1.C.14 The Cytohemolysin (CHL) Family
The CHL family consists of hemolytic cytotoxins from various species of Vibrio, Aeromonas and Listonella. The proteins act on a variety of target animal cells such as enterocytes and immune cells. During secretion of the V. cholerae cytolysin, the N-terminal 25 residue leader peptide is cleaved off yielding an extracellular 79 kDa procytolysin which must be proteolytically activated. Removal of an N-terminal 14 kDa fragment of the procytolysin followed by further proteolytic cleavage in the C-terminal region yields an active 50 kDa species which oligomerizes in the presence of cholesterol-sphingolipid-containing membranes to generate a transmembrane water-filled pore of about 1.5 nm diameter. The complex is probably a homoheptamer (Olson & Gonaux, 2005). This family is distantly related to the αHL family (#1.C.3) of heptameric toxins from Gram-positive bacteria.
Vibrio cholerae cytolysin (VCC; 1.C.14.1.1) is an oligomerizing pore-forming toxin that is related to cytolysins of many other Gram-negative organisms. VCC contains six cysteine residues, of which two are present in free sulphydryl form. Two intramolecular disulphide bonds are present, and one is essential for correct folding of protoxin. The pore-forming domain starts at residue 311, and forms a β-barrel in the assembled oligomer with the subsequent odd-numbered residues facing the lipid bilayer and even-numbered residues facing the lumen. The pore-forming domain of VCC is homologous to the β-barrel-forming sequence of staphylococcal cytolysins (TC# 1.C.3) (Valeva et al., 2005). The crystal structure of the heptamer reveals common features among disparate pore-forming toxins (De and Olson, 2011). A ring of tryptophan residues forms the narrowest constriction in the transmembrane channel reminiscent of the phenylalanine clamp identified in anthrax protective antigen (Krantz et al., 2005).
Vibrio cholerae cytolysin (VCC) is essential for high enterotoxicity and apoptosis induction (Saka et al., 2007). The crystal structure of the protoxin has been reported (1 XEZ_A) (Olson & Gonaux, 2005). Formation of an oligomeric Vibrio cholerae cytolysin (VCC) prepore may precede membrane insertion of the pore-forming amino acid sequence (Löhner et al., 2009). Pore formation by VCC follows the same archetypical pathway as beta-barrel cytolysins of gram-positive organisms such as staphylococcal alpha-toxin. Unfolding distinguishes the Vibrio cholerae cytolysin precursor from the mature form of the toxin (Paul and Chattopadhyay, 2011).
VCC) exhibits lectin-like activity by interacting with β1-galactosyl-terminated glycoconjugates. Apart from the cytolysin domain, VCC harbors two lectin-like domains: the β-Trefoil and the β-Prism domains. Rai et al (2012) showed that the β-Prism domain of VCC acts as the structural scaffold to determine the lectin activity of the protein toward β1-galactosyl-terminated glycoconjugates, and the presence of the β-Prism domain-mediated lectin activity is crucial for an efficient interaction of the toxin toward the target cells. Such lectin activity may regulate oligomerization of the membrane-bound toxin.
The HlyA monomer self-assembles on the target cell surface to the more stable beta-barrel amphipathic heptamer which inserts into the membrane bilayer to form a diffusion channel. Deletion of the 15-kDa beta-prism lectin domain at the C-terminus generates a 50-kDa hemolysin variant (HlyA50) with approximately 1000-fold decrease in hemolytic activity. Because functional differences are eventually dictated by structural differences, Dutta et al. (2009) determined three-dimensional structures of 65 and 50-kDa HlyA oligomers using cryo-electron microscopy and single particle methods. Their study shows that the HlyA oligomer has 7-fold symmetry, but the HlyA50 oligomer is an asymmetric molecule. The HlyA oligomer has bowl-like, arm-like and ring-like domains. Although a central channel is present in both HlyA and HlyA50 oligomers, they differ in pore-size as well as in shapes of the molecules and channel.
Vibrio vulnificus is an etiological agent causing systemic infections in immunocompromised humans and cultured eels (Miyoshi et al., 2011). It produces a hemolytic toxin consisting of the cytolysin domain and the lectin-like domain. For hemolysis, the lectin (ricin) domain specifically binds to cholesterol in the erythrocyte membrane. The toxin assembles on the membrane, and the cytolysin domain is essential for formation of a hollow oligomer. A three-dimensional structure model revealed that the two domains connect linearly, and the C-terminus is located near to the joint of the two domains. Insertion of amino acyl residues between these domains caused inactivation of the toxin, and deletions, substitutions or additions of residue also reduce activity. However, the cholesterol-binding ability was not affected by the mutations.
The generalized transport reaction catalyzed by members of the CHL family is:
ions and solutes (in) ions and solutes (out).