Abstract
Streptococcus pneumoniae (pneumococcus) is an opportunistic bacterial pathogen responsible for causing several human diseases including pneumonia, meningitis, and otitis media. Pneumococcus is also a major cause of human ocular infections and is commonly isolated in cases of bacterial keratitis, an infection of the cornea. The ocular pathology that occurs during pneumococcal keratitis is partly due to the actions of pneumolysin (Ply), a cholesterol-dependent cytolysin produced by pneumococcus. The lytic mechanism of Ply is a three step process beginning with surface binding to cholesterol. Multiple Ply monomers then oligomerize to form a prepore. The prepore then undergoes a conformational change that creates a large pore in the host cell membrane, resulting in cell lysis. We engineered a collection of single amino acid substitution mutants at residues (A370, A406, W433, and L460) that are crucial to the progression of the lytic mechanism and determined the effects that these mutations had on lytic function. Both PlyWT and the mutant Ply molecules (PlyA370G, PlyA370E, PlyA406G, PlyA406E, PlyW433G, PlyW433E, PlyW433F, PlyL460G, and PlyL460E) were able to bind to the surface of human corneal epithelial cells (HCECs) with similar efficiency. Additionally, PlyWT localized to cholesterol-rich microdomains on the HCEC surface, however, only one mutant (PlyA370G) was able to duplicate this behavior. Four of the 9 mutant Ply molecules (PlyA370E, PlyW433G, PlyW433E, and PlyL460E) were deficient in oligomer formation. Lastly, all of the mutant Ply molecules, except PlyA370G, exhibited significantly impaired lytic activity on HCECs. The other 8 mutants all experienced a reduction in lytic activity, but 4 of the 8 retained the ability to oligomerize. A thorough understanding of the molecular interactions that occur between Ply and the target cell, could lead to targeted treatments aimed to reduce the pathology observed during pneumococcal keratitis.
Highlights
Streptococcus pneumoniae is a worldwide pathogen responsible for both invasive and noninvasive infections, including pneumonia, meningitis, bacteremia, and otitis media [1,2,3,4,5]
Previous studies that focused on Pfo, a related cholesterol-dependent cytolysin (CDC) that shares 42% amino acid homology with Ply, have pinpointed several important amino acids that are involved in interacting with the lipid environment of the host membrane during initial binding [26,45]
The fact that 8 of the 9 mutant Ply variants exhibit a reduction in cytotoxicity when compared to PlyWT, but none of the mutants are deficient in human corneal epithelial cells (HCECs) surface binding indicates that the domain 4 loops are, in some capacity, involved in initiating oligomerization and/or the prepore to pore conversion
Summary
Streptococcus pneumoniae (pneumococcus) is a worldwide pathogen responsible for both invasive and noninvasive infections, including pneumonia, meningitis, bacteremia, and otitis media [1,2,3,4,5]. Pneumococcus is typically among the top three most commonly isolated species from cases of bacterial keratitis, an infection of the cornea of the eye [13,15,16]. Ply is a member of the cholesterol-dependent cytolysin (CDC) family of proteins, a group of pore-forming proteins from several gram positive bacterial genera including Streptococcus, Listeria, Bacillus, and Clostridium [24]. All CDCs are thought to share a common lytic mechanism which is 100% dependent on the presence of cholesterol in the target cell membrane [24]. In the case of Ply, cholesterol serves as the initial binding ligand which anchors Ply to the host cell surface [25]. Surfacebound Ply monomers are oriented perpendicular to the cell surface and begin to diffuse laterally across the host membrane [26]. The b-hairpins collectively form a large b-barrel pore approximately 25 nm in diameter that traverses the cell membrane resulting in osmotic disregulation and cell death [28,29]
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