The recognition and binding of cholesterol is an important feature of many eukaryotic, viral, and prokaryotic proteins, but the molecular details of such interactions are understood only for a few proteins. The pore-forming cholesterol-dependent cytolysins (CDCs) contribute to the pathogenic mechanisms of a large number of Grampositive bacteria. Cholesterol dependence of the CDC mechanism is a hallmark of these toxins, yet the identity of the CDC cholesterol recognition motif has remained elusive. A detailed analysis of membrane interactive structures at the tip of perfringolysin O (PFO) domain 4 reveals that a threonine-leucine pair mediates CDC recognition of and binding to membrane cholesterol. This motif is conserved in all known CDCs and conservative changes in its sequence or order are not well tolerated. Thus, the Thr-Leu pair constitutes a common structural basis for mediating CDC-cholesterol recognition and binding, and defines a unique paradigm for membrane cholesterol recognition by surface-binding proteins.M embrane cholesterol is important to a variety of pathogenic processes that include virus fusion and budding (1) and the mechanisms of eukaryotic (2, 3) and prokaryotic toxins (4-7). Whether cholesterol is bound directly by these proteins as a receptor or it indirectly influences the binding or activity of the protein at the membrane surface remains unknown. The cholesterol-dependent cytolysins (CDCs) use cholesterol as their receptor at the membrane surface (7) and contribute to the pathogenesis of a large number of Gram-positive bacterial pathogens (8). The CDC-sterol interaction initiates a cascade of secondary and tertiary structural changes that lead to the formation of a large oligomeric complex, and ultimately a pore in the membrane of eukaryotic cells (9-13). Although significant progress has been made in understanding the assembly of the CDC pore complex, the structural basis for recognition and binding to cholesterol-rich membranes remains elusive.Early studies with the Clostridium perfringens perfringolysin O (PFO) suggested that the highly conserved tryptophan-rich undecapeptide sequence at the base of domain 4 (14, 15) (Fig. S1) mediated the PFO-cholesterol interaction. However, recent studies by Soltani et al. (16) uncoupled cholesterol binding from the undecapeptide and showed that the membrane insertion of loops L1-L3 at the base of domain 4 was cholesterol dependent (Fig. S1). These observations are also consistent with a lack of conservation of the 3D structures of the undecapeptide in the closely related CDCs PFO (17) and Bacillus anthracis anthrolysin O (ALO) (18) (Fig. S1). These studies suggest the residues that comprise the cholesterol recognition motif are located within L1-L3 because these loops and the undecapeptide are the only structures at the tip of domain 4 exposed to the nonpolar bilayer core; the rest of the domain 4 surface is surrounded by water (19).Cholesterol was thought to function as the sole CDC receptor until the discovery of intermedilysin (ILY), a CDC fr...

Toxoplasma gondii is an obligate intracellular protozoan parasite that invades and replicates within most nucleated cells of warm-blooded animals. The basis for this wide host cell tropism is unknown but could be because parasites invade host cells using distinct pathways and/or repertoires of host factors. Using synchronized parasite invasion assays, we found that host microtubule disruption significantly reduces parasite invasion into host cells early after stimulating parasite invasion but not at later time points. Host microtubules are specifically associated with the moving junction, which is the site of contact between the host cell and the invading parasite. Host microtubules are specifically associated with the moving junction of those parasites invading early after stimulating invasion but not with those invading later. Disruption of host microtubules has no effect on parasite contact, attachment, motility, or rate of penetration. Rather, host microtubules hasten the time before parasites commence invasion. This effect on parasite invasion is distinct from the role that host microtubules play in bacterial and viral infections, where they function to traffic the pathogen or pathogenderived material from the host cell's periphery to its interior. These data indicate that the host microtubule cytoskeleton is a structure used by Toxoplasma to rapidly infect its host cell and highlight a novel function for host microtubules in microbial pathogenesis.Toxoplasma gondii is an obligate intracellular protozoan parasite that is capable of causing disease in fetuses and immunocompromised individuals (23). The parasite infects a wide range of nucleated cells of most warm-blooded animals. The mechanisms underlying this wide tropism are not known but could be due to either the parasite infecting cells using a ubiquitously expressed host receptor and associated machinery, inserting its own receptor into the host cell's plasma membrane, or using multiple host cell receptors/machinery (5).Toxoplasma invasion is a multistep, complex process consisting of parasite contact to host cells, intimate attachment, parasite motility, and then penetration (5). Host cell contact is a loose, low-affinity interaction that is mediated by parasite surface antigens. An unknown signal then triggers the release of proteins from a specialized secretory organelle called micronemes whose contents include proteins that function as adhesins. This is then followed by parasite gliding motility on the host cell surface. At some point, proteins from a second secretory organelle, named rhoptries, are exocytosed. Among these rhoptry proteins, several (RON2, RON4, RON5, and RON8) are part of a preformed complex that binds the previously secreted AMA1 microneme protein (1,2,20,33). Together, these proteins form the moving junction complex, which defines the parasite entry site on the host cell plasma membrane. Parasite penetration occurs by the parasite propel-

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 C8alpha and C9.

Tumors that express the cell surface enzyme gamma-glutamyl transpeptidase (GGT) have increased resistance to chemotherapy and radiation. GGT cleaves the gamma-glutamyl bond of extracellular glutathione, initiating the cleavage of glutathione into glutamate, cysteine and glycine. Glutathione can not be taken up intact by tumor cells. Cleavage of extracellular glutathione increases the supply of cysteine to the cell, which is essential to increasing intracellular glutathione levels. Glutathione detoxifies chemotherapy drugs, and elevated levels of intracellular glutathione can block apoptosis. We are developing inhibitors of GGT that can be used to sensitize tumors to therapy. The standard assay for GGT activity uses p-nitroanilide derivatives as substrates, millimolar concentrations of dipeptides as acceptors and is buffered at pH 8.6. Under these assay conditions several groups of investigators have shown that GGT cleaves the gamma-glutamyl bond of the substrate and transfers the gamma-glutamyl group to an acceptor by a Ping-Pong mechanism. However, the standard assay for GGT activity does not measure the reaction catalyzed by GGT in vivo. We have assayed GGT activity under physiologic conditions. We have found that some compounds that inhibit GGT activity in the standard reaction do not inhibit under physiologic conditions. Our data reveal that, at physiologic pH and osmolarity with reduced concentrations of acceptor, the enzyme does cleave the gamma-glutamyl bond by a Ping-Pong mechanism, but hydrolysis of the intermediate of the gamma-glutamyl enzyme intermediate is significant, and competition between water and the peptide acceptor for the intermediate makes it appear Sequential. Several compounds that inhibit GGT in the standard assay actually accelerate activity as the concentration of the acceptor is reduced to physiologic levels. To identify inhibitors that are optimized for clinical use, we have developed an assay that evaluates GGT cleavage of glutathione, the physiologic substrate, at physiologic pH and osmolarity. We are using our previously reported inhibitor OU749 and other lead compounds to further characterize the physiologic GGT reaction and to identify essential substructural components for inhibition of GGT activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 760.