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:

Alm, R.A., U.H. Stroeher, and P.A. Manning. (1988). Extracellular proteins of Vibrio cholerae: Nucleotide sequence of the structural gene (hlyA) for the haemolysin of the haemolytic El Tor strain 017 and characterization of the hlyA mutation in the non-haemolytic classical strain 569B. Mol. Microbiol. 2: 481-488.

Chattopadhyay, K., D. Bhattacharyya, and K.K. Banerjee. (2002). Vibrio cholerae hemolysin. Eur. J. Biochem. 269: 4351-4358.

Cyr, N. (2018). Piercing the lipid raft: the case of cytolysin. Biochem. J. 475: 3917-3919.

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.

De, S. and R. Olson. (2011). Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins. Proc. Natl. Acad. Sci. USA 108: 7385-7390.

Dutta S., Mazumdar B., Banerjee KK. and Ghosh AN. (2010). Three-dimensional structure of different functional forms of the Vibrio cholerae hemolysin oligomer: a cryo-electron microscopic study. J Bacteriol. 192(1):169-78.

Elluri, S., C. Enow, S. Vdovikova, P.K. Rompikuntal, M. Dongre, S. Carlsson, A. Pal, B.E. Uhlin, and S.N. Wai. (2014). Outer Membrane Vesicles Mediate Transport of Biologically Active Vibrio cholerae Cytolysin (VCC) from V. cholerae Strains. PLoS One 9: e106731.

Kathuria, R. and K. Chattopadhyay. (2018). Vibrio cholerae cytolysin: Multiple facets of the membrane interaction mechanism of a β-barrel pore-forming toxin. IUBMB Life 70: 260-266.

Khilwani, B. and K. Chattopadhyay. (2015). Signaling beyond Punching Holes: Modulation of Cellular Responses by Vibrio cholerae Cytolysin. Toxins (Basel) 7: 3344-3358.

Krantz, B.A., R.A. Melnyk, S. Zhang, S.J. Juris, D.B. Lacy, Z. Wu, A. Finkelstein, and R.J. Collier. (2005). A phenylalanine clamp catalyzes protein translocation through the anthrax toxin pore. Science 309: 777-781.

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.

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.

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]

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.

Pantano, S. and C. Montecucco. (2006). A molecular model of the Vibrio cholerae cytolysin transmembrane pore. Toxicon 47: 35-40.

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.

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.

Paul, K. and K. Chattopadhyay. (2011). Unfolding distinguishes the Vibrio cholerae cytolysin precursor from the mature form of the toxin. Biochemistry 50: 3936-3945.

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.

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.

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.

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.

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.

Rivas, A.J., G. von Hoven, C. Neukirch, M. Meyenburg, Q. Qin, S. Füser, K. Boller, M.L. Lemos, C.R. Osorio, and M. Husmann. (2015). Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae. Infect. Immun. 83: 4335-4348.

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.

Sengupta, N., A.K. Mondal, S. Mishra, K. Chattopadhyay, and S. Dutta. (2021). Single-particle cryo-EM reveals conformational variability of the oligomeric VCC β-barrel pore in a lipid bilayer. J. Cell Biol. 220:.

Valeva, A., I. Walev, F. Boukhallouk, T.M. Wassenaar, N. Heinz, J. Hedderich, S. Lautwein, M. Möcking, S. Weis, A. Zitzer, and S. Bhakdi. (2005). Identification of the membrane penetrating domain of Vibrio cholerae cytolysin as a β-barrel structure. Mol. Microbiol. 57: 124-131.

von Hoven, G., A.J. Rivas, C. Neukirch, M. Meyenburg, Q. Qin, S. Parekh, N. Hellmann, and M. Husmann. (2017). Repair of a Bacterial Small β-Barrel Toxin Pore Depends on Channel Width. MBio 8:.

Zitzer, A., O. Zitzer, S. Bhakdi, and M. Palmer. (1999). Oligomerization of Vibrio cholerae cytolysin yields a pentameric pore and has a dual specificity for cholesterol and sphingolipids in the target membrane. J. Biol. Chem. 274: 1375-1380.

Examples:

TC#NameOrganismal TypeExample
1.C.14.1.1

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).

Gram-negative bacteria

HlyA (VCC) precursor of Vibrio cholerae

 
1.C.14.1.2Cytohemolysin 1 precursor, Hly1 Gram-negative bacteria Hly1 of Aeromonas hydrophila
 
1.C.14.1.3

Vibrio vulnificus hemolysin (VVH-A).  Consists of three domains:  Hemolysin N (residues 1 - 200), Leukocidin (residues 220 - 480) and Ricin (690 - 600).

Bacteria

VVH-A of Vibrio vulnificus (P19247)

 
1.C.14.1.4

β-barrel pore-forming Cytotoxin of 663 aas from the leukocidin family.

Toxin of Algicola sagamiensis

 
1.C.14.1.5

β-barrel pore-forming Toxin of 612 aas, it contains a Ricin-type beta-trefoil lectin domain.

Toxin of Thalassomonas viridans

 
1.C.14.1.6

β-barrel pore-forming toxin of 597 aas with a ricin-type beta-trefoil lectin domain.

Toxin of Pseudomonas mediterranea

 
1.C.14.1.7

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)