphylococcus aureus causes many infections, such as skin and soft tissue, pneumonia, osteomyelitis, and infective endocarditis (IE). IE is an endovascular infection of native and prosthetic valves and the lining of the heart; it is characterized by the formation of cauliflower-like "vegetations" composed of fibrin, platelets, other host factors, bacteria, and bacterial products. β-Toxin is an S. aureus virulence factor that contributes to the microorganism's ability to cause IE. This cytolysin has two enzymatic activities: sphingomyelinase (SMase) and biofilm ligase. Although both activities have functions in a rabbit model of IE, the mechanism(s) by which β-toxin directly affects human cells and is involved in the infectious process has not been elucidated. Here, we compared the in vitro effects of purified recombinant wild-type β-toxin, SMase-deficient β-toxin (H289N), and biofilm ligase-deficient β-toxin (H162A and/or D163A) on human aortic endothelial cells (HAECs) and platelets. β-Toxin was cytotoxic to HAECs and inhibited the production of interleukin 8 (IL-8) from these cells by both SMase and biofilm ligase activities. β-Toxin altered HAEC surface expression of CD40 and vascular cell adhesion molecule 1 (VCAM-1). HAECs treated with β-toxin displayed granular membrane morphology not seen in treatment with the SMase-deficient mutant. The altered morphology resulted in two possibly separable activities, cell rounding and redistribution of cell membranes into granules, which were not the result of endosome production from the Golgi apparatus or lysosomes. β-Toxin directly aggregated rabbit platelets via SMase activity.IMPORTANCE Each year there are up to 100,000 cases of infective endocarditis (IE) in the United States. S. aureus is the most common pathogen in patients with health care-associated IE and the leading cause of community-associated IE in the developed world. Multiple clonal group strains as defined by the Centers for Disease Control and Prevention, particularly USA200 and other clones encoding β-toxin, are highly associated with IE. Considering the strong association and established contribution of β-toxin in animal models of IE, determining how β-toxin directly affects human cell types, including endothelial cells and platelets, is important. In this study, we demonstrate that β-toxin functions to modulate endothelial cells and platelets by both toxin sphingomyelinase and biofilm ligase activities. Our data suggest that these activities modulate inflammation and increase infection severity.

Keywords: Staphylococcus aureus; beta-toxin; biofilm ligase; endothelial cells; sphingomyelinase.

FIG 1

β-Toxin is cytotoxic to HAECs in a dose-dependent manner. (A to E) MTS cytotoxicity assays were performed with wild-type β-toxin (A), SMase-deficient mutant H289N (B), ceramide (C), and biofilm ligase-deficient mutants H162A (D) and D163A (E). Asterisks denote significance in relation to PBS-treated cells. Absorbance at 490 nm is shown on the y axes. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 2

SMase and biofilm ligase activities of β-toxin inhibit the ability to induce production of IL-8 by HAECs. (A to E) ELISAs for IL-8 were performed on supernatants from HAECs treated with wild-type β-toxin (A), β-toxin lacking SMase activity (B), ceramide (C), and β-toxin mutants lacking biofilm ligase activity (D and E). Asterisks denote significance in relation to PBS-treated cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

β-Toxin changes HAEC expression of CD40 and VCAM-1. In-cell Western immunoblotting was performed with HAECs. (A to C) Changes in CD40 expression (A), VCAM-1 expression (B), and ICAM-1 expression (C) from PBS-treated cells after treatment with wild-type β-toxin, SMase-deficient mutant, biofilm ligase-deficient mutants, and ceramide. Asterisks denote significance in relation to PBS-treated cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 4

β-Toxin causes membrane alterations of HAECs. Images of HAECs under a dissecting microscope after treatment with β-toxin for 24 h are shown. (A) PBS-treated cells showing no change in cell morphology and retaining a cobblestone shape and HAECs treated with wild-type (WT) β-toxin, mutants, and ceramide-inducing cell rounding. (B) HAECs treated with β-toxin displaying granular distribution of wheat germ agglutinin (WGA) staining, PBS-treated cells show smooth even WGA staining throughout the cells, HAECs treated with wild-type or biofilm ligase mutants showing altered (speckled) cell membrane staining, treatment with the SMase mutant or ceramide appearing not to alter WGA cell membrane staining. (C) β-Toxin-induced speckling of HAECs resulting in the change to cell membrane morphology not due to the formation of endosomes from the Golgi apparatus or lysosomes. The top panel shows WGA does not colocalize with GS15 in β-toxin-treated cells differently from PBS-treated control HAECs. The bottom panel shows that LAMP-1 does not colocalize with WGA speckles formed from β-toxin treatment in HAECs.

FIG 5

SMase activity of β-toxin induces platelet aggregation through integrin activation. (A) Representative aggregation curves showing treatment of platelets with wild-type β-toxin or H162A, D163A, and H289N mutants. H162A and H165A mutants induce robust aggregation response. However, treatment with the H289N mutant does not induce aggregation. (B) Pretreatment with EGTA inhibits the aggregation response invoked by wild-type β-toxin or the D163A mutant.