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

Alfa Herrera,Katarina Kulhankova,Sanjana Dayal,Aloysius J Klingelhutz,Wilmara Salgado-Pabón,Patrick M Schlievert,Vijay K Sonkar,Eric A Johnson

doi:10.1128/mbio.00273-17

Abstract

ABSTRACTStaphylococcus 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.

Highlights

Staphylococcus aureus causes many infections, such as skin and soft tissue, pneumonia, osteomyelitis, and infective endocarditis (IE)
To begin to assess the effects of ␤-toxin on human aortic endothelial cells (HAECs) and by which mechanisms of action, we completed 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium (MTS) cytotoxicity assays for HAECs treated individually with purified recombinant wild-type ␤-toxin, SMase-deficient ␤-toxin (H289N), and biofilm ligasedeficient ␤-toxin (H162A and D163A) [17, 20]
Previous data demonstrate that ␤-toxin inhibits interleukin 8 (IL-8) production in human umbilical vein endothelial cells (HUVECs), and we investigated the ability of ␤-toxin and mutants to inhibit IL-8 production in HAECs [21]

Summary

Introduction

Staphylococcus aureus causes many infections, such as skin and soft tissue, pneumonia, osteomyelitis, and infective endocarditis (IE). Endothelial cells line heart valve surfaces, and do they serve to maintain structural integrity and act as barriers but they function in regulating the immune system [14] These cells are affected in many diseases related to endothelial injury, dysfunction, and activation and influence atherosclerosis, hypertension, sepsis, and inflammatory syndromes [14]. When damage occurs to endothelial valve surfaces, their structure is altered significantly This in turn leads to altered blood flow, and sterile vegetations can form due to platelet and fibrin deposition and deposition of other host factors, recruited to repair damage. S. aureus virulence factors contribute to host cell damage, thereby continuing to promote vegetation formation; this occurs by increasing platelet and fibrin deposition, as well as amplifying injury through recruitment of immune cells and modification of the inflammatory state [15]

Methods

Results

Conclusion