Cholesterol supplementation protects hepatocytes against the cholesterol-dependent pore-forming toxin pneumolysin

Introduction: Pneumolysin (PLY) is a major virulence factor of Streptococcus pneumonia and crucial for the pathogenesis of invasive pneumococcal disease and sepsis following community-acquired pneumonia (CAP). The b-pore-forming toxin recognizes membrane cholesterol leading to dysintegration of the alveolar epithelial barrier and impairment of innate immunity. Remotely, pneumococcal CAP triggers a hepatic activation of the sterol biosynthesis through a yet unknown mechanism that depends on PLY.Objectives: The direct impact of PLY on hepatocytes is determined. Supplementation of cholesterol species is evaluated as candidate for adjuvant therapy to counteract pore formation. The underlying principle of a protective effect of cholesterol supplementation is investigated. Methods: Membrane integrity and pore formation is analyzed by LDH-release assay and PI staining using dynamic live cell imaging techniques. Stress and sterol signaling adaption of HepG2 cells towards PLY challenge is examined by Western blot and quantitative RT-PCR. Cellular cholesterol of HepG2 cells is quantified by mass spectrometry and Filipin III staining. Membrane viscosity is characterized by applying live-cell two-photon fluorescence lifetime imaging of a bimodal dye sensing the cholesterol environment. In a clinical trial at the Jena University Hospital, a link between patient plasma cholesterol level and CAP score was assessed. Results: Supplementation of cholesterol resulted in amelioration of PLY toxicity, increased membrane integrity and a delay of pore formation. PLY directly activates key regulators of the sterol homeostasis on the protein level whereas gene expression is downregulated globally in HepG2 cells. Intriguingly, cells respond to PLY by increasing their cholesterol content despite cholesterol being the toxin’s receptor for cell membrane recognition. By supplementation of cholesterol the membrane is shifted to a more rigid phase, hindering pore formation. In the clinic, CAP patients with elevated plasma cholesterol are significantly less prone to the progression of CAP. Conclusions: We conclude that PLY pore formation preferentially targets cholesterol in a highly flexible membrane resulting in shedding and cell death. Together, we highlight the role of a hepatic induction of cholesterol biosynthesis following PLY stress and outline the biophysical reasons for a protective effect of cholesterol supplementation against a cholesterol-dependent pore forming toxin.