Cholesterol-dependent cytolysin

The Thiol-activated Cholesterol-dependent Cytolysin (CDC) family (TC# 1.C.12) is a member of the MACPF superfamily. Cholesterol dependent cytolysins are a family of β-barrel pore-forming exotoxins that are secreted by gram-positive bacteria. CDCs are secreted as water-soluble monomers of 50-70 kDa, that when bound to the target cell, form a circular homo-oligomeric complex containing as many as 40 (or more) monomers. Through multiple conformational changes, the β-barrel transmembrane structure (~250 Å in diameter depending on the toxin) is formed and inserted into the target cell membrane. The presence of cholesterol in the target membrane is required for pore formation, though the presence of cholesterol is not required by all CDCs for binding. For example, Intermedilysin (ILY; TC# 1.C.12.1.5) secreted by Streptococcus intermedius will bind only to target membranes containing a specific protein receptor, independent of the presence of cholesterol, but cholesterol is required by intermedilysin (ILY; TC# 1.C.12.1.5) for pore formation. While the lipid environment of cholesterol in the membrane can affect toxin binding, the exact molecular mechanism that cholesterol regulates the cytolytic activity of the CDC is not fully understood. The Thiol-activated Cholesterol-dependent Cytolysin (CDC) family (TC# 1.C.12) is a member of the MACPF superfamily. Cholesterol dependent cytolysins are a family of β-barrel pore-forming exotoxins that are secreted by gram-positive bacteria. CDCs are secreted as water-soluble monomers of 50-70 kDa, that when bound to the target cell, form a circular homo-oligomeric complex containing as many as 40 (or more) monomers. Through multiple conformational changes, the β-barrel transmembrane structure (~250 Å in diameter depending on the toxin) is formed and inserted into the target cell membrane. The presence of cholesterol in the target membrane is required for pore formation, though the presence of cholesterol is not required by all CDCs for binding. For example, Intermedilysin (ILY; TC# 1.C.12.1.5) secreted by Streptococcus intermedius will bind only to target membranes containing a specific protein receptor, independent of the presence of cholesterol, but cholesterol is required by intermedilysin (ILY; TC# 1.C.12.1.5) for pore formation. While the lipid environment of cholesterol in the membrane can affect toxin binding, the exact molecular mechanism that cholesterol regulates the cytolytic activity of the CDC is not fully understood. Once the pore is formed within the target cell membrane, the regulation of the intracellular environment and what enters and leaves the cell is lost. The pore being ~250 Å in diameter is large enough to allow the loss of amino acids, nucleotides, small and large proteins, as well as ions (Ca2+,Na+, K+, etc.). The loss of calcium in particular, which is involved in multiple molecular pathways, will have a large impact on cell survival. The pore will also lead to an influx of water, which may lead to blebbing and cell death. Bacteria invest energy into creating these toxins because they act as virulence factors. By targeting immune cells such as macrophages the bacteria will be protected against phagocytosis and destruction by respiratory burst. At the level of the primary structure, cholesterol dependent cytolysins (CDCs) display a high degree of sequence similarity ranging from 40% to 80%. This is mainly reflected in the conserved core of about 471 amino acids shared by all CDCs, which essentially corresponds to the sequence of pneumolysin, the shortest member of the family. CDCs with longer sequences usually display variations in the N-terminus, the functions of which are unknown for many members but for some are believed to serve various functions unrelated to secretion. For instance, Listeriolysin O (LLO; TC# 1.C.12.1.7) from Listeria monocytogenes exhibits a proline rich sequence at its amino terminus that plays a role in the stability of LLO. The extreme case is lectinolysin (LLY; UniProt: B3UZR3) from some strains of Streptococcus mitis and S. pseudopneumoniae contain a functional fucose-binding lectin at the amino terminus. Furthermore, all CDCs contain a highly conserved undecapeptide, which is thought to be critical for cholesterol-mediated membrane recognition. The CDC monomer consist of 4 structural domains, with domain 4 (D4) being involved with membrane binding. Multiple CDC monomers will oligomerize once bound to the target cell membrane forming a β-barrel structure which will be inserted into the target cell membrane. The core section of amino acids, which are required for pore formation, are more conserved between CDCs, which is illustrated by similar three-dimensional structures and pore-forming mechanisms. The structurally conserved domain 4 of CDC contains four conserve loops L1-L3 and an undecapeptide region, which is believed to be involved in cholesterol dependent recognition. Single amino acid modifications in these loops prevented Perfingolysin O (PFO; TC# 1.C.12.1.1), which is a CDC secreted by Clostridium perfringens from binding to cholesterol rich liposomes. More recently, Farrand et al. has shown that two amino acids, a threonine-leucine pair in loop L1, comprise the cholesterol binding motif and is conserved in all known CDCs.