Abstract
Neuronal damage is a major consequence of bacterial meningitis, but little is known about mechanisms of bacterial interaction with neurons leading to neuronal cell death. Streptococcus pneumoniae (pneumococcus) is a leading cause of bacterial meningitis and many survivors develop neurological sequelae after the acute infection has resolved, possibly due to neuronal damage. Here, we studied mechanisms for pneumococcal interactions with neurons. Using human primary neurons, pull-down experiments and mass spectrometry, we show that pneumococci interact with the cytoskeleton protein β-actin through the pilus-1 adhesin RrgA and the cytotoxin pneumolysin (Ply), thereby promoting adhesion and invasion of neurons, and neuronal death. Using our bacteremia-derived meningitis mouse model, we observe that RrgA- and Ply-expressing pneumococci co-localize with neuronal β-actin. Using purified proteins, we show that Ply, through its cholesterol-binding domain 4, interacts with the neuronal plasma membrane, thereby increasing the exposure on the outer surface of β-actin filaments, leading to more β-actin binding sites available for RrgA binding, and thus enhanced pneumococcal interactions with neurons. Pneumococcal infection promotes neuronal death possibly due to increased intracellular Ca2+ levels depending on presence of Ply, as well as on actin cytoskeleton disassembly. STED super-resolution microscopy showed disruption of β-actin filaments in neurons infected with pneumococci expressing RrgA and Ply. Finally, neuronal death caused by pneumococcal infection could be inhibited using antibodies against β-actin. The generated data potentially helps explaining mechanisms for why pneumococci frequently cause neurological sequelae.
Highlights
The Gram-positive bacterium Streptococcus pneumoniae is the main cause of bacterial meningitis worldwide [1, 2]
Streptococcus pneumoniae is the leading etiological cause of bacterial meningitis, yet how pneumococci interact with neurons and cause neuronal death is poorly understood
Using human neurons in vitro and our established bacteremia-derived meningitis mouse model in vivo, we found that pneumococci use the pilus-1 adhesin RrgA and the cytotoxin pneumolysin (Ply) to interact with neuronal β-actin expressed on the plasma membrane
Summary
The Gram-positive bacterium Streptococcus pneumoniae (the pneumococcus) is the main cause of bacterial meningitis worldwide [1, 2]. Pneumococcal infections usually start with pneumococcal colonization of the upper respiratory tract. From this location bacteria may reach the blood stream and interact and bypass the blood-brain barrier (BBB) endothelium to cause meningitis [9, 10]. It has been shown that the RrgA protein of the pneumococcal pilus-1 interacts with the endothelial receptors pIgR and PECAM-1, thereby promoting pneumococcal spread through the BBB and infection of the brain [1]. Pilus-1, which is present in *30% of pneumococcal isolates, has previously been shown to promote colonization, virulence, and pro-inflammatory responses in mouse models [9, 11], and it is composed of three structural proteins, RrgA, RrgB and RrgC, where the tip pilin protein RrgA has been found to be the major adhesin to epithelial cells [12], and RrgB to be the major stalk protein [11]. Ply is a cholesterol-dependent and pore-forming toxin that has been found to induce pro-inflammatory responses, but recently we showed that in certain cell types such as alveolar macrophages and dendritic cells, Ply may induce antiinflammatory responses and effect T-cell responses [15]
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