The single D380 amino acid substitution increases pneumolysin cytotoxicity towards neuronal cells
Description
Bacterial meningitis, frequently caused by Streptococcus pneumoniae (the pneumococcus), is a major cause of permanent neurological disorders such as cognitive delay, motor disabilities, and hearing loss, which are due to a neuronal damage caused by the infection. Pneumolysin (PLY), a cholesterol-binding toxin that, once released in the host environment, can damage a variety of eukaryotic cells, including neurons, through its pore-forming activity. Interestingly, we observed that pneumococcal meningitis clinical isolates release significantly higher amounts of pneumolysin both in laboratory conditions and in human blood most likely due to the incredibly higher expression of the autolysin LytA. PLY is largely conserved among pneumococcal strains. However, based on the original PLY amino acid (aa) sequence of the laboratory/reference strain D39, we have identified a single aa substitution in the PLY sequence of pneumococcal meningitis clinical isolates of serotypes 6A, 15A and 16F. Strikingly, the switch from Aspartate to Asparagine in the D4 domain significantly enhances PLY capability of binding to cholesterol which increases the cytotoxicity of PLY towards neuronal cells due to a more acute pore-forming activity as revealed by the scanning electron microscopy analysis showing the degree of pore formation on neuronal plasma membrane. Furthermore, the higher neuronal cytotoxicity by the PLY of the clinical isolates with the single aa substitution was also observed in patch clamp electrophysiological recordings using mouse brain slices ex vivo. In conclusion, a single aa substitution in the PLY D4 domain is sufficient to significantly alter the pore-forming activity of PLY and enhance its cytotoxicity, making pneumococcal strains more neuro-toxic, thereby increasing the risks of neurological sequelae.