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.
Highlights
Streptococcus pneumoniae is a human pathogen that causes life-threatening diseases, such as pneumonia, sepsis, and the most common form of bacterial meningitis
To clarify the role of pore formation in the reorganization of the actin cytoskeleton, as well as to translate the finding to primary brain cells, we investigated the effects of WT-PLY and the two non-lytic mutants delta6-PLY and W433F-PLY in primary astrocytes
The astrocytic nature of the cells was further verified by an anti-GFAP immunostaining
Summary
Streptococcus pneumoniae (pneumococcus) is a human pathogen that causes life-threatening diseases, such as pneumonia, sepsis, and the most common form of bacterial meningitis. Disease rates are high in young children, elderly people and immunosuppressed patients [1]. Developing and highly developed countries are confronted with the threat of S. pneumoniae infection [2]. Bacterial meningitis is associated with high lethality and neurological disability in the surviving patient. 30% of the infected patients overcome the disease, and 30% of these survivors are affected by long term sequelae [3], including mental retardation, learning impairment and focal neurological deficits (e.g., hearing loss). The acute infection is accompanied by blood vessel inflammation, CNS necrosis, neuronal loss and general inflammation of brain tissue [4]
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