The Cholesterol-Dependent Cytolysin Pneumolysin from Streptococcus pneumoniae Binds to Lipid Raft Microdomains in Human Corneal Epithelial Cells

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.

The STX6-VTI1B-VAMP3 complex facilitates xenophagy by regulating the fusion between recycling endosomes and autophagosomes

“…Intriguingly, STX6 is a cholesterol-binding protein, 29 and alteration of the cholesterol level at the TGN/endosome boundary causes STX6 accumulation on VAMP3-positive REs. 30 Bacterial pathogens carry secreted toxins that interact with cholesterol and affect its intracellular balance, 31,32 and streptolysion O, a cytolysin secreted by GAS, also interacts with cholesterol molecules in target bilayers and could affect cholesterol dynamics. 33 This raises the possibility that the STX6-VAMP3-VTI1B complex is activated in response to changes in cholesterol levels during bacterial infection.…”

The Cytolytic Activity of Vaginolysin Strictly Depends on Cholesterol and Is Potentiated by Human CD59

“…Our data on VLY cytolytic activity thus support the attribution of VLY together with Sm-hPAF to a novel group (group III) of atypical CDCs in the classification based on the mode of receptor recognition introduced Tabata et al [ 23 ]. It has been suggested that CDCs preferentially bind to cholesterol and sphingolipid-enriched membrane microdomains known as lipid rafts [ 31 , 32 , 33 ]. Other studies demonstrated that exposure of cholesterol and toxin binding are not limited to the presence of the particular membrane domain [ 8 , 34 , 35 ].…”

Red Blood Cell Susceptibility to Pneumolysin

“…For example, studies using PFO revealed that membrane binding depends on the availability of "free cholesterol" at the membrane surface and does not correlate with the presence of detergent-resistant domains (93). In contrast, PLY has been reported to bind to cholesterol-rich lipid raft microdomains in corneal epithelial cells (95). Such differences suggest that the composition and organization (and hence physical properties) of the lipid bilayer itself is likely to be a key regulator of the interaction of CDCs with the membrane.…”

“…This could be due to the chemical modification of PLY receptors on the membrane surface (40) and/or oxidation of cholesterol that could modulate the conformation of glycans necessary for the successful binding of the toxin to the RBC membrane (102). Direct cholesterol oxidation (103) will also affect the lateral microdomain organization of the lipid membrane (104 -106), which could impair membrane insertion of the toxin, because PLY has been reported to bind to cholesterol-rich lipid raft microdomains (95). As a result, the membrane is rendered more impervious to toxin.…”