Keratitis [6?4]. 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]. Pneumococcal keratitis can be a sight-threatening infection if left untreated or if treatment is delayed. Corneal ulceration occurs during the course of the infection and often results in an opaque scarification of the corneal surface after the infection is cleared. The lytic action of pneumolysin (Ply), a 53 kilodalton (kDa) virulence protein produced by S. pneumoniae, is responsible for the formation of corneal ulcers and is a major contributor to pneumococcal virulence as a whole [17?3]. Ply is a member of the cholesterol-dependent cytolysin (CDC) family of proteins, a group of pore-forming proteins from several gram positivebacterial genera including Streptococcus, Listeria, Bacillus, and Clostridium [24]. All CDCs are thought to share a common lytic mechanism which is 100 dependent on the Felypressin 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]. After binding to cholesterol, surfacebound Ply monomers are oriented perpendicular to the cell surface and begin to diffuse laterally across the host membrane [26]. Eventually Ply monomers will interact with one another and oligomerize to form a large multimeric 11089-65-9 prepore structure consisting of 34?0 monomers [24,27]. The prepore structure then undergoes a synchronized conformational change that causes a vertical collapse of the entire complex and the insertion of two bhairpin structures from each monomer into the host membrane [28]. The b-hairpins collectively form a large b-barrel 15481974 pore approximately 25 nm in diameter that traverses the cell 11967625 membrane resulting in osmotic disregulation and cell death [28,29]. Much of what is known about Ply has been extrapolated from previous findings focusing on the lytic mechanism of perfringolysin (Pfo), the CDC produced by Clostridium perfringens. The tertiaryPneumolysin Binds to Lipid Rafts of Corneal Cellsstructure of domain 4 of Pfo contains 4 peptide loops that were found to directly enter the lipid environment upon interaction with cholesterol containing membranes [26]. One of these loops is commonly referred to as the undecapeptide sequence and was originally hypothesized to interact directly with cholesterol and facilitate membrane 
anchoring since the sequence is highly conserved in most CDCs [30,31]. However, intermedilysin (Ily), of Streptococcus intermedius, has been found to have an altered undecapeptide sequence which directly targets human CD59 (protectin) as the initial binding target [32]. Despite the modified undecapeptide, Ily is similar to Pfo in that it still contains the other 3 hydrophobic loops, commonly referred to as L1 3, and these L1 3 loops enter the lipid environment of cholesterol-containing membranes in the same manner as seen in Pfo. The behavior of the L1 3 loops indicates that one or more of these loops likely interact with cholesterol in the host membrane [33]. Recently, two residues found within the L1 loop (T490 and L491 in Pfo) have been 
proposed to be the cholesterol recognition motif for all CDCs, as the two residues are 100 conserved across the CDC family and mutagenesis of these two residues results in drastic reductions in cholesterol binding [34]. Within the host cell membrane, cholesterol is found at a higher concentra.Keratitis [6?4]. 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]. Pneumococcal keratitis can be a sight-threatening infection if left untreated or if treatment is delayed. Corneal ulceration occurs during the course of the infection and often results in an opaque scarification of the corneal surface after the infection is cleared. The lytic action of pneumolysin (Ply), a 53 kilodalton (kDa) virulence protein produced by S. pneumoniae, is responsible for the formation of corneal ulcers and is a major contributor to pneumococcal virulence as a whole [17?3]. Ply is a member of the cholesterol-dependent cytolysin (CDC) family of proteins, a group of pore-forming proteins from several gram positivebacterial 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]. After binding to cholesterol, surfacebound Ply monomers are oriented perpendicular to the cell surface and begin to diffuse laterally across the host membrane [26]. Eventually Ply monomers will interact with one another and oligomerize to form a large multimeric prepore structure consisting of 34?0 monomers [24,27]. The prepore structure then undergoes a synchronized conformational change that causes a vertical collapse of the entire complex and the insertion of two bhairpin structures from each monomer into the host membrane [28]. The b-hairpins collectively form a large b-barrel 15481974 pore approximately 25 nm in diameter that traverses the cell 11967625 membrane resulting in osmotic disregulation and cell death [28,29]. Much of what is known about Ply has been extrapolated from previous findings focusing on the lytic mechanism of perfringolysin (Pfo), the CDC produced by Clostridium perfringens. The tertiaryPneumolysin Binds to Lipid Rafts of Corneal Cellsstructure of domain 4 of Pfo contains 4 peptide loops that were found to directly enter the lipid environment upon interaction with cholesterol containing membranes [26]. One of these loops is commonly referred to as the undecapeptide sequence and was originally hypothesized to interact directly with cholesterol and facilitate membrane anchoring since the sequence is highly conserved in most CDCs [30,31]. However, intermedilysin (Ily), of Streptococcus intermedius, has been found to have an altered undecapeptide sequence which directly targets human CD59 (protectin) as the initial binding target [32]. Despite the modified undecapeptide, Ily is similar to Pfo in that it still contains the other 3 hydrophobic loops, commonly referred to as L1 3, and these L1 3 loops enter the lipid environment of cholesterol-containing membranes in the same manner as seen in Pfo. The behavior of the L1 3 loops indicates that one or more of these loops likely interact with cholesterol in the host membrane [33]. Recently, two residues found within the L1 loop (T490 and L491 in Pfo) have been proposed to be the cholesterol recognition motif for all CDCs, as the two residues are 100 conserved across the CDC family and mutagenesis of these two residues results in drastic reductions in cholesterol binding [34]. Within the host cell membrane, cholesterol is found at a higher concentra.