Intermedilysin-Receptor Interactions during Assembly of the Pore Complex: ASSEMBLY INTERMEDIATES INCREASE HOST CELL SUSCEPTIBILITY TO COMPLEMENT-MEDIATED LYSIS*

Intermedilysin (ILY) is an unusual member of the family of cholesterol-dependent cytolysins because it binds to human CD59 (hCD59) rather than directly to cholesterol-rich membranes. Binding of ILY to hCD59 initiates a series of conformational changes within the toxin that result in the conversion of the soluble monomer into an oligomeric membrane-embedded pore complex. In this study the association of ILY with its membrane receptor has been examined throughout the assembly and formation of the pore complex. Using ILY mutants trapped at various stages of pore assembly, we show ILY remains engaged with hCD59 throughout the assembly of the prepore oligomer, but it disengages from the receptor upon the conversion to the pore complex. We further show that the assembly intermediates increase the sensitivity of the host cell to lysis by its complement membrane attack complex, apparently by blocking the hCD59-binding site for complement proteins C8α and C9.The cholesterol-dependent cytolysins (CDC)² are a family of structurally related pore-forming toxins that are important virulence factors for a variety of Gram-positive pathogens (1–4). The CDCs are secreted by the bacterium as soluble monomers and then bind to cholesterol-rich eukaryotic cell membranes (5). Once bound, the monomers laterally diffuse and interact with one another to form a large oligomeric prepore structure comprised of 35–40 CDC monomers. One of the hallmarks of this family of toxins is the absolute requirement of their pore-forming mechanism on membrane cholesterol (1). Membrane cholesterol serves to target the CDCs to the eukaryotic cell membrane and is necessary to convert the prepore oligomer to the inserted pore complex (6). Two classes of CDCs currently exist. The first class is typified by perfringolysin O (PFO) from Clostridium perfringens that appears to bind directly to cholesterol-rich membranes, an interaction mediated by three short loops in domain 4 (7). The second group includes intermedilysin (ILY) from Streptococcus intermedius and vaginolysin from Gardnerella vaginalis (8). These CDCs bind to the glycosylphosphatidylinositol-anchored protein human CD59 (hCD59). It has been shown for ILY that it first binds hCD59 and then inserts its domain 4 loops in a cholesterol-dependent fashion (7). Why the latter two CDCs have evolved to specifically bind hCD59 and whether they remain engaged with this receptor throughout the assembly of the pore complex remains unclear. S. intermedius is a pathogen frequently associated with abscesses of the oral cavity as well as with life-threatening abscesses of the head, neck, and liver (9, 10). ILY appears to be important in establishing these deep-seated abscesses as S. intermedius isolated from these sites produces levels of ILY 6–10 times greater than strains isolated from peripheral site infections or the oropharynx (9). ILY binds only human cells, whereas other CDCs, such as PFO, bind to most cholesterol-rich eukaryotic membranes. The species selectivity of ILY is because of its specificity for human hCD59 and appears to be encoded in domain 4 of the toxin (11, 12).CD59 is an 18–20-kDa surface-expressed glycoprotein tethered to the cell membrane via a glycosylphosphatidylinositol anchor. It is widely distributed on most human and nonhuman cell types. It is associated with a number of important cellular functions that include serving as an adaptor molecule for a candidate C1q receptor (C1qRO^–₂) (13, 14) and acting as a cell-signaling molecule (15). Its primary role, however, is regulating the terminal pathway of complement by inhibiting the formation of the membrane attack complex (MAC) on host cells by binding to C8α and C9, thus preventing the formation of the MAC pore (16–18). In various autoimmune diseases and inflammatory conditions, excessive complement activation can saturate the available CD59 resulting in MAC-mediated host cell injury (19). CD59 exhibits species selectivity such that it most effectively inhibits only the homologous MAC (20). ILY recognition of the same or similar structural differences in CD59 is the basis for its species selective activity (11).ILY binding to hCD59 triggers a series of conformational changes in ILY leading to its membrane oligomerization into the prepore complex (6). This is accompanied by the cholesterol-dependent insertion of three loops at the base of domain 4 and the insertion of the undecapeptide, events that are necessary for the conversion of the prepore to a pore complex (7). It is not known, however, whether ILY remains engaged with hCD59 throughout its assembly into the pore complex. Whether ILY remains engaged during and after the assembly of the pore complex may also impact the ability of the eukaryotic cell to protect itself from the host MAC because a previous study suggested the ILY-binding site on hCD59 overlaps that for complement proteins C8α and C9 (11). To address these questions, we investigated the interaction of ILY with hCD59 during the assembly of the ILY pore complex. We further determined whether nonlytic assembly intermediates of ILY increase MAC-mediated damage to host cells by short circuiting the protective function of hCD59. These studies show ILY remains engaged during the assembly of its prepore complex and disengages from its receptor upon pore formation. In addition, we show that engagement of hCD59 by ILY prior to pore formation significantly increases the host cell sensitivity to the host MAC-mediated lysis.