1.C.3 The α-Hemolysin Channel-forming Toxin (αHL) Family
The α-hemolysin (αHL; α-toxin, alpha-toxin) of the human pathogen Staphylococcus aureus is secreted as a 33 kDa monomer. This monomeric species associates with animal cell membranes to form a 232 kDa homoheptameric transmembrane β-barrel pore that promotes cell death by allowing bilayer permeability to ions, water and small solutes, thereby promoting cell lysis. The three-dimensional structure of αHL has been solved by x-ray crystallography to 1.9 Å resolution (Song et al., 1996). Imaging αHL with molecular dynamics has provided information about its ionic conductance and osmotic permeability (Aksimentiev and Schulten 2005). αHL forms a solvent-filled channel with a length of 100 Å, that runs along the seven-fold axis of the protein and ranges from 14 to 46 Å in diameter. The transmembrane domain of the mushroom-shaped heptamer is the lower portion of the mushroom, consisting of a 14-strand antiparallel β-barrel to which each protomer contributes two α-strands, each 65 Å long. The interior of the β-barrel is primarily hydrophilic, and the exterior has a hydrophobic belt 28 Å wide. The pore can transport peptides, and charged residues in the pore influences the rate of passage (Wolfe et al., 2007; Hammerstein et al., 2011). αHL has been used to create a system with directional control of a processive molecular hopper (Qing et al. 2018). Sphingomyelin depletion from the plasma membranes of human airway epithelial cells completely abrogates the deleterious actions of alpha-toxin (Ziesemer et al. 2019). The beta-barrel pore-formation mechanism has been reviewed (Mondal and Chattopadhyay 2019). Chimeric mutants of staphylococcal hemolysin, which act as both one-component and two-component hemolysins, have been created by grafting the stem domain (Ghanem et al. 2022).
Several Staphylococcal toxins of this family are two-component cytolysins. These toxins include α-hemolysin (Hlg: Hlg1 (LukF) Hlg2), leukocidin (Luk: LukF LukS) and Pantone-Valentine leukocidin (Luk-PV: LukF-PV LukS-PV). They have 7 subunits arranged in a ring with alternate subunit arrangements (subunit stoichiometries of 3:4 or 4:3 (Sugawara-Tomita et al., 2002). Each toxin has specificity for different mammalian cell types and hosts. LukF (also called Hlg1) and LukF-PV comprise class F while Hlg2, LukS and LukS-PV comprise class S. Proteins of each class are 70% identical, but the proteins are about 30% identical between classes. They are 20-30% identical to the single component Staphylococcal α-hemolysin described above. Because the two components (LukS and LukF) comprise two distinct subfamilies of the αHL family, they are listed under two different TC numbers (#1.C.3.3.1 and #1.C.3.4.1).
The αHL family consists of pore-forming toxins from Staphylococcal species, Bacillus cereus, B. anthracis and Clostridium perfringens. They are distantly related to the CHL family of pentameric toxins from Gram-negative bacteria (TC #1.C.14). The S. aureus protein monomers are 308-326 residues in length, while the B. cereus protein is of 412 residues and the C. perfringens monomers is of 336 residues. α-HL forms two subpopulations of ion conducting channels. Furini et al. (2008) provided evidence that this toxin can form both hexameric and heptameric pores, accounting for the ion conuctance results.
The phylogenetic tree for the αHL family reveals four clusters (Saier et al., 1999). The Staphylococcus α-hemolysin for which the three-dimensional structure is available comprises one branch, the B. cereus and C. perfringens proteins comprise a second, and all other members of the family fall into the remaining two clusters.
α-haemotoxins are members of the αHL family that form heterooligomeric (bicomponent) toxins (HlgA · HlgB or HlgB · HlgC). Both form pores in lipid membranes with conductances, current-voltage characeristics and stability properties similar to α-toxin. However, they are cation selective rather than anion selective. There is a conserved region at the pore entrance with four basic residues in α-toxin but either basic or acidic residues in α-haemolysins. These residues form an entrance electrostatic filter (Comai et al. 2002). A variety of peptides interact with αHL (Movileanu et al. 2005).
Cracknell et al. (2013) described the translocation of ssRNA heteropolymers (91-6083 bases) through the α-hemolysin nanopore. Translocation of these long ssRNAs is characterized by surprisingly long, almost complete ionic current blockades with durations averaging milliseconds per base (at +180 mV). The event durations decrease exponentially with increased transmembrane potential but are largely unaffected by the presence of urea. When the ssRNA is coupled at the 3' end to streptavidin, which cannot translocate through the pore, permanent blockades are observed, supporting the conclusion that the transient blockage of current arises from ssRNA translocation. Asandei et al. 2016 have described the dynamics of a single peptide as it passes across a voltage-biased alpha-hemolysin nanopore.
PLEKHA7 and other junctional proteins are host factors mediating death by S. aureus alpha-toxin. ADAM10 is docked to junctions by its transmembrane partner Tspan33, whose cytoplasmic C-terminus binds to the WW domain of PLEKHA7 in the presence of PDZD11. ADAM10 is locked at junctions through binding of its cytoplasmic C terminus to afadin. Junctionally clustered ADAM10 supports the efficient formation of stable toxin pores. Disruption of the PLEKHA7-PDZD11 complex inhibits ADAM10 and toxin junctional clustering. This promotes toxin pore removal from the cell surface through an actin- and macropinocytosis-dependent process, resulting in cell recovery from initial injury and survival. Thus, a dock-and-lock molecular mechanism targets ADAM10 to junctions, providing a paradigm for how junctions may regulate transmembrane receptors through their clustering (Shah et al. 2018).
The generalized transport reaction catalyzed by these pore-forming toxins is:
Small molecules (in) Small molecules (out)
α-Hemolysin (alpha haemolysin; Hly; Hla; α-toxin). Fragments (13-293 aas) form heptamers like the native full length protein, but a fragment with aas 72-293 formed heptamers, octamers and nonamers. All formed Cl- permeable β-barrel channels (Vécsey-Semjén et al., 2010). The 3-d structure is available (PDB#7AHL). Both symmetry and size of cyclodextrin inhibitors and the toxin pore are important for effective inhibition (Yannakopoulou et al., 2011). Oxoxylin A inhibits hemolysis by hindering self assembly of the hepatmeric pore in which two β-strands are contributed by each subunit (Song et al. 1996; Dong et al. 2013). Applications of pore-forming α-haemolysin include small- and macromolecule-sensing, targeted cancer therapy, and drug delivery (Gurnev and Nestorovich 2014). Sugawara et al. 2015 studied pore formation. Structural comparisons among monomer, prepore and pore revealed a series of motions in which the N-terminal amino latch released upon oligomerization destroys its own key hydrogen bond betweem Asp45 and Try118. This action initiates the protrusion of the prestem. A Y118F mutant and the N-terminal truncated mutant markedly decreased the hemolytic activity, indicating the importance of the key hydrogen bond and the N-terminal amino latch for pore formation. A dynamic molecular mechanism of pore formation was proposed (Sugawara et al. 2015). Release of ATP from cells may occur directly through transmembrane pores formed by α-toxin (Baaske et al. 2016). The amino latch of staphylococcal alpha-hemolysin functions in pore formation via an co-operative interaction between the N terminus and position 217 (Jayasinghe et al. 2006).
PLEKHA7 and other junctional proteins are host factors mediating death by S. aureus alpha-toxin. ADAM10 is docked to junctions by its transmembrane partner Tspan33, whose cytoplasmic C-terminus binds to the WW domain of PLEKHA7 in the presence of PDZD11. ADAM10 is locked at junctions through binding of its cytoplasmic C terminus to afadin. Junctionally clustered ADAM10 supports the efficient formation of stable toxin pores. Disruption of the PLEKHA7-PDZD11 complex inhibits ADAM10 and toxin junctional clustering. This promotes toxin pore removal from the cell surface through an actin- and macropinocytosis-dependent process, resulting in cell recovery from initial injury and survival. Thus, a dock-and-lock molecular mechanism targets ADAM10 to junctions, providing a paradigm for how junctions may regulate transmembrane receptors through their clustering (Shah et al. 2018). Airway epithelial cells are sensitivity to S. aureus α-Toxin, but the toxin heptamers are removed by extracellular vesicle formation and lysosomal degradation (Möller et al. 2021). The effect of electroosmotic solvent flow on the binding of a neutral molecule [beta-cyclodextrin (betaCD)] to sites within alpha-hemolysin pore was investigated. Mutant α-hemolysin pores were used to which betaCD can bind from either entrance and through which the direction of water flow can be controlled by choosing the charge selectivity of the pore and the polarity of the applied potential. The Kd values for betaCD for individual mutant pores varied by >100-fold with the applied potential over a range of -120 to +120 mV (Gu et al. 2003). Alpha-hemolysin can be incorporated into bicelles (Dziubak and Sęk 2023). It exhibits long-term memory with respect to ion channel kinetics (Silva et al. 2023).
α-hemolysin of Staphylococcus aureus
Hemolysin II of Bacillus cereus
Cytotoxin of 336 aas and 1 N-terminal TMS.
Cytotoxin CytK of Bacillus cereus
Necrotic enteritis toxin B precursor, NetB (Keyburn et al., 2008)
CctA (Clostridium chauvoei toxin A; 317 aas) is the main cytotoxic and haemolytic substance secreted by C. chauvoei. Vaccination of guinea pigs with CctA in the form of a fusion protein with the E. coli heat labile toxin B subunit (rCctA::LTB) as a peptide adjuvant protected the animals against challenge with spores of virulent C. chauvoei., (Frey et al. 2012).
Cytotoxin of Clostridium chauvoei
Necrotizing enteritis toxin, NetF, of 305 aas. NetF-producing type A Clostridium perfringens is an important cause of canine and foal necrotizing enteritis. NetF, related to the β-sheet pore-forming Leukocidin/Hemolysin superfamily, is considered a major virulence factor for this disease. The NetF receptor is probably a sialic acid-containing glycoprotein (Mehdizadeh Gohari et al. 2018).
NetF of Clostridium perfringens
Leucocidin/Hemolysin toxin family member. 90% identical to a Leukocidin of Vibrio proteolyticus of 305 aas that plays an important role in virulence (Ray et al. 2016).
V12G01_16082 of Vibrio alginolyticus (Q1V718)
Leucocidin chain F. 3-D structures of the prepore revealed that this is substantially different from the pore structure. The structures revealed a disordered bottom half of the beta-barrel corresponding to the transmembrane region, and a rigid upper extramembrane half (Yamashita et al. 2014). LukF can form an octameric pore with 4 subunits of LukF and 4 subunits of LukS (TC# 1.C.3.4.3) (Jayasinghe and Bayley 2005). Panton-Valentine leukocidin (PVL, encoded by lukSF-PV genes) is a bi-component and pore-forming toxin carried by different staphylococcal bacteriophages (Zhao et al. 2016). The gamma-hemolysin protein is used by the pathogen to escape the immune system of the host, by assembling into octameric transmembrane pores on the surface of the target immune cell, leading to its death by leakage or apoptosis. The interactions between the individual monomers that lead to the formation of a dimer on the cell membrane, which represents the unit for further oligomerization, has not been defined. Paternoster et al. 2023 determined the stabilizing contacts that guide formation of a functional dimer.
Leucocidin chain F (LukF) of Staphylococcus aureus (Q53747)
Two component β-barrel γ-haemolysin, HlgA·HlgB. Tomita et al. (2011) reported that Hlg2 and LukF form a complex, and that Hlg pores form clusters that release hemoglobin from erythrocytes. The crystal structure of this octameric pore (PDB# 3B07; 2QK7) reveals the beta-barrel pore formation mechanism by the two components (Yamashita et al., 2011). Dominant-negative mutant toxins may provide novel therapeutics to combat S. aureus infection (Reyes-Robles et al. 2016). S. aureus beta-barrel pore-forming cytotoxins, including the identification of the toxin receptors on host cells, and their roles in pathogenesis have been reviewed (Reyes-Robles and Torres 2016).
HlgA·HlgB of Staphylococcus aureus
Two component β-barrel γ-haemolysin, HlgC·HlgB. HglC is identical to Leucocidin chain S (LukS) (P31716), and HlgB is identical to the HlgB protein listed under TC# 1.C.3.4.2 (Roblin et al. 2008). The pore-forming regions are initially folded up on the surfaces of the soluble precursors. To create the transmembrane pores, these regions must extend and refold into membrane-inserted beta-barrels (Tilley and Saibil 2006).
HlgC·HlgB of Staphylococcus aureus
Equid-adapted leukocidin PQ, LukPQ, of 311 (LukP) and 326 aas (LukQ), respectively (Koop et al. 2017).
LukPQ of Staphylococcus aureus
Beta-channel-forming cytolysin, the synergohymenotropic toxin, of 310 aas. Bacterial infections from Staphylococcus pseudintermedius are the most common cause of skin infections (pyoderma) affecting dogs. Two component pore-forming leukocidins are a family of potent toxins secreted by staphylococci and consist of S (slow) and F (fast) components. They impair the innate immune system, the first line of defense against these pathogens. Seven different leukocidins have been characterized in Staphylococcus aureus, some of which are host and cell specific. Abouelkhair et al. 2018 identified two proteins, named "LukS-I" and "LukF-I", encoded on a degenerate prophage contained in the genome of S. pseudintermedius isolates. The killing effect of recombinant S. pseudintermedius LukS-I together with LukF-I on canine polymorphonuclear leukocytes depended on both constituents of the two-component pore-forming leukocidin.
LukS-I/LukF-I of Staphylococcus pseudintermedius
Beta-channel forming cytolysin, LukNF (HlyII, hlgB, lukD, lukDv) of 327 aas. Geraniol had the highest ligand efficiency and was the most potent phyto-constituent interacting with the HlyII cytotoxin (Mohapatra et al. 2021).
LukNF of Staphylococcus aureus
Leukotoxin domain protein B (plasmid) of 329 aas and 1 N-terminal TMS.
Leukotoxin of Clostridium perfringens
Prostaglandin-H2 D-isomerase, PTGDS (PDS) of 190 aas and 1 N-terminal TMS. It regulates the calcium channel forming VIC family member with TC# 1.A.1.11.9 (Gomez et al. 2023). It catalyzes the conversion of PGH2 to PGD2, a prostaglandin involved in smooth muscle contraction/relaxation and a potent inhibitor of platelet aggregation (Zhou et al. 2010). It is also involved in a variety of CNS functions, such as sedation, NREM sleep and PGE2-induced allodynia, and may have an anti-apoptotic role in oligodendrocytes. Binds small non-substrate lipophilic molecules, including biliverdin, bilirubin, retinal, retinoic acid and thyroid hormone, and may act as a scavenger for harmful hydrophobic molecules and as a secretory retinoid and thyroid hormone transporter.
PTGDS of Homo sapiens