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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)

This family belongs to the: Aerolysin Superfamily.

References associated with 1.C.14 family:

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De S., Bubnys A., Alonzo F 3rd., Hyun J., Lary JW., Cole JL., Torres VJ. and Olson R. (2015). The Relationship between Glycan Binding and Direct Membrane Interactions in Vibrio cholerae Cytolysin, a Channel-forming Toxin. J Biol Chem. 290(47):28402-15. 26416894
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Lohner S., Walev I., Boukhallouk F., Palmer M., Bhakdi S. and Valeva A. (2009). Pore formation by Vibrio cholerae cytolysin follows the same archetypical mode as beta-barrel toxins from gram-positive organisms. FASEB J. 23(8):2521-8. 19276173
Miyoshi, S., Y. Abe, M. Senoh, T. Mizuno, Y. Maehara, and H. Nakao. (2011). Inactivation of Vibrio vulnificus hemolysin through mutation of the N- or C-terminus of the lectin-like domain. Toxicon 57: 904-908. 21426913
Mondal, A.K., N. Sengupta, M. Singh, R. Biswas, K. Lata, I. Lahiri, S. Dutta, and K. Chattopadhyay. (2022). Glu289 residue in the pore-forming motif of Vibrio cholerae cytolysin is important for efficient β-barrel pore formation. J. Biol. Chem. 298: 102441. [Epub: Ahead of Print] 36055404
Mondal, A.K., P. Verma, N. Sengupta, S. Dutta, S.B. Pandit, and K. Chattopadhyay. (2020). Tyrosine in the hinge region of the pore-forming motif regulates oligomeric β-barrel pore formation by Vibrio cholerae cytolysin. Mol. Microbiol. [Epub: Ahead of Print] 33089544
Olson R, Gouaux E. (2005). Crystal structure of the Vibrio cholerae cytolysin (VCC) pro-toxin and its assembly into a heptameric transmembrane pore. J Mol Biol. 350: 997-1016. 15978620
Pantano, S. and C. Montecucco. (2006). A molecular model of the Vibrio cholerae cytolysin transmembrane pore. Toxicon 47: 35-40. 16330061
Paul K. and Chattopadhyay K. (2012). Single point mutation in Vibrio cholerae cytolysin compromises the membrane pore-formation mechanism of the toxin. FEBS J. 279(21):4039-51. 22934938
Paul K. and Chattopadhyay K. (2014). Pre-pore oligomer formation by Vibrio cholerae cytolysin: insights from a truncated variant lacking the pore-forming pre-stem loop. Biochem Biophys Res Commun. 443(1):189-93. 24291710
Paul, K. and K. Chattopadhyay. (2011). Unfolding distinguishes the Vibrio cholerae cytolysin precursor from the mature form of the toxin. Biochemistry 50: 3936-3945. 21491932
Rai AK. and Chattopadhyay K. (2014). Trapping of Vibrio cholerae cytolysin in the membrane-bound monomeric state blocks membrane insertion and functional pore formation by the toxin. J Biol Chem. 289(24):16978-87. 24794872
Rai AK. and Chattopadhyay K. (2015). Revisiting the membrane interaction mechanism of a membrane-damaging beta-barrel pore-forming toxin Vibrio cholerae cytolysin. Mol Microbiol. 97(6):1051-62. 26059432
Rai AK., Kundu N. and Chattopadhyay K. (2015). Physicochemical constraints of elevated pH affect efficient membrane interaction and arrest an abortive membrane-bound oligomeric intermediate of the beta-barrel pore-forming toxin Vibrio cholerae cytolysin. Arch Biochem Biophys. 583:9-17. 26235489
Rai, A.K. and K. Chattopadhyay. (2016). Revisiting the oligomerization mechanism of Vibrio cholerae cytolysin, a β-barrel pore-forming toxin. Biochem. Biophys. Res. Commun. 474: 421-427. 27150630
Rai, A.K., K. Paul, and K. Chattopadhyay. (2013). Functional mapping of the lectin activity site on the β-prism domain of vibrio cholerae cytolysin: implications for the membrane pore-formation mechanism of the toxin. J. Biol. Chem. 288: 1665-1673. 23209283
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Saka H.A., C. Bidinost, C. Sola, P. Carranza, C. Collino, S. Ortiz, J.R. Echenique, J.L. Bocco. (2008). Vibrio cholerae cytolysin is essential for high enterotoxicity and apoptosis induction produced by a cholera toxin gene-negative V. cholerae non-O1, non-O139 strain. Microb Pathog. 44: 118-128. 17919878
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