1.C.4 The Aerolysin Channel-forming Toxin (Aerolysin) Family
The aerolysins are a closely related group of channel-forming toxins secreted by members of the family Aeromonas, important human and animal pathogens. They are activated by host and bacterial proteases which remove a C-terminal fragment of about 40 amino acyl residues. The activated monomeric toxin then binds to a receptor glycosyl phosphatidylinositol (GPI)-anchored protein on the surface of the target cell. Because GPI anchored proteins are incorporated into the envelope membrane of human immunodeficiency virus type I (HIV-1), aerolysin can neutralize the virus in a process that depends on channel formation. The dual chaperone role of the C-terminal propeptide of aerolysin participates in folding and oligomerization of the pore-forming toxin (Iacovache et al., 2011). Monomer activation is possibly the rate-limiting step for the entire pore formation process, probably through release of a propeptide (Bischofberger et al. 2016). A loop that lines the aerolysin channel has an alternating pattern of charged and uncharged residues, suggesting that this transmembrane region has a beta-barrel configuration, as observed for Staphylococcal alpha-toxin. The turn of the beta-hairpin is composed of a stretch of five hydrophobic residues which drives membrane insertion of the developing channel. Possibly once the lipid bilayer has been crossed, it folds back parallel to the plane of the membrane in a rivet-like fashion (Iacovache et al. 2006).
Aerolysin-like pore-forming proteins are characterized by a domain organization and mechanism of action that involves extensive conformational rearrangements. The structures of the membrane integraed pores, well-defined beta-barrels, and their mechanism of assembly are fairly well understood (Podobnik et al. 2017). The cell surface-binding domains present high variability within the family to provide membrane receptor specificity (Cirauqui et al. 2017). However, the novel concentric double β-barrel structure found in aerolysin is highly conserved in terms of sequence, structure and conformational dynamics, which likely contribute to preserve a common transition mechanism from the prepore to the mature pore within the family. The key role of several amino acids in the conformational changes needed for oligomerization and further pore formation, include Y221, W227, P248, Q263 and L277, which may be involved in the release of the stem loop and the two adjacent β-strands to form the transmembrane β-barrel (Cirauqui et al. 2017).
Membrane binding of the monomeric toxin promotes oligomerization to a stable heptamer (as is known for the homologous α-hemolysin (αHL) family (TC #1.C.3)). Heptamerization converts the protein from a soluble form to a membrane insertion-competent form, and the oligomer penetrates the membrane producing channels that destroy the permeability barrier of the membrane, thereby killing the cell. The membrane-associated channel-forming protein may comprise a β-barrel. The three-dimensional structure of the soluble form of aerolysin from the Gram-negative bacterium, Aeromonas hydrophila, has been determined by x-ray crystallography (2.8 Å resolution) (Parker et al., 1994, 1996). The closely related aerolysins are distantly related to many other toxins including the α-toxin of the Gram-positive bacterium, Clostridium septicum, enterolobin, a cytolysin of the plant, Enterolobium contortisiliquum, the ε-toxin of Clostridium perfringens (1.C.5.1.1), and the α-hemolysin of Staphylococcus aureus (1.C.3.1.1). Members of the aerolysin family are therefore found in both bacteria and eukaryotes.
Hydralysins (1.C.4.2.1) are β pore-forming toxins in cnidaria, venomous animals such as Hydra vulgaris, and Chlorohydra viridissima (Sher et al., 2005). The soluble monomers are rich in β-structure and bind to erythrocyte membranes to form pores with an inner diameter of about 1.2 nm (Sher et al., 2005). Cytolysis is cell type-specific, suggesting the involvement of specific receptors. These toxins share some motif similarity around the pore-forming domains of the toxins. They induce immediate fast muscle contraction followed by flaccid paralysis when injected into blowfly larvae (Zhang et al., 2003). They have strong hemolytic activity against certain insect cells. Other toxins, including the pore-forming actinoporins, but not hydralysins, are stored in sting cells called nematocytes.
The binary toxin (Bin), produced by Lysinibacillus (Bacillus) sphaericus, is composed of BinA (42 kDa) and BinB (51 kDa) proteins, which are both required for full toxicity against Culex and Anopheles mosquito larvae. Specificity of Bin toxin is determined by the binding of BinB to a receptor present on the midgut epithelial membranes, while BinA is proposed to be a toxic component. Srisucharitpanit et al. 2014 determined the crystal structure of the active form of BinB at a resolution of 1.75 A. It possesses two distinct structural domains in its N- and C-termini. The globular N-terminal domain has a beta-trefoil scaffold which is a highly conserved architecture of some sugar binding lectins, suggesting a role of this domain in receptor-binding. The BinB beta-rich C-terminal domain shares similar three-dimensional folding with aerolysin type beta-pore forming toxins, despite a low sequence identity. The BinB structure, therefore, is a new member of the aerolysin-like toxin family, with probably similarities in the cytolytic mechanism that takes place via pore formation.
The generalized transport reaction catalyzed by members of the aerolysin family is:
Small molecules (in) small molecules (out)
Aerolysin (β-hemolysin; cytolytic enterotoxin) precursor (Parker et al., 1994). Upon transition from the prepore to pore, the aerolysin heptamer shows a unique concerted swirling movement, accompanied by a vertical collapse of the complex, ultimately leading to the insertion of a transmembrane beta-barrel (Degiacomi et al. 2013). Multiple conformational states lead to rotation of the core lysin to unleash the membrane spanning regions (Whisstock and Dunstone 2013). Monomer activation, dependent on proteolysis, is the rate-limiting step for pore formation (Bischofberger et al. 2016). Cryo-electron microscopy structures of three conformational intermediates and the final aerolysin pore provide insight into the conformational changes that allow pore formation. The structures reveal a protein fold consisting of two concentric beta-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry (Iacovache et al. 2016).
Gram-negative bacteria of the Aeromonas family
Aerolysin precursor of Aeromonas hydrophila
α-toxin forms large ion permeable (slightly anion-selective) pores with no lipid specificity. It induces rapid cell necrosis in many cell types (Knapp et al., 2009).
α-toxin of Clostridium septicum (BAC54147)
Hydralysin (Sher et al., 2005; Zhang et al., 2003). Hydrolysins comprise a family of pore-forming proteins that are secreted into the gastrovascular cavity during feeding, probably helping in disintegration of the prey (Sher and Zlotkin 2009). Induces an immediate fast muscle contraction followed by flaccid paralysis when injected into blowfly larvae. The paralytic effect is lower in mice and fish. Has strong cytolytic activity against insect Sf9 cells and human HeLa cells. Binds to erythrocyte membranes and has weak hemolytic activity by mediating oligomerization and pore formation (Zhang et al. 2003; Sher et al. 2008).
Hydralysin of Hydra viridis (Q86LR2)
Hemolytic lectin LSLc exhibits hemolytic and hemagglutinating activities. The structure at 2.6 Å resolution has been determined (Mancheño et al., 2005). The protein is hexameric. The monomer (35kDa) consists of two distinct modules: an N-terminal lectin module (a β-trefoil scaffold) and a pore-forming module (composed of domains 2 and 4) which resemble the β-pore-forming domains of aerolysin and ε-toxin (Mancheño et al., 2005).
LSLc of Laetiporus sulphureus (BAC78490)
Parasporin-2 β-toxin (crystal structures are known) (Akiba et al., 2009; Akiba and Okumura 2016).
Paraspora-2 of Bacillus thuringiensis (Q7WZI1)
Fhb1 protein (PFT gene product) of 478 aas with two agglutinin domains followed by a DON (ETX/MTX2) domain that has the toxin activity (Rawat et al. 2016). Counteracts Fusarium head blight (FHB), caused by Fusarium graminearum, a devastating disease of wheat and barley.
Fhb1 of Triticum aestivum
Amaranthin agglutinin of 304 aas and 0 TMSs. The x-ray structure at 2.2 Å resolution of the homodimeric protein is available (1JLX) (Transue et al. 1997) Sequences containing amaranthin domains are widely distributed in plants (Dang et al. 2017).
Amaranthin agglutinin of Amaranthus caudatus (Love-lies-bleeding) (Inca-wheat)
Natterin-like precursor of 315 aas from zebra fish, Dln1 or Aep1. Aep1 is an innate immune molecule that prevents zebrafish from bacterial infections. Thus, Aep1 may be a pro-inflammatory protein that triggers the antimicrobial immune responses (Chen et al. 2018).
Natterin-like protein of Danio rerio
The Bin binary toxin, BinAB. BinA is a toxic P42 protein (protein of 42 KDa) of 362 aas. The 3-d structure of BinB (448 aas; 1.75 Å resolution) is available; it has two domains, an N-terminal sugar-binding lectin-like domain, and a C-terminal aerolysin-like β-barrel pore-forming domain. Although it shows low sequence identity with other members of the family, it is a member of the Aerolysin Family (Srisucharitpanit et al. 2014). Protoxin subunits only form monomers, but in vitro activated toxin forms heterodimers. Maximal toxicity to mosquito larvae is achieved when the two subunits, BinA and BinB, are in a 1:1 molar ratio (Surya et al. 2016). An aromatic residue cluster in the C-terminal domain of BinB is critical for toxin insertion in membranes (Chooduang et al. 2018).
BinAB of Lysinibacillus (Bacillus) sphaericus
Cry35 of 385 aas. Shares a common strucure with ε-toxin, ETX (Moar et al. 2016).
Cry35 of Bacillus thuringiensis