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A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes
Published on: February 5, 2018
Changes in astrocyte shape induced by sublytic concentrations of the cholesterol-dependent cytolysin pneumolysin
Christina Förtsch1, Sabrina Hupp, Jiangtao Ma
1DFG Membrane, Cytoskeleton Interaction Group, Institute of Pharmacology and Toxicology & Rudolf Virchow Center for Experimental Medicine, University of Würzburg, Versbacherstr. 9, 97078 Würzburg, Germany. foertsch@toxi.uni-wuerzburg.de
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.
Insights
Pneumolysin, a toxin from Streptococcus pneumoniae, causes cell shape changes in astrocytes. Its pore-forming ability is crucial for this, even at non-lytic concentrations, suggesting a complex interaction with the actin cytoskeleton.
Area of Science:
- Microbiology
- Cell Biology
- Toxicology
Background:
- Streptococcus pneumoniae is a major human pathogen.
- Pneumolysin is a key virulence factor, causing cell lysis and apoptosis.
- Sublytic pneumolysin induces actin cytoskeleton changes in neuroblastoma cells.
Purpose of the Study:
- To investigate the role of pneumolysin's pore-forming capacity in actin-dependent cell shape changes in primary astrocytes.
- To analyze the mechanism by which pneumolysin induces cell retraction and shape alterations.
Main Methods:
- Live imaging of primary astrocytes challenged with wild-type and mutant pneumolysin.
- Analysis of permeabilized and non-permeabilized cell populations.
- Assessment of the impact of actin depolymerization on cell shape changes.
Main Results:
- Wild-type pneumolysin rapidly permeabilized a portion of astrocytes, but non-lytic populations showed shape changes.
- Actin depolymerization inhibited pneumolysin-induced cell retraction.
- Non-lytic pneumolysin mutants failed to induce cell shape changes, highlighting the importance of pore formation.
- Cell shape changes were independent of ion influx and membrane depolarization.
Conclusions:
- Pore formation by pneumolysin is critical for initiating actin-dependent cell shape changes in astrocytes, even at non-lytic concentrations.
- The mechanism involves a complex interplay between pneumolysin pore formation and actin cytoskeleton reorganization, distinct from major pore-related phenomena.
