1.C.97 The Pleurotolysin Pore-Forming (Pleurotolysin) Family
Pleurotolysin, a sphingomyelin-specific cytolysin consisting of A (17 kDa) and B (59 kDa) components from the basidiomycete Pleurotus ostreatus, assembles into a transmembrane pore complex. Sakurai et al. 2004 cloned complementary and genomic DNAs encoding pleurotolysin, and studied pore-forming properties of recombinant proteins. Recombinant pleurotolysin A lacking the first methionine was purified as a 17-kDa protein with sphingomyelin-binding activity. The cDNA for pleurotolysin B encoded a precursor consisting of 523 amino acid residues, of which N-terminal 48 amino acid residues were absent in natural pleurotolysin B. Mature and precursor forms of pleurotolysin B were expressed as insoluble 59- and 63-kDa proteins, respectively. Although neither recombinant pleurotolysin A nor B alone was hemolytically active at higher concentrations of up to 100 mg/ml, they cooperatively assembled into a membrane pore complex on human erythrocytes and lysed the cell.
Tomita et al. 2004 purified pleurotolysin, a sphingomyelin-specific two-component cytolysin from the basidiocarps of Pleurotus ostreatus and studied pore-formation. Pleurotolysin caused leakage of potassium ions from human erythrocytes and formed membrane pores with a functional diameter of 4-5 nm. Pleurotolysin-induced lysis of human erythrocytes was inhibited by the addition of sphingomyelin-cholesterol liposomes to the extracellular space. Pleurotolysin A specifically bound to sphingomyelin-cholesterol liposomes and caused leakage of the internal carboxyfluorescein in concert with pleurotolysin B. Pleurotolysin A and B bound to human erythrocytes in this sequence and assembled into an SDS-stable, 700-kDa complex. Ring-shaped structures with outer and inner diameters of 14 and 7 nm, respectively, were isolated from the solubilized erythrocyte membranes.
Another two-component hemolysin, erylysin A and B (EryA and EryB), was isolated from an edible mushroom, Pleurotus eryngii (Shibata et al. 2010). Hemolytic activity was exhibited only by the EryA and EryB mixture. EryA showed one band on SDS-PAGE while EryB showed two bands at 15 kDa (EryB1) and 37 kDa (EryB2). At pH 7.2, EryA exists as a homodimer whereas EryB exists as a heterodimer of B1 and B2. CD spectrum analysis showed T(m) values of 47°C and 37°C for EryA and EryB, respectively. EryB was particularly unstable.
While Pleurotolysin B is in the MACPF superfamily (1.C.39), Pleurotolysin A is in the Aegerolysin superfamily. Several members of the Aegerolysin family have been used as tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates but not in animals (Bhat et al. 2015). It may be distantly related to members of the Equinatoxin Family (1.C.38).
Proteins with membrane-attack complex/perforin (MACPF) domains have a variety of biological roles, including defence and attack, organismal development, and cell adhesion and signalling. The distribution of these proteins in fungi appears to be restricted to some Pezizomycotina and Basidiomycota species only, in correlation with the aegerolysins. These two protein groups coincide in only a few species, and they operate as cytolytic bi-component pore-forming agents (Ota et al. 2014). Representative proteins include pleurotolysin B, which has a MACPF domain, and the aegerolysin-like protein pleurotolysin A, and the very similar ostreolysin A, which have been purified from oyster mushroom (Pleurotus ostreatus). These act in concert to perforate natural and artificial lipid membranes with high cholesterol and sphingomyelin contents. The complex has a 13-meric rosette-like structure with a central lumen that is ~4-5 nm in diameter. The opened transmembrane pore is non-selectively permeable to ions and smaller neutral solutes, and is a cause of cytolysis of a colloid-osmotic type. The biological significance of these proteins for the fungal life-style has been discussed (Ota et al. 2014).
The aegerolysin family consists of several bacterial and eukaryotic aegerolysin-like proteins. It has been found that aegerolysin and ostreolysin are expressed during formation of primordia and fruiting bodies and possibly play a role in the initial phase of fungal fruiting. The bacterial members of this family are expressed during sporulation. Ostreolysin is cytolytic to various erythrocytes and tumor cells because of pore formation (Berne et al. 2002; Berne et al. 2002; Berne et al. 2009).
In this TC family, both constituents of pleurotolysin and ostreolysin (A and B) are included under TC#s 1.C.97.1.1 and 1.2, respectively. However, homologues of Pleurotolysin B are found under TC #s 1.C.97.1.3 - 1.9 while homologues of Pleurotolysin A are found under TC #s 1/C/97.2.1 - 2.4 and 3.1 - 3.8. Pleurotolysins A are not homologous to Pleurotolysins B. While some homologues depend on the presence of both constituents for pore formation, as noted for both pleurotolysin and ostreolysin, some homologues of both A and B can form pores without the other.
Aegerolysin proteins, ostreolysin A6 (OlyA6), pleurotolysin A2 (PlyA2) and erylysin A (EryA), produced by the mushroom genus Pleurotus bind strongly to an invertebrate-specific membrane sphingolipid, and often together with a protein partner pleurotolysin B (PlyB), form transmembrane pore complexes. Pore formation is the basis for the selective insecticidal activity of aegerolysin/PlyB complexes against two economically important coleopteran pests: the Colorado potato beetle and the western corn rootworm. Panevska et al. 2021 evaluated the toxicities of these aegerolysin/PlyB complexes using feeding tests with two ecologically important non-target arthropod species: the woodlouse and the honey bee. The mammalian toxicity of the EryA/PlyB complex was also evaluated after intravenous administration to mice. None of the aegerolysin/PlyB complexes were toxic against woodlice, but OlyA6/PlyB and PlyA2/PlyB were toxic to honeybees, with 48 h mean lethal concentrations (LC50) of 0.22 and 0.39 mg/mL, respectively, in their food. EryA/PlyB was also tested intravenously in mice up to 3 mg/kg body mass, without showing toxicity. With no toxicity seen for EryA/PlyB for environmentally beneficial arthropods and mammals at the tested concentrations, these EryA/PlyB complexes may be used for the development of new bioinsecticides for control of selected coleopteran pests (Panevska et al. 2021).
Pleurotolysin A/B pore-forming toxin. Pleurotolysin A (PlyA; also called ostreolysin A, OlyA) binds first in a sphingomyelin-dependent process; Pleurotolysin B (PlyS) binds to A in the membrane and inserts (Kondos et al., 2011). The binary cytolytic pore-forming complex forms non-selective ion conducting pores of variable size (Schlumberger et al. 2013) to promote fruiting (Ota et al. 2014). Conformational changes accompanying pore formation have been reported (Lukoyanova et al. 2015). In these systems, the aegerolysin-like proteins provide the membrane cholesterol/sphingomyelin selectivity and recruit oligomerised pleurotolysin B molecules, to create a membrane-inserted pore complex. The resulting protein structure has been imaged with electron microscopy, and it has a 13-meric rosette-like structure, with a central lumen that is ~4-5 nm in diameter. The opened transmembrane pore is non-selectively permeable for ions and smaller neutral solutes, and is a cause of cytolysis of a colloid-osmotic type (Ota et al. 2014). Ostreolysin A6 (OlyA6) is a protein produced by the oyster mushroom (Pleurotus ostreatus). It binds to membrane sphingomyelin/cholesterol domains, and together with its protein partner, pleurotolysin B (PlyB), it forms 13-meric transmembrane pore complexes. OlyA6 binds 1000 times more strongly to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with PlyB, OlyA6 has potent and selective insecticidal activity against the western corn rootworm. Milijaš Jotić et al. 2021 analysed the histological alterations of the midgut wall columnar epithelium of western corn rootworm larvae fed with OlyA6/PlyB, which showed vacuolisation of the cell cytoplasm, swelling of the apical cell surface into the gut lumen, and delamination of the basal lamina underlying the epithelium. Cryo-EM was used to explore the membrane interactions of the OlyA6/PlyB complex using lipid vesicles composed of artificial lipids containing CPE, and western corn rootworm brush border membrane vesicles. Multimeric transmembrane pores were formed in both vesicle preparations, similar to those described for sphingomyelin/cholesterol membranes. Thus, the molecular mechanism of insecticidal action of OlyA6/PlyB arises from specific interactions of OlyA6 with CPE, and the consequent formation of transmembrane pores in the insect midgut (Milijaš Jotić et al. 2021).
Pleurotolysin A/B of Pleurotus ostreatus
Pleurotolysin A (138aas) (Q8X1M9)
Pleurotolysin B precursor (Q5W9E8)
Erylysin A/B pore-forming toxin (Shibata et al., 2010). Erylysin B is 96% identical to Pleurotolysin B)
Erylysin A/B of Pleurotus eryngii
Erylysin A (138aas) (D0FZZ2)
Erylysin B (522aas) (D0FZZ3)
MACPF protein homologue of Erylysin B
MACPF protein of Dictyostelium discoideum (Q54I05)
Hypothetical protein homologous to Erylysin B of 924aas with a MACPF domain.
Hypothetical protein of Chaetomium globosum (Q2GRU1)
Uncharacterized protein of 557 aas
UP of Aspergillus oryzae
Uncharacterized protein homologous to Erylysin B (892aas) Residues 85-337aas of Erylysin B align with residues 242-477 of the Chlorobium sequence.
Hypothetical protein of Chlorobium limicola (B3EDT0)
Uncharacterized protein of 1165 aas
UP of Tistrella mobilis
Uncharacterized protein of 800 aas with an N-terminal 280 aa soluble PQ-rich domain that shows similarity with TC# 1.B.27.1.9 and a C-terminal domain rich in predicted β-structure that shows similarity to Pleurotolysin snf its homologues.
UP of Hypocrea virens (Gliocladium virens) (Trichoderma virens)
Uncharacterized protein of 668 aas
UP of Piriformospora indica
Putative hemolysin of 224 aas
Putative hemolysin of Ajellomyces dermatitidis (Blastomyces dermatitidis)
Uncharacterized protein of 282 aas
UP of Coccidioides posadasii (Valley fever fungus)
Uncharacterized protein of 244 aas
UP of Ajellomyces capsulatus (Darling's disease fungus) (Histoplasma capsulatum)
Uncharacterized protein of 190 aas
UP of Cyphellophora europaea
Hemolysin of 198 aas
Hemolysin of Neurospora crassa
Pore-forming ostreolysin (Berne et al., 2005). 97% identical to Erylysin A (1.C.97.1.2).
Ostreolysin of Pleurotus osteatus (P83467)
Aegerolysin (135aas) (Homologous to Pleurotolysin A). Aegerolysin-like proteins provide the membrane cholesterol/sphingomyelin selectivity and recruit oligomerized pleurotolysin B molecules to create a membrane-inserted pore complex (Ota et al. 2014).
Aegerolysin of Spirosoma linguale (D2QTE8)
Aegerolysin (121aas) (Homologous to Pleurotolysin A)
Aegerolysin of Pseudomonas aeruginosa (A6UXQ8)
Putative hemolysin of 140 aas
Hemolysin of Penicillium oxalicum (Penicillium decumbens)
Putative hemolysin of 199 aas
Putative hemolysin of Ajellomyces capsulatus (Darling's disease fungus) (Histoplasma capsulatum)
Putative hemolysin of 222 aas
Putative hemolysin of Trichoplusia ni ascovirus 2c
Putative hemolysin of 204 aas
Putative hemolysin of Selaginella moellendorffii (Spikemoss)