The Cholesterol-Dependent Cytolysin Signature Motif: A Critical Element in the Allosteric Pathway that Couples Membrane Binding to Pore Assembly

The cholesterol-dependent cytolysins (CDCs) constitute a family of pore-forming toxins that contribute to the pathogenesis of a large number of Gram-positive bacterial pathogens.The most highly conserved region in the primary structure of the CDCs is the signature undecapeptide sequence (ECTGLAWEWWR). The CDC pore forming mechanism is highly sensitive to changes in its structure, yet its contribution to the molecular mechanism of the CDCs has remained enigmatic. Using a combination of fluorescence spectroscopic methods we provide evidence that shows the undecapeptide motif of the archetype CDC, perfringolysin O (PFO), is a key structural element in the allosteric coupling of the cholesterol-mediated membrane binding in domain 4 (D4) to distal structural changes in domain 3 (D3) that are required for the formation of the oligomeric pore complex. Loss of the undecapeptide function prevents all measurable D3 structural transitions, the intermolecular interaction of membrane bound monomers and the assembly of the oligomeric pore complex. We further show that this pathway does not exist in intermedilysin (ILY), a CDC that exhibits a divergent undecapeptide and that has evolved to use human CD59 rather than cholesterol as its receptor. These studies show for the first time that the undecapeptide of the cholesterol-binding CDCs forms a critical element of the allosteric pathway that controls the assembly of the pore complex.     

Monomer-monomer interactions propagate structural transitions necessary for pore formation by the cholesterol-dependent cytolysins

The assembly of the cholesterol-dependent cytolysin (CDC) oligomeric pore complex requires a complex choreography of secondary and tertiary structural changes in domain 3 (D3) of the CDC monomer structure. A point mutation was identified in the archetype CDC, perfringolysin O, that blocks detectable D3 structural changes and traps the membrane-bound monomers in an early and reversible stage of oligomer assembly. Using this and other mutants we show that specific D3 structural changes are propagated from one membrane-bound monomer to another. Propagation of these structural changes results in the exposure of a β-strand in D3 that allows it to pair and form edge-on interactions with a second β-strand of a free membrane-bound monomer. Pairing of these strands establishes the final geometry of the pore complex and is necessary to drive the formation of the β-barrel pore. These studies provide new insights into how structural information is propagated between membrane-bound monomers of a self-assembling system and the interactions that establish the geometry of the final pore complex.     

The MACPF/CDC family of pore-forming toxins

Pore-forming toxins (PFTs) are commonly associated with bacterial pathogenesis. In eukaryotes, however, PFTs operate in the immune system or are deployed for attacking prey (e.g. venoms). This review focuses upon two families of globular protein PFTs: the cholesterol-dependent cytolysins (CDCs) and the membrane attack complex/perforin superfamily (MACPF). CDCs are produced by Gram-positive bacteria and lyse or permeabilize host cells or intracellular organelles during infection. In eukaryotes, MACPF proteins have both lytic and non-lytic roles and function in immunity, invasion and development. The structure and molecular mechanism of several CDCs are relatively well characterized. Pore formation involves oligomerization and assembly of soluble monomers into a ring-shaped pre-pore which undergoes conformational change to insert into membranes, forming a large amphipathic transmembrane β-barrel. In contrast, the structure and mechanism of MACPF proteins has remained obscure. Recent crystallographic studies now reveal that although MACPF and CDCs are extremely divergent at the sequence level, they share a common fold. Together with biochemical studies, these structural data suggest that lytic MACPF proteins use a CDC-like mechanism of membrane disruption, and will help understand the roles these proteins play in immunity and development.     

Characterization of a streptococcal cholesterol-dependent cytolysin with a lewis y and b specific lectin domain

The cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins that often exhibit distinct structural changes that modify their pore-forming activity. A soluble platelet aggregation factor from Streptococcus mitis (Sm-hPAF) was characterized and shown to be a functional CDC with an amino-terminal fucose-binding lectin domain. Sm-hPAF, or lectinolysin (LLY) as renamed herein, is most closely related to CDCs from Streptococcus intermedius (ILY) and Streptococcus pneumoniae (pneumolysin or PLY). The LLY gene was identified in strains of S. mitis, S. pneumoniae, and Streptococcus pseudopneumoniae. LLY induces pore-dependent changes in the light scattering properties of the platelets that mimic those induced by platelet aggregation but does not induce platelet aggregation. LLY monomers form the typical large homooligomeric membrane pore complex observed for the CDCs. The pore-forming activity of LLY on platelets is modulated by the amino-terminal lectin domain, a structure that is not present in other CDCs. Glycan microarray analysis showed the lectin domain is specific for difucosylated glycans within Lewis b (Le (b)) and Lewis y (Le (y)) antigens. The glycan-binding site is occluded in the soluble monomer of LLY but is apparently exposed after cell binding, since it significantly increases LLY pore-forming activity in a glycan-dependent manner. Hence, LLY represents a new class of CDC whose pore-forming mechanism is modulated by a glycan-binding domain.     

Pre-pore oligomer formation by Vibrio cholerae cytolysin: Insights from a truncated variant lacking the pore-forming pre-stem loop

Vibrio cholerae cytolysin (VCC), a β-barrel pore-forming toxin (β-PFT), induces killing of the target eukaryotic cells by forming heptameric transmembrane β-barrel pores. Consistent with the β-PFT mode of action, binding of the VCC toxin monomers with the target cell membrane triggers formation of pre-pore oligomeric intermediates, followed by membrane insertion of the β-strands contributed by the pre-stem motif within the central cytolysin domain of each protomer. It has been shown previously that blocking of membrane insertion of the VCC pre-stem motif arrests conversion of the pre-pore state to the functional transmembrane pore. Consistent with the generalized β-PFT mechanism, it therefore appears that the VCC pre-stem motif plays a critical role toward forming the structural scaffold of the transmembrane β-barrel pore. It is, however, still not known whether the pre-stem motif plays any role in the membrane interaction process, and subsequent pre-pore structure formation by VCC. In this direction, we have constructed a recombinant variant of VCC deleting the pre-stem region, and have characterized the effect(s) of physical absence of the pre-stem motif on the distinct steps of the membrane pore-formation process. Our results show that the deletion of the pre-stem segment does not affect membrane binding and pre-pore oligomer formation by the toxin, but it critically abrogates the functional pore-forming activity of VCC. Present study extends our insights regarding the structure–function mechanism associated with the membrane pore formation by VCC, in the context of the β-PFT mode of action.     

Mouse,but Not Human,ApoB-100 Lipoprotein Cholesterol Is a Potent Innate Inhibitor of Streptococcus pneumoniae Pneumolysin

Streptococcus pneumoniae produces the pore-forming toxin pneumolysin (PLY), which is a member of the cholesterol-dependent cytolysin (CDC) family of toxins. The CDCs recognize and bind the 3β-hydroxyl group of cholesterol at the cell surface, which initiates membrane pore formation. The cholesterol transport lipoproteins, which carry cholesterol in their outer monolayer, are potential off-pathway binding targets for the CDCs and are present at significant levels in the serum and the interstitial spaces of cells. Herein we show that cholesterol carried specifically by the ApoB-100-containing lipoprotein particles (CH-ApoB-100) in the mouse, but not that carried by human or guinea pig particles, is a potent inhibitor of the PLY pore-forming mechanism. Cholesterol present in the outer monolayer of mouse ApoB-100 particles is recognized and bound by PLY, which stimulates premature assembly of the PLY oligomeric complex thereby inactivating PLY. These studies further suggest that the vast difference in the inhibitory capacity of mouse CH-ApoB-100 and that of the human and the guinea pig is due to differences in the presentation of cholesterol in the outer monolayer of their ApoB-100 particles. Therefore mouse CH-ApoB-100 represents a significant innate CDC inhibitor that is absent in humans, which may underestimate the contribution of CDCs to human disease when utilizing mouse models of disease.     

Incomplete pneumolysin oligomers form membrane pores

Pneumolysin is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming proteins that are produced as water-soluble monomers or dimers, bind to target membranes and oligomerize into large ring-shaped assemblies comprising approximately 40 subunits and approximately 30 nm across. This pre-pore assembly then refolds to punch a large hole in the lipid bilayer. However, in addition to forming large pores, pneumolysin and other CDCs form smaller lesions characterized by low electrical conductance. Owing to the observation of arc-like (rather than full-ring) oligomers by electron microscopy, it has been hypothesized that smaller oligomers explain smaller functional pores. To investigate whether this is the case, we performed cryo-electron tomography of pneumolysin oligomers on model lipid membranes. We then used sub-tomogram classification and averaging to determine representative membrane-bound low-resolution structures and identified pre-pores versus pores by the presence of membrane within the oligomeric curve. We found pre-pore and pore forms of both complete (ring) and incomplete (arc) oligomers and conclude that arc-shaped oligomeric assemblies of pneumolysin can form pores. As the CDCs are evolutionarily related to the membrane attack complex/perforin family of proteins, which also form variably sized pores, our findings are of relevance to that class of proteins as well.     

β-Barrel membrane protein folding and structure viewed through the lens of α-hemolysin

The β-barrel is a transmembrane structural motif commonly encountered in bacterial outer membrane proteins and pore-forming toxins (PFTs). α-Hemolysin (αHL) is a cytotoxin secreted by Staphylococcus aureus that assembles from a water-soluble monomer to form a membrane-bound heptameric β-barrel on the surface of susceptible cells, perforating the cell membranes, leading to cell death and lysis. The mechanism of heptamer assembly, which has been studied extensively, occurs in a stepwise manner, and the structures of the initial, monomeric form and final, membrane-embedded pore are known. The toxin's ability to assemble from an aqueous, hydrophilic species to a membrane-inserted oligomer is of interest in understanding the assembly of PFTs in particular and the folding and structure of β-barrel membrane proteins in general. Here we review the structures of the monomeric and heptamer states of LukF and αHL, respectively, the mechanism of toxin assembly, and the relationships between αHL and nontoxin β-barrel membrane proteins.     

Cholesterol-dependent cytolysins: from water-soluble state to membrane pore

The cholesterol-dependent cytolysins (CDCs) are a family of bacterial toxins that are important virulence factors for a number of pathogenic Gram-positive bacterial species. CDCs are secreted as soluble, stable monomeric proteins that bind specifically to cholesterol-rich cell membranes, where they assemble into well-defined ring-shaped complexes of around 40 monomers. The complex then undergoes a concerted structural change, driving a large pore through the membrane, potentially lysing the target cell. Understanding the details of this process as the protein transitions from a discrete monomer to a complex, membrane-spanning protein machine is an ongoing challenge. While many of the details have been revealed, there are still questions that remain unanswered. In this review, we present an overview of some of the key features of the structure and function of the CDCs, including the structure of the secreted monomers, the process of interaction with target membranes, and the transition from bound monomers to complete pores. Future directions in CDC research and the potential of CDCs as research tools will also be discussed.     

Specific protein-membrane contacts are required for prepore and pore assembly by a cholesterol-dependent cytolysin

Three short hydrophobic loops and a conserved undecapeptide at the tip of domain 4 (D4) of the cholesterol-dependent cytolysins (CDCs) mediate the binding of the CDC monomers to cholesterol-rich cell membranes. But intermedilysin (ILY), from Streptococcus intermedius, does not bind to cholesterol-rich membranes unless they contain the human protein CD59. This observation suggested that the D4 loops, which include loops L1-L3 and the undecapeptide, of ILY were no longer required for its cell binding. However, we show here that membrane insertion of the D4 loops is required for the cytolysis by ILY. Receptor binding triggers changes in the structure of ILY that are necessary for oligomerization, but membrane insertion of the D4 loops is critical for oligomer assembly and pore formation. Defects that prevent membrane insertion of the undecapeptide also block assembly of the prepore oligomer, while defects in the membrane insertion of the L1-L3 loops prevent the conversion of the prepore oligomer to the pore complex. These studies reveal that pore formation by ILY, and probably other CDCs, is affected by an intricate and coupled sequence of interactions between domain 4 and the membrane.     

The mechanism of pore assembly for a cholesterol-dependent cytolysin: formation of a large prepore complex precedes the insertion of the transmembrane beta-hairpins

Perfringolysin O (PFO) is a member of the cholesterol-dependent cytolysin (CDC) family of membrane-penetrating toxins. The CDCs form large homooligomers (estimated to be comprised of up to 50 CDC monomers) that are responsible for generating a large pore in cholesterol-containing membranes of eukaryotic cells. The assembly of the PFO cytolytic complex was examined to determine whether it forms an oligomeric prepore complex on the membrane prior to the insertion of its membrane-spanning beta-sheet. A PFO oligomeric complex was formed on liposomes at both 4 degrees C and 37 degrees C and shown by SDS-agarose gel electrophoresis to be comprised of a large, comparatively homogeneous complex instead of a distribution of oligomer sizes. At low temperature, the processes of oligomerization and membrane insertion could be resolved, and PFO was found to form an oligomer without significant membrane insertion of its beta-hairpins. Furthermore, PFO was found to increase the ion conductivity through a planar bilayer by large and discrete stepwise changes in conductance that are consistent with the insertion of a preassembled pore complex into the bilayer. The combined results of these analyses strongly support the hypothesis that PFO forms a large oligomeric prepore complex on the membrane surface prior to the insertion of its transmembrane beta-sheet.     

The Cholesterol-dependent Cytolysin Membrane-binding Interface Discriminates Lipid Environments of Cholesterol to Support β-Barrel Pore Insertion

The majority of cholesterol-dependent cytolysins (CDCs) utilize cholesterol as a membrane receptor, whereas a small number are restricted to the GPI-anchored protein CD59 for initial membrane recognition. Two cholesterol-binding CDCs, perfringolysin O (PFO) and streptolysin O (SLO), were found to exhibit strikingly different binding properties to cholesterol-rich natural and synthetic membranes. The structural basis for this difference was mapped to one of the loops (L3) in the membrane binding interface that help anchor the toxin monomers to the membrane after receptor (cholesterol) binding by the membrane insertion of its amino acid side chains. A single point mutation in this loop conferred the binding properties of SLO to PFO and vice versa. Our studies strongly suggest that changing the side chain structure of this loop alters its equilibrium between membrane-inserted and uninserted states, thereby affecting the overall binding affinity and total bound toxin. Previous studies have shown that the lipid environment of cholesterol has a dramatic effect on binding and activity. Combining this data with the results of our current studies on L3 suggests that the structure of this loop has evolved in the different CDCs to preferentially direct binding to cholesterol in different lipid environments. Finally, the efficiency of β-barrel pore formation was inversely correlated with the increased binding and affinity of the PFO L3 mutant, suggesting that selection of a compatible lipid environment impacts the efficiency of membrane insertion of the β-barrel pore.     

Vertical collapse of a cytolysin prepore moves its transmembrane beta-hairpins to the membrane

Perfringolysin O (PFO) is a prototype of the large family of pore-forming cholesterol-dependent cytolysins (CDCs). A central enigma of the cytolytic mechanism of the CDCs is that their membrane-spanning beta-hairpins (the transmembrane amphipathic beta-hairpins (TMHs)) appear to be approximately 40 A too far above the membrane surface to cross the bilayer and form the pore. We now present evidence, using atomic force microscopy (AFM), of a significant difference in the height by which the prepore and pore protrude from the membrane surface: 113+/-5 A for the prepore but only 73+/-5 A for the pore. Time-lapse AFM micrographs show this change in height in real time. Moreover, the monomers in both complexes exhibit nearly identical surface features and these results in combination with those of spectrofluorimetric analyses indicate that the monomers remain in a perpendicular orientation to the bilayer plane during this transition. Therefore, the PFO undergoes a vertical collapse that brings its TMHs to the membrane surface so that they can extend across the bilayer to form the beta-barrel pore.     

A New Model for Pore Formation by Cholesterol-Dependent Cytolysins

Cholesterol Dependent Cytolysins (CDCs) are important bacterial virulence factors that form large (200–300 Å) membrane embedded pores in target cells. Currently, insights from X-ray crystallography, biophysical and single particle cryo-Electron Microscopy (cryo-EM) experiments suggest that soluble monomers first interact with the membrane surface via a C-terminal Immunoglobulin-like domain (Ig; Domain 4). Membrane bound oligomers then assemble into a prepore oligomeric form, following which the prepore assembly collapses towards the membrane surface, with concomitant release and insertion of the membrane spanning subunits. During this rearrangement it is proposed that Domain 2, a region comprising three β-strands that links the pore forming region (Domains 1 and 3) and the Ig domain, must undergo a significant yet currently undetermined, conformational change. Here we address this problem through a systematic molecular modeling and structural bioinformatics approach. Our work shows that simple rigid body rotations may account for the observed collapse of the prepore towards the membrane surface. Support for this idea comes from analysis of published cryo-EM maps of the pneumolysin pore, available crystal structures and molecular dynamics simulations. The latter data in particular reveal that Domains 1, 2 and 4 are able to undergo significant rotational movements with respect to each other. Together, our data provide new and testable insights into the mechanism of pore formation by CDCs.     

Structural Basis for the Function of the β-Barrel Assembly-Enhancing Protease BepA

The β-barrel assembly machinery (BAM) complex mediates the assembly of β-barrel membrane proteins in the outer membrane. BepA, formerly known as YfgC, interacts with the BAM complex and functions as a protease/chaperone for the enhancement of the assembly and/or degradation of β-barrel membrane proteins. To elucidate the molecular mechanism underlying the dual functions of BepA, its full-length three-dimensional structure is needed. Here, we report the crystal structure of full-length BepA at 2.6-Å resolution. BepA possesses an N-terminal protease domain and a C-terminal tetratricopeptide repeat domain, which interact with each other. Domain cross-linking by structure-guided introduction of disulfide bonds did not affect the activities of BepA in vivo, suggesting that the function of this protein does not involve domain rearrangement. The full-length BepA structure is compatible with the previously proposed docking model of BAM complex and tetratricopeptide repeat domain of BepA.     

The bacterial outer membrane β-barrel assembly machinery

β-Barrel proteins found in the outer membrane of Gram-negative bacteria serve a variety of cellular functions. Proper folding and assembly of these proteins are essential for the viability of bacteria and can also play an important role in virulence. The β-barrel assembly machinery (BAM) complex, which is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, has been the focus of many recent studies. This review summarizes the significant progress that has been made toward understanding the structure and function of the bacterial BAM complex.     

Assembly of β-barrel proteins into bacterial outer membranes

Membrane proteins with a β-barrel topology are found in the outer membranes of Gram-negative bacteria and in the plastids and mitochondria of eukaryotic cells. The assembly of these membrane proteins depends on a protein folding reaction (to create the barrel) and an insertion reaction (to integrate the barrel within the outer membrane). Experimental approaches using biophysics and biochemistry are detailing the steps in the assembly pathway, while genetics and bioinformatics have revealed a sophisticated production line of cellular components that catalyze the assembly pathway in vivo. This includes the modular BAM complex, several molecular chaperones and the translocation and assembly module (the TAM). Recent screens also suggest that further components of the pathway might remain to be discovered. We review what is known about the process of β-barrel protein assembly into membranes, and the components of the β-barrel assembly machinery. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Dynamic Association of BAM Complex Modules Includes Surface Exposure of the Lipoprotein BamC

The β-barrel assembly machinery (BAM) complex drives the assembly of β-barrel proteins into the outer membrane of gram-negative bacteria. It is composed of five subunits: BamA, BamB, BamC, BamD, and BamE. We find that the BAM complex isolated from the outer membrane of Escherichia coli consists of a core complex of BamA:B:C:D:E and, in addition, a BamA:B module and a BamC:D module. In the absence of BamC, these modules are destabilized, resulting in increased protease susceptibility of BamD and BamB. While the N-terminus of BamC carries a highly conserved region crucial for stable interaction with BamD, immunofluorescence, immunoprecipitation, and protease-sensitivity assays show that the C-terminal domain of BamC, composed of two helix-grip motifs, is exposed on the surface of E. coli. This unexpected topology of a bacterial lipoprotein is reminiscent of the analogous protein subunits from the mitochondrial β-barrel insertion machinery, the SAM complex. The modular arrangement and topological features provide new insight into the architecture of the BAM complex, towards a better understanding of the mechanism driving β-barrel membrane protein assembly.     

Autotransporter β-domains have a specific function in protein secretion beyond outer-membrane targeting

Autotransporters (ATs) of Gram-negative bacteria contain an N-proximal passenger domain that is transported to the extracellular milieu and a C-terminal β-domain that inserts into the outer membrane (OM) in a β-barrel conformation. This β-domain facilitates translocation of the passenger domain across the OM and has long been considered to be the translocation pore. However, available crystal structures of β-domains show that the β-barrel pore is too narrow for the observed transport of folded elements within the passenger domains. ATs have recently been shown to interact with the β-barrel assembly machinery. These findings questioned a direct involvement of the β-domain in passenger translocation and suggested that it may only target the passenger to the β-barrel assembly machinery pore. To address the function of the β-domain in more detail, we have replaced the β-domain of the Escherichia coli AT hemoglobin protease by β-domains originating from other OM proteins. Furthermore, we have modified the diameter of the β-domain pore. The mutant proteins were analyzed for their capacity to insert into the OM and for surface display of the passenger. Our results show that efficient passenger secretion requires a specific β-domain that not only functions as a targeting device but also is directly involved in the translocation of the passenger to the cell surface.     

Neisserial Omp85 protein is selectively recognized and assembled into functional complexes in the outer membrane of human mitochondria

As a consequence of their bacterial origin, mitochondria contain β-barrel proteins in their outer membrane (OMM). These proteins require the translocase of the outer membrane (TOM) complex and the conserved sorting and assembly machinery (SAM) complex for transport and integration into the OMM. The SAM complex and the β-barrel assembly machinery (BAM) required for biogenesis of β-barrel proteins in bacteria are evolutionarily related. Despite this homology, we show that bacterial β-barrel proteins are not universally recognized and integrated into the OMM of human mitochondria. Selectivity exists both at the level of the TOM and the SAM complex. Of all of the proteins we tested, human mitochondria imported only β-barrel proteins originating from Neisseria sp., and only Omp85, the central component of the neisserial BAM complex, integrated into the OMM. PorB proteins from different Neisseria, although imported by the TOM, were not recognized by the SAM complex and formed membrane complexes only when functional Omp85 was present at the same time in mitochondria. Omp85 alone was capable of integrating other bacterial β-barrel proteins in human mitochondria, but could not substitute for the function of its mitochondrial homolog Sam50. Thus, signals and machineries for transport and assembly of β-barrel proteins in bacteria and human mitochondria differ enough to allow only a certain type of β-barrel proteins to be targeted and integrated in mitochondrial membranes in human cells.