Changes in Astrocyte Shape Induced by Sublytic Concentrations of the Cholesterol-Dependent Cytolysin Pneumolysin Still Require Pore-Forming Capacity

Abstract

Streptococcus pneumoniae is a common pathogen that causes various infections, such as sepsis and meningitis. A major pathogenic factor of S. pneumoniae is the cholesterol-dependent cytolysin, pneumolysin. It produces cell lysis at high concentrations and apoptosis at lower concentrations. We have shown that sublytic amounts of pneumolysin induce small GTPase-dependent actin cytoskeleton reorganization and microtubule stabilization in human neuroblastoma cells that are manifested by cell retraction and changes in cell shape. In this study, we utilized a live imaging approach to analyze the role of pneumolysin’s pore-forming capacity in the actin-dependent cell shape changes in primary astrocytes. After the initial challenge with the wild-type toxin, a permeabilized cell population was rapidly established within 20-40 minutes. After the initial rapid permeabilization, the size of the permeabilized population remained unchanged and reached a plateau. Thus, we analyzed the non-permeabilized (non-lytic) population, which demonstrated retraction and shape changes that were inhibited by actin depolymerization. Despite the non-lytic nature of pneumolysin treatment, the toxin’s lytic capacity remained critical for the initiation of cell shape changes. The non-lytic pneumolysin mutants W433F-pneumolysin and delta6-pneumolysin, which bind the cell membrane with affinities similar to that of the wild-type toxin, were not able to induce shape changes. The initiation of cell shape changes and cell retraction by the wild-type toxin were independent of calcium and sodium influx and membrane depolarization, which are known to occur following cellular challenge and suggested to result from the ion channel-like properties of the pneumolysin pores. Excluding the major pore-related phenomena as the initiation mechanism of cell shape changes, the existence of a more complex relationship between the pore-forming capacity of pneumolysin and the actin cytoskeleton reorganization is suggested.

Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue

“…Permeabilization occurs exponentially and remains constant within the first 20–30 minutes [20]. Here, we challenged primary mouse glial cells with 0.1 μg/mL PLY in physiological calcium concentrations (2 mM).…”

Direct Transmembrane Interaction between Actin and the Pore-Competent, Cholesterol-Dependent Cytolysin Pneumolysin

“…For experimental and immunisation purposes, a non-pore-forming mutant (Δ6-PLY) 4 and the fragments D4-PLY (containing domain 4) and D123-PLY (containing domains 1, 2 and 3) have been created. Δ6-PLY has been shown to preserve membrane-binding activity, 5 but it lacks the amino acids alanine 146 and arginine 147 at the transition interface from domain 1 to domain 3 (Fig. 1b), rendering the toxin non-lytic due to a compromised refolding and penetration capacity of domain 3 (Fig.…”

Neutralizing Antibodies Elicited by a Novel Detoxified Pneumolysin Derivative, PlyD1, Provide Protection against Both Pneumococcal Infection and Lung Injury

“…The toxicity of PLY is associated with its ability to induce pores in cholesterolcontaining membranes (10,12,36). The protein is highly conserved in both amino acid sequence and antigenicity among clinical isolates (16), thus satisfying some basic criteria for its use as a vaccine antigen.…”