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). Membrane pore formation by VCC involves four key steps: (i) membrane binding, (ii) formation of a pre-pore oligomeric intermediate, (iii) membrane insertion of the pore-forming motifs, and (iv) formation of the functional transmembrane pore, determined in part by the pH (Rai et al. 2015).
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)
Cytohemolysin precursor, HlyA (Vibrio cholerae cytolysin, VCC) is a beta-barrel pore-forming toxin (beta-PFT). A cryo-electron microscopic study revealed low resolution structures for different functional forms (Dutta et al., 2009). Crystal structures of the soluble and transmembrane heptamer reveal common features among disparate pore-forming toxins (De and Olson, 2011). The toxin forms transmembrane heptameric β-barrel channels with two lectin activities on the β-prism and the β-trefoil (Rai et al. 2013). 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). A single point mutation prevents membrane integration and pore formation (Paul and Chattopadhyay 2012). The deletion of the pre-stem segment does not affect membrane binding and pre-pore oligomer formation, but it critically abrogates the functional pore-forming activity of VCC (Paul and Chattopadhyay 2013). The membrane-bound monomer can not form pores (Rai and Chattopadhyay 2014). VCC can be delivered to host cells via extracellular bacterial vesicles (Elluri et al. 2014). Loops within the membrane-proximal region of VCC play critical roles in determining the functional interactions of the toxin with the membrane lipids that allow pore formation (Rai and Chattopadhyay 2015). VCC may interfer with signalling in the target cell as well as form pores (Khilwani and Chattopadhyay 2015). A functional map of the VCC membrane-binding surface has been published (De et al. 2015). Residues involved in oligomerization have been identified (Rai and Chattopadhyay 2016). The multiple membrane interaction mechanisms of VCC have been reviewed (Kathuria and Chattopadhyay 2018). A model of the transmembrane pore has been presented that accounts for some of its properties (Pantano and Montecucco 2006). An overview of the understanding regarding the membrane interaction mechanisms of VCC and their functional implications for the pore-forming activity of the toxin have been reviewed (Kathuria and Chattopadhyay 2018). The specific cholesterol-binding ability of VCC does not appear to dictate its association with the cholesterol-rich micro-domains on human erythrocytes. Rather, targeting of VCC toward the membrane micro-domains of human erythrocytes possibly acts to facilitate the cholesterol-dependent pore-formation mechanism of the toxin (Cyr 2018). Tyrosine in the hinge region of the pore-forming motif regulates oligomeric beta-barrel pore formation (Mondal et al. 2020). Single-particle cryo-EM was used to characterize the structure of the VCC oligomer in large unilamellar vesicles. The rim domain amino acid residues of VCC interacting with lipid membrane were visualized. Cryo-EM views of lipid bilayer-embedded VCC suggested interesting conformational variabilities, especially in the transmembrane channel, which could have a potential impact on the pore architecture and assist in understanding the pore formation mechanism (Sengupta et al. 2021).
HlyA (VCC) precursor of Vibrio cholerae
Vibrio vulnificus hemolysin (VVH-A). Consists of three domains: Hemolysin N (residues 1 - 200), Leukocidin (residues 220 - 480) and Ricin (690 - 600).
VVH-A of Vibrio vulnificus (P19247)
β-barrel pore-forming Cytotoxin of 663 aas from the leukocidin family.
Toxin of Algicola sagamiensis
β-barrel pore-forming Toxin of 612 aas, it contains a Ricin-type beta-trefoil lectin domain.
Toxin of Thalassomonas viridans
β-barrel pore-forming toxin of 597 aas with a ricin-type beta-trefoil lectin domain.
Toxin of Pseudomonas mediterranea
Phobalysin (Cytolysin; Hemolysin; HlyA, PhlyP ("photobacterial lysin encoded on a plasmid") of 603 aas. 48% identical to The Vibrio cholerae hemolysin (1.C.14.1.1). Forms small β-barrel pores in eukaryotic membranes causing efflux of K+ and ATP but not proteins and entry of Ca2+ and dyes (Rivas et al. 2015; von Hoven et al. 2017).
Phobalysin of Photobacterium damselae (Listonella damsela)