Pore-forming toxins (PFTs) are potent cytolytic agents secreted by pathogenic bacteria that protect microbes against the cellmediated immune system (by targeting phagocytic cells), disrupt epithelial barriers, and liberate materials necessary to sustain growth and colonization. Produced by gram-positive and gramnegative bacteria alike, PFTs are released as water-soluble monomeric or dimeric species, bind specifically to target membranes, and assemble transmembrane channels leading to cell damage and/or lysis. Structural and biophysical analyses of individual steps in the assembly pathway are essential to fully understanding the dynamic process of channel formation. To work toward this goal, we solved by X-ray diffraction the 2.9-Å structure of the 450-kDa heptameric Vibrio cholerae cytolysin (VCC) toxin purified and crystallized in the presence of detergent. This structure, together with our previously determined 2.3-Å structure of the VCC water-soluble monomer, reveals in detail the architectural changes that occur within the channel region and accessory lectin domains during pore formation including substantial rearrangements of hydrogen-bonding networks in the pore-forming amphipathic loops. Interestingly, a ring of tryptophan residues forms the narrowest constriction in the transmembrane channel reminiscent of the phenylalanine clamp identified in anthrax protective antigen [Krantz BA, et al. (2005) Science 309:777-781]. Our work provides an example of a β-barrel PFT (β-PFT) for which soluble and assembled structures are available at high-resolution, providing a template for investigating intermediate steps in assembly.hemolysin | membrane protein | X-ray crystallography | virulence factor
Pathogens selectively target host cells using adhesion molecules and secreted virulence factors that may utilize protein, lipid, or carbohydrate ligands on the cell surface. The human intestinal pathogen Vibrio cholerae secretes a pore-forming toxin, Vibrio cholerae cytolysin (VCC), which contains two domains that are structurally similar to known carbohydrate-binding proteins. These tandem domains are attached to the carboxy-terminus of the cytolytic domain and contain a β-trefoil fold and a β-prism fold. VCC has been shown to bind glycosylated proteins, and removal of the β-prism domain leads to a large decrease in lytic activity against rabbit erythrocytes. Despite these clues, the identity of the glycan receptors of VCC and the role of glycan binding in toxin activity remains unknown. To better understand this specificity, we used a combination of structural and functional approaches to characterize the carbohydrate-binding activity of the VCC toxin. We first probed the monosaccharide-binding activity of VCC and demonstrated that the toxin exhibits millimolar affinity for aldohexoses. To understand this specificity, we solved the crystal structure of the VCC β-prism domain bound to methyl-α-mannose. Next, we utilized a mammalian glycan screen to determine that the β-prism domain preferentially binds complex N-glycans with a heptasaccharide GlcNAc4 Man3 core (NGA2). Fluorescence anisotropy and surface plasmon resonance indicated an approximately 100-nanomolar affinity of the β-prism domain for the heptasaccharide core. Our results suggest that carbohydrate-binding domains on the VCC toxin facilitate high-affinity targeting of mammalian cell membranes, which may contribute to the ability of VCC to lyse cells at picomolar concentrations.
Vibrio cholerae is an aquatic gram-negative microbe responsible for cholera, a pandemic disease causing life-threatening diarrheal outbreaks in populations with limited access to health care. Like most pathogenic bacteria, V. cholerae secretes virulence factors to assist colonization of human hosts, several of which bind carbohydrate receptors found on cell-surfaces. Understanding how pathogenic virulence proteins specifically target host cells is important for the development of treatment strategies to fight bacterial infections. Vibrio cholerae cytolysin (VCC) is a secreted pore-forming toxin with a carboxy-terminal β-prism domain that targets complex N-glycans found on mammalian cell-surface proteins. To investigate glycan selectivity, we studied the VCC β-prism domain and two additional β-prism domains found within the V. cholerae biofilm matrix protein RbmC. We show that the two RbmC β-prism domains target a similar repertoire of complex N-glycan receptors as VCC and find through binding and modeling studies that a branched pentasaccharide core (GlcNAc2-Man3) represents the likely footprint interacting with these domains. To understand the structural basis of V. cholerae β-prism selectivity, we solved high-resolution crystal structures of fragments of the pentasaccharide core bound to one RbmC β-prism domain and conducted mutagenesis experiments on the VCC toxin. Our results highlight a common strategy for cell-targeting utilized by both toxin and biofilm matrix proteins in Vibrio cholerae and provide a structural framework for understanding the specificity for individual receptors. Our results suggest that a common strategy for disrupting carbohydrate interactions could affect multiple virulence factors produced by V. cholerae, as well as similar β-prism domains found in other vibrio pathogens.
In addition to multiple virulence factors, Bacillus cereus a pathogen that causes food poisoning and life-threatening wound infections, secretes the pore-forming toxin hemolysin II (HlyII). The HlyII toxin has a unique 94 amino acid C-terminal domain (HlyIIC). HlyIIC exhibits splitting of NMR resonances due to cis/trans isomerization of a single proline near the C-terminus. To overcome heterogeneity, we solved the structure of P405M-HlyIIC, a mutant that exclusively stabilizes the trans state. The NMR structure of HlyIIC reveals a novel fold, consisting of two subdomains αA-β1-β2 and β3-β4-αB-β5, that come together in a barrel-like structure. The barrel core is fastened by three layers of hydrophobic residues. The barrel end opposite the HlyIIC-core has a positively charged surface, that by binding negatively charged moieties on cellular membranes, may play a role in target-cell surface recognition or stabilization of the heptameric pore complex. In the WT domain, dynamic flexibility occurs at the N-terminus and the first α-helix that connects the HlyIIC domain to the HlyII-core structure. In the destabilizing P405M mutant, increased flexibility is evident throughout the first subdomain, suggesting that the HlyIIC structure may have arisen through gene fusion.The soil-dwelling, spore-forming B. cereus bacterium 1-3 produces a number of virulence factors 4 including several secreted pore-forming toxins (PFTs) that form lytic channels in the membranes of target cells 5 . One of these toxins, hemolysin II (HlyII), is present in several closely related Bacillus species including B. cereus, B. thuringiensis (a bacterium that parasitizes insects and has insecticide applications), and B. anthracis (the cause of anthrax) 6, 7 . In B. cereus, expression of HlyII is under the control of the HlyIIR protein 8 , and the Fur system that regulates iron homeostasis 9,10 . Expression of HlyII is greater under oxic conditions than under conditions mimicking the intestinal tract, suggesting the toxin may not play a major role in gastrointestinal disease 11 . Although the physiological target of HlyII is not known, the purified toxin lyses rabbit and human erythrocytes 12 as well as other cultured mammalian cells 13,14 . In addition, the toxin can attack species like algae 15 and insects. Studies in mice and insects suggest that HlyII is involved in virulence, and that the toxin causes apoptosis of macrophages in vitro and in vivo 16 . HlyII belongs to a larger family of secreted toxins with similar predicted core structures including the B. cereus cytotoxin K (CytK) 17 , Staphylococcal hemolysins/leukocidins 18,19 , and toxins secreted by a variety of Vibrio species 20 . Similar to other family members, HlyII is secreted as a water-soluble monomer that assembles into a heptameric pore following binding to cell membranes 12,21 . A unique feature of HlyII is the attachment of a C-terminal domain consisting of 94 amino acids that shows no sequence or structural homology to other known proteins 18 . The C-terminal domain, henceforth r...
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