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...

During infection, vertebrates limit access to manganese and zinc, starving invading pathogens, such as Staphylococcus aureus, of these essential metals in a process termed "nutritional immunity." The manganese and zinc binding protein calprotectin is a key component of the nutrient-withholding response, and mice lacking this protein do not sequester manganese from S. aureus liver abscesses. One potential mechanism utilized by S. aureus to minimize host-imposed manganese and zinc starvation is the expression of the metal transporters MntABC and MntH. We performed transcriptional analyses of both mntA and mntH, which revealed increased expression of both systems in response to calprotectin treatment. MntABC and MntH compete with calprotectin for manganese, which enables S. aureus growth and retention of manganese-dependent superoxide dismutase activity. Loss of MntABC and MntH results in reduced staphylococcal burdens in the livers of wild-type but not calprotectin-deficient mice, suggesting that these systems promote manganese acquisition during infection. During the course of these studies, we observed that metal content and the importance of calprotectin varies between murine organs, and infection leads to profound changes in the anatomical distribution of manganese and zinc. In total, these studies provide insight into the mechanisms utilized by bacteria to evade host-imposed nutrient metal starvation and the critical importance of restricting manganese availability during infection. Staphylococcus aureus is a commensal organism that asymptomatically colonizes nearly one-third of the population (1). However, once S. aureus breaches the epithelial barrier, the bacterium is capable of infecting nearly every organ despite the robust defenses elaborated by the host (2). This adaptability contributes to the significant morbidity and mortality associated with S. aureus infections. The emergence of methicillin-and vancomycin-resistant isolates has compounded the threat of this organism, highlighting the need to identify new therapeutics (3-7). This is of particular importance, as antibiotic resistance is prevalent in both hospital-and community-acquired isolates (4,6,8).Metals are essential for all forms of life due to their critical contributions to protein structure and enzymatic function (9-12). To combat invading pathogens, vertebrates leverage the essentiality of transition metals by restricting their availability, a process termed "nutritional immunity" (10, 13). While the most prominent example of nutritional immunity is the restriction of iron (Fe) by the host, it has recently been discovered that vertebrates also limit manganese (Mn) and zinc (Zn) availability during infection (10,(13)(14)(15)(16)(17). In fact, examination of abscesses formed during S. aureus infection has revealed that these lesions are Mn and Zn depleted (14). It was subsequently determined that the Mnand Zn-binding S100 protein calprotectin (CP) is a critical component of this nutrient-withholding response (10,14,18,19). CPdeficient mice...

CD59 is a glycosylphosphatidylinositol-anchored protein that inhibits the assembly of the terminal complement membrane attack complex (MAC) pore, whereas Streptococcus intermedius intermedilysin (ILY), a pore forming cholesterol-dependent cytolysin (CDC), specifically binds to human CD59 (hCD59) to initiate the formation of its pore. The identification of the residues of ILY and hCD59 that form their binding interface revealed a remarkably deep correspondence between the hCD59 binding site for ILY and that for the MAC proteins C8␣ and C9. ILY disengages from hCD59 during the prepore to pore transition, suggesting that loss of this interaction is necessary to accommodate specific structural changes associated with this transition. Consistent with this scenario, mutants of hCD59 or ILY that increased the affinity of this interaction decreased the cytolytic activity by slowing the transition of the prepore to pore but not the assembly of the prepore oligomer. A signature motif was also identified in the hCD59 binding CDCs that revealed a new hCD59-binding member of the CDC family. Although the binding site on hCD59 for ILY, C8␣, and C9 exhibits significant homology, no similarity exists in their binding sites for hCD59. Hence, ILY and the MAC proteins interact with common amino acids of hCD59 but lack detectable conservation in their binding sites for hCD59.The cholesterol-dependent cytolysins (CDCs) 4 are a family of pore-forming toxins produced by a diverse group of Grampositive pathogens. Recently crystal structures of the membrane attack complex/perforin proteins complement C8␣, a C9-like protein from Photorhabdus luminescens, and mouse perforin (1-4) suggested that they are structurally and mechanistically related the CDCs (5-7) and may be ancient ancestors (8). Interestingly, this relationship between the CDCs and membrane attack complex/perforin proteins extends to other features of the complement system. Two members of the CDC family, intermedilysin (ILY) secreted by Streptococcus intermedius and vaginolysin (VLY) secreted by Gardnerella vaginalis, specifically bind to the human form of CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-anchored terminal inhibitor of the mammalian complement membrane attack complex (MAC) (9), rather than cholesterol (10, 11). These CDCs bind to hCD59 to initiate the assembly of their oligomeric pore complex on the membrane of human cells (10), whereas the main function of CD59 is to block the assembly of the MAC pore on host cells, thereby protecting them from the lytic effects of activated complement MAC. Domain 4 of the CDCs mediates its interaction with cholesterol-rich membranes (12)(13)(14). Most CDCs use cholesterol as their receptor and specifically recognize its 3-␤-hydroxy group. Recently Farrand et al. (15) defined the cholesterol recognition motif as a threonine/leucine pair located in loop 1 that is conserved in all members of the CDC family, including ILY and VLY. However, ILY initiates its interaction with the cell by binding to hCD59 (10) rather than chole...

Background:The cholesterol-dependent cytolysins (CDCs) undergo a complex set of structural transitions to form the homo-oligomeric pore complex. Results: Structural transitions are propagated between monomers of the oligomeric complex. Conclusion: Specific structural changes establish the geometry of the oligomeric pore complex and promote the completion of existing oligomers. Significance: CDCs use membrane binding and ordered intermolecular interactions to drive assembly of their ␤-barrel pore.

Pore-forming proteins are weapons often used by bacterial pathogens to breach the membrane barrier of target cells. Despite their critical role in infection important structural aspects of the mechanism of how these proteins assemble into pores remain unknown. Streptococcus pneumoniae is the world’s leading cause of pneumonia, meningitis, bacteremia and otitis media. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae and a target for both small molecule drug development and vaccines. PLY is a member of the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins that form gigantic pores in cell membranes. Here we present the structure of PLY determined by X-ray crystallography and, in solution, by small-angle X-ray scattering. The crystal structure reveals PLY assembles as a linear oligomer that provides key structural insights into the poorly understood early monomer-monomer interactions of CDCs at the membrane surface.

Background:The cholesterol-dependent cytolysins (CDCs) have an identical cholesterol-binding motif but exhibit different binding parameters. Results: Binding affinity is altered by the loop three (L3) structure, which impacts pore-forming efficiency. Conclusion: The L3 structure affects its equilibrium between stabilizing (inserted) and destabilizing (uninserted) membrane interactions. Significance: The L3 structure provides CDCs with cellular selectivity by discriminating lipid environments surrounding membrane cholesterol.

Early intravitreal injection of vancomycin or gatifloxacin improved the therapeutic outcome of B. cereus endophthalmitis. The addition of dexamethasone to antibiotic treatment did not provide a therapeutic benefit over antibiotics alone and appeared to reduce the antibiotic efficacy of vancomycin 6 hours after infection. In this model, delay in treatment past 6 hours significantly reduced the potential for salvaging useful vision.

Protein turnover is a key process for bacterial survival mediated by intracellular proteases. Proteolytic degradation reduces the levels of unfolded and misfolded peptides that accumulate in the cell during stress conditions. Three intracellular proteases, ClpP, HslV, and FtsH, have been identified in the Gram-positive bacterium Staphylococcus aureus, a pathogen responsible for significant morbidity and mortality worldwide. Consistent with their crucial role in protein turnover, ClpP, HslV, and FtsH affect a number of cellular processes, including metabolism, stress responses, and virulence. The ClpP protease is believed to be the principal degradation machinery in S. aureus. This study sought to identify the effect of the Clp protease on the iron-regulated surface determinant (Isd) system, which extracts heme-iron from host hemoglobin during infection and is critical to S. aureus pathogenesis. Inactivation of components of the Clp protease alters abundance of several Isd proteins, including the hemoglobin receptor IsdB. Furthermore, the observed changes in IsdB abundance are the result of transcriptional regulation, since transcription of isdB is decreased by clpP or clpX inactivation. In contrast, inactivation of clpC enhances isdB transcription and protein abundance. Loss of clpP or clpX impairs host hemoglobin binding and utilization and results in severe virulence defects in a systemic mouse model of infection. These findings suggest that the Clp proteolytic system is important for regulating nutrient iron acquisition in S. aureus. The Clp protease and Isd complex are widely conserved in bacteria; therefore, these data reveal a novel Clp-dependent regulation pathway that may be present in other bacterial pathogens.