Ostreolysin A6 (OlyA6) is a protein produced by the oyster mushroom (
). It binds to membrane sphingomyelin/cholesterol domains, and together with its protein partner, pleurotolysin B (PlyB), it ...forms 13-meric transmembrane pore complexes. Further, 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. We 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. Additionally, cryo-electron microscopy 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. These results strongly suggest that 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.
Fungi are the most common pathogens of insects and thus important regulators of their populations. Lipid-binding aegerolysin proteins, which are commonly found in the fungal kingdom, may be involved ...in several biologically relevant processes including attack and defense against other organisms. Aegerolysins act alone or together with membrane-attack-complex/perforin (MACPF)-like proteins to form transmembrane pores that lead to cell lysis. We performed an in-depth bioinformatics analysis of aegerolysins in entomopathogenic fungi and selected a candidate aegerolysin, beauveriolysin A (BlyA) from
. BlyA was expressed as a recombinant protein in
, and purified to further determine its functional and structural properties, including lipid-binding ability. Aegerolysins were found to be encoded in genomes of entomopathogenic fungi, such as
,
,
and
. Detailed bioinformatics analysis revealed that they are linked to MACPF-like genes in most genomes. We also show that BlyA interacts with an insect-specific membrane lipid. These results were placed in the context of other fungal and bacterial aegerolysins and their partner proteins. We believe that aegerolysins play a role in promoting the entomopathogenic and antagonistic activity of
, which is an active ingredient of bioinsecticides.
Aegerolysin proteins ostreolysin A6 (OlyA6), pleurotolysin A2 (PlyA2) and erylysin A (EryA) produced by the mushroom genus
bind strongly to an invertebrate-specific membrane sphingolipid, and ...together with a protein partner pleurotolysin B (PlyB), form transmembrane pore complexes. This 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. In this study, we 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 (LC
) 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 are of particular interest for development of new bioinsecticides for control of selected coleopteran pests.
Aegerolysins are small lipid-binding proteins particularly abundant in fungi. Aegerolysins from oyster mushrooms interact with an insect-specific membrane lipid and, together with MACPF proteins ...produced by the same organism, form pesticidal pore-forming complexes. The specific interaction with the same membrane lipid was recently demonstrated for nigerolysin A2 (NigA2), an aegerolysin from
. In
species, the aegerolysins were frequently found as secreted proteins, indicating their function in fungal defense. Using immunocytochemistry and live-cell imaging we investigated the subcellular localization of the nigerolysins A in
, while their secretion was addressed by secretion prediction and Western blotting. We show that both nigerolysins A are leaderless proteins that reach the cell exterior by an unconventional protein secretion. NigA proteins are evenly distributed in the cytoplasm of fungal hyphae. A detailed bioinformatics analysis of
aegerolysins suggests that the same function occurs only in a limited number of aegerolysins. From alignment, analysis of chromosomal loci, orthology, synteny, and phylogeny it follows that the same or a similar function described for pairs of pesticidal proteins of
sp. can be expected in species of the subgenus
, section
, series
, and some other species with adjacent pairs of putative pesticidal proteins.
Chlamydiae are major human pathogens and the etiological agents of several diseases in humans, other mammals and birds. Depending on the serotype, infection with Chlamydiae trachomatis can result in ...trachoma, sexually-transmitted diseases or inclusion conjunctivitis. A second chlamydial species, C. pneumoniae, is associated with upper respiratory tract infections. Following internalisation by eukaryotic cells, chlamydiae are enveloped within membrane-bound compartments, or inclusions, that do not fuse with lysosomes. Multiple chlamydiae in a eukaryotic cell fuse their inclusions into a single large inclusion that contains the infectious but metabolically-inactive elementary body (EB). EBs differentiate into replicative, reticulate bodies (RBs) 6-8 hours postinfection. RBs eventually revert to EBs and are released from the host cell to continue the infectious process. The complete genome sequences of C. trachomatis and C. pneumoniae have been recently determined. After the predicted protein sequences of C. trachomatis were deposited in the database, the web-based annotation resource database SMART (http://coot.emblheidelberg.de/SMART/) automatically predicted that the C. trachomatis hypothetical protein CT153 contains a MACPF domain. This domain is present in mammalian membrane-attack complex and perforin proteins, but had not been previously identified in non-eukaryotic proteins. The associated statistical significance of this prediction (E = 7.5 x 10 super(-3)) was corroborated using PSI-BLAST database searches with an E-value inclusion threshold of 0.001. For example, two rounds of a PSI-BLAST search using the human complement component 9 MACPF domain sequence (amino acid residues 138-503) as the query resulted in identification of the C. trachomatis CT153 protein as significantly similar to this domain with an E-value of 3 x 10 super(-7).
The vertebrate complement cascade is an essential host protection system that functions at the intersection of adaptive and innate immunity. However, it was originally assumed that complement was ...present only in vertebrates because it was activated by antibodies and functioned with adaptive immunity. Subsequently, the identification of the key component, SpC3, in sea urchins plus a wide range of other invertebrates significantly expanded the concepts of how complement functions. Because there are few reports on the echinoid complement system, an alternative approach to identify complement components in echinoderms is to search the deduced proteins encoded in the genomes. This approach identified known and putative members of the lectin and alternative activation pathways, but members of the terminal pathway are absent. Several types of complement receptors are encoded in the genomes. Complement regulatory proteins composed of complement control protein (CCP) modules are identified that may control the activation pathways and the convertases. Other regulatory proteins without CCP modules are also identified, however regulators of the terminal pathway are absent. The expansion of genes encoding proteins with Macpf domains is noteworthy because this domain is a signature of perforin and proteins in the terminal pathway. The results suggest that the major functions of the echinoid complement system are detection of foreign targets by the proteins that initiate the activation pathways resulting in opsonization by SpC3b fragments to augment phagocytosis and destruction of the foreign targets by the immune cells.
•Proteins encoded in the Strongylocentrotus purpuratus genome identifies the lectin and alternative pathways of complement.•Associated serine proteases of the lectin pathway are absent in echinoids but identified in asteroids.•The terminal pathway is missing and may be replaced by an expansion of Macpf-containing proteins.•Many proteins with multiple CCP modules may have regulatory functions.•The major activity of the complement system in echinoids may be opsonization.
PDF
