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Aegerolysins are a family of proteins that recognize and bind to specific membrane lipids or lipid domains; hence they can be used as membrane lipid sensors. Although aegerolysins are distributed throughout the tree of life, the most studied are those produced by the fungal genus Pleurotus. Most of the aegerolysin-producing mushrooms code also for proteins containing the membrane attack complex/perforin (MACPF)-domain. The combinations of lipid-sensing aegerolysins and MACPF protein partners are lytic for cells harboring the aegerolysin membrane lipid receptor and can be used as ecologically friendly bioinsecticides. In this work, we have recombinantly expressed four novel aegerolysin/MACPF protein pairs from the mushrooms Heterobasidion irregulare, Trametes versicolor, Mucidula mucida, and Lepista nuda, and compared these proteins with the already studied aegerolysin/MACPF protein pair ostreolysin A6–pleurotolysin B from P. ostreatus. We show here that most of these new mushroom proteins can form active aegerolysin/MACPF cytolytic complexes upon aegerolysin binding to membrane sphingolipids. We further disclose that these mushroom aegerolysins bind also to selected glycerophospholipids, in particular to phosphatidic acid and cardiolipin; however, these interactions with glycerophospholipids do not lead to pore formation. Our results indicate that selected mushroom aegerolysins show potential as new molecular biosensors for labelling phosphatidic acid. [ABSTRACT FROM AUTHOR]

The intricate voltage regulation presented by lysenin channels reconstituted in artificial lipid membranes leads to a strong hysteresis in conductance, bistability, and memory. Prior investigations on lysenin channels indicate that the hysteresis is modulated by multivalent cations which are also capable of eliciting single-step conformational changes and transitions to stable closed or sub-conducting states. However, the influence on voltage regulation of Cu2+ ions, capable of completely closing the lysenin channels in a two-step process, was not sufficiently addressed. In this respect, we employed electrophysiology approaches to investigate the response of lysenin channels to variable voltage stimuli in the presence of small concentrations of Cu2+ ions. Our experimental results showed that the hysteretic behavior, recorded in response to variable voltage ramps, is accentuated in the presence of Cu2+ ions. Using simultaneous AC/DC stimulation, we were able to determine that Cu2+ prevents the reopening of channels previously closed by depolarizing potentials and the channels remain in the closed state even in the absence of a transmembrane voltage. In addition, we showed that Cu2+ addition reinstates the voltage gating and hysteretic behavior of lysenin channels reconstituted in neutral lipid membranes in which lysenin channels lose their voltage-regulating properties. In the presence of Cu2+ ions, lysenin not only regained the voltage gating but also behaved like a long-term molecular memory controlled by electrical potentials. [ABSTRACT FROM AUTHOR]

We exploit single‐molecule tracking and optical single channel recording in droplet interface bilayers to resolve the assembly pathway and pore formation of the archetypical cholesterol‐dependent cytolysin nanopore, Perfringolysin O. We follow the stoichiometry and diffusion of Perfringolysin O complexes during assembly with 60 ms temporal resolution and 20 nm spatial precision. Our results suggest individual nascent complexes can insert into the lipid membrane where they continue active assembly. Overall, these data support a model of stepwise irreversible assembly dominated by monomer addition, but with infrequent assembly from larger partial complexes. [ABSTRACT FROM AUTHOR]

Perforin‐2 (PFN2, MPEG1) is a key pore‐forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane‐bound pre‐pore complex that converts to a pore‐forming structure upon acidification; but its mechanism of conformational transition has been debated. Here we used cryo‐electron microscopy, tomography and subtomogram averaging to determine structures of PFN2 in pre‐pore and pore conformations in isolation and bound to liposomes. In isolation and upon acidification, the pre‐assembled complete pre‐pore rings convert to pores in both flat ring and twisted conformations. On membranes, in situ assembled PFN2 pre‐pores display various degrees of completeness; whereas PFN2 pores are mainly incomplete arc structures that follow the same subunit packing arrangements as found in isolation. Both assemblies on membranes use their P2 β‐hairpin for binding to the lipid membrane surface. Overall, these structural snapshots suggest a molecular mechanism for PFN2 pre‐pore to pore transition on a targeted membrane, potentially using the twisted pore as an intermediate or alternative state to the flat conformation, with the capacity to cause bilayer distortion during membrane insertion. Synopsis: Perforin‐2 is an innate immune effector protein that forms pores in the membranes of phagocytosed bacteria. Here, cryo‐EM imaging of pre‐pore and pore complexes of perforin‐2 in isolation and after self‐assembly on membranes reveal new insights into membrane‐binding and pore formation. Pre‐pore complexes formed on membranes bind in a distinct conformation to those observed when complexes form in isolation, before membrane binding.Two types of pore structure can assemble: a closed flat ring and a twisted form that has the capacity to distort membrane bilayers.Pores formed on membranes are mostly open arcs of subunits rather than closed rings.The twisted pore conformation suggests a mechanism enabling a 180° rotation of the pore‐forming domain during membrane insertion and leading to clockwise propagation of subunit conformational change from pre‐pore to pore states. [ABSTRACT FROM AUTHOR]

Pore-forming toxins are an important component of the venom of many animals. Actinoporins are potent cytolysins that were first detected in the venom of sea anemones; however, they are occasionally found in animals other than cnidarians and are expanded in a few predatory gastropods. Here, we report the presence of 27 unique actinoporin-like genes with monophyletic origin in Mytilus galloprovincialis , which we have termed mytiporins. These mytiporins exhibited a remarkable level of molecular diversity and gene presence–absence variation, which warranted further studies aimed at elucidating their functional role. We structurally and functionally characterized mytiporin-1 and found significant differences from the archetypal actinoporin fragaceatoxin C. Mytiporin-1 showed weaker permeabilization activity, no specificity towards sphingomyelin, and weak activity in model lipid systems with negatively charged lipids. In contrast to fragaceatoxin C, which forms octameric pores, functional mytiporin-1 pores on negatively charged lipid membranes were hexameric. Similar hexameric pores were observed for coluporin-26 from Cumia reticulata and a conoporin from Conus andremenezi. This indicates that also other molluscan actinoporin-like proteins differ from fragaceatoxin C. Although the functional role of mytiporins in the context of molluscan physiology remains to be elucidated, the lineage-specific gene family expansion event that characterizes mytiporins indicates that strong selective forces acted on their molecular diversification. Given the tissue distribution of mytiporins, this process may have broadened the taxonomic breadth of their biological targets, which would have important implications for digestive processes or mucosal immunity. [ABSTRACT FROM AUTHOR]

Lipids are complex organic compounds made up of carbon, oxygen, and hydrogen. These play a diverse and intricate role in cellular processes like membrane trafficking, protein sorting, signal transduction, and bacterial infections. Both Gram-positive bacteria (Staphylococcus sp., Listeria monocytogenes, etc.) and Gram-negative bacteria (Chlamydia sp., Salmonella sp., E. coli, etc.) can hijack the various host-lipids and utilize them structurally as well as functionally to mount a successful infection. The pathogens can deploy with various arsenals to exploit host membrane lipids and lipid-associated receptors as an attachment for toxins' landing or facilitate their entry into the host cellular niche. Bacterial species like Mycobacterium sp. can also modulate the host lipid metabolism to fetch its carbon source from the host. The sequential conversion of host membrane lipids into arachidonic acid and prostaglandin E2 due to increased activity of cPLA-2 and COX-2 upon bacterial infection creates immunosuppressive conditions and facilitates the intracellular growth and proliferation of bacteria. However, lipids' more debatable role is that they can also be a blessing in disguise. Certain host-lipids, especially sphingolipids, have been shown to play a crucial antibacterial role and help the host in combating the infections. This review shed light on the detailed role of host lipids in bacterial infections and the current understanding of the lipid in therapeutics. We have also discussed potential prospects and the need of the hour to help us cope in this race against deadly pathogens and their rapidly evolving stealthy virulence strategies. [ABSTRACT FROM AUTHOR]

Lipids are complex organic compounds made up of carbon, oxygen, and hydrogen. These play a diverse and intricate role in cellular processes like membrane trafficking, protein sorting, signal transduction, and bacterial infections. Both Gram-positive bacteria (Staphylococcus sp., Listeria monocytogenes, etc.) and Gram-negative bacteria (Chlamydia sp., Salmonella sp., E. coli, etc.) can hijack the various host-lipids and utilize them structurally as well as functionally to mount a successful infection. The pathogens can deploy with various arsenals to exploit host membrane lipids and lipid-associated receptors as an attachment for toxins' landing or facilitate their entry into the host cellular niche. Bacterial species like Mycobacterium sp. can also modulate the host lipid metabolism to fetch its carbon source from the host. The sequential conversion of host membrane lipids into arachidonic acid and prostaglandin E2 due to increased activity of cPLA-2 and COX-2 upon bacterial infection creates immunosuppressive conditions and facilitates the intracellular growth and proliferation of bacteria. However, lipids' more debatable role is that they can also be a blessing in disguise. Certain host-lipids, especially sphingolipids, have been shown to play a crucial antibacterial role and help the host in combating the infections. This review shed light on the detailed role of host lipids in bacterial infections and the current understanding of the lipid in therapeutics. We have also discussed potential prospects and the need of the hour to help us cope in this race against deadly pathogens and their rapidly evolving stealthy virulence strategies. [ABSTRACT FROM AUTHOR]

Summary: Proteins of the aegerolysin family have a high abundance in Fungi. Due to their specific binding to membrane lipids, and their membrane‐permeabilization potential in concert with protein partner(s) belonging to a membrane‐attack‐complex/perforin (MACPF) superfamily, they were proposed as useful tools in different biotechnological and biomedical applications. In this work, we performed functional studies on expression of the genes encoding aegerolysin and MACPF‐like proteins in Aspergillus niger. Our results suggest the sporulation process being crucial for strong induction of the expression of all these genes. However, deletion of either of the aegerolysin genes did not influence the growth, development, sporulation efficiency and phenotype of the mutants, indicating that aegerolysins are not key factors in the sporulation process. In all our expression studies we noticed a strong correlation in the expression of one aegerolysin and MACPF‐like gene. Aegerolysins were confirmed to be secreted from the fungus. We also showed the specific interaction of a recombinant A. niger aegerolysin with an invertebrate‐specific membrane sphingolipid. Moreover, using this protein labelled with mCherry we successfully stained insect cells membranes containing this particular sphingolipid. Our combined results suggest, that aegerolysins in this species, and probably also in other aspergilli, could be involved in defence against predators. [ABSTRACT FROM AUTHOR]

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 Beauveria bassiana. BlyA was expressed as a recombinant protein in Escherichia coli, 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 Beauveria, Cordyceps, Metarhizium and Ophiocordyceps. 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 B. bassiana, which is an active ingredient of bioinsecticides. [ABSTRACT FROM AUTHOR]

Lysenin is a pore-forming protein extracted from the earthworm Eisenia fetida, which inserts large conductance pores in artificial and natural lipid membranes containing sphingomyelin. Its cytolytic and hemolytic activity is rather indicative of a pore-forming toxin; however, lysenin channels present intricate regulatory features manifested as a reduction in conductance upon exposure to multivalent ions. Lysenin pores also present a large unobstructed channel, which enables the translocation of analytes, such as short DNA and peptide molecules, driven by electrochemical gradients. These important features of lysenin channels provide opportunities for using them as sensors for a large variety of applications. In this respect, this literature review is focused on investigations aimed at the potential use of lysenin channels as analytical tools. The described explorations include interactions with multivalent inorganic and organic cations, analyses on the reversibility of such interactions, insights into the regulation mechanisms of lysenin channels, interactions with purines, stochastic sensing of peptides and DNA molecules, and evidence of molecular translocation. Lysenin channels present themselves as versatile sensing platforms that exploit either intrinsic regulatory features or the changes in ionic currents elicited when molecules thread the conducting pathway, which may be further developed into analytical tools of high specificity and sensitivity or exploited for other scientific biotechnological applications. [ABSTRACT FROM AUTHOR]

Potential nanodomains in cellular membranes are widely believed to be targeted by proteins and other biomolecules to enable execution of critical cellular functions. However, characterization of these nanodomains remains elusive, primarily due to the diffraction limit of a conventional optical microscope. Herein using super-resolution STED microscopy coupled with fluorescence correlation spectroscopy (STED-FCS), we provide experimental evidence of lipid nanodomains present in model membranes comprised of phosphocholine-cholesterol binary mixture, and its reorganization induced by pore-forming toxins (PFTs). In this study, we used two different types of PFTs, namely α-PFT (cytolysin A) and β-PFT (listeriolysin O), that preferentially associate as well as reorganize cholesterol-containing saturated and unsaturated phosphocholine membranes. The emergence of nanodomains due to the lipid–lipid and lipid-PFT interactions was quantified at a length scales of ~50–150 nm using FCS diffusion law enabled by variation of spot sizes in the super-resolution STED microscopy. Our results shed light on the usefulness of super-resolution microscopy to quantify the underlying nanoscale domains in model membranes with implications for a wide variety of membrane-mediated cellular events observed in real cell membranes. [ABSTRACT FROM AUTHOR]

Lysenin is a pore-forming toxin of the aerolysin family, which is derived from coelomic fluid of the earthworm Eisenia fetida. Upon binding to sphingomyelin (SM)-containing membranes, lysenin undergoes a series of structural changes promoting the conversion of water-soluble monomers into oligomers, leading to its insertion into the membrane and the formation of a lytic β-barrel pore. The soluble monomer and transmembrane pore structures were recently described, but the underlying structural details of oligomerization remain unclear. To investigate the molecular mechanisms controlling the conformational rearrangements accompanying pore formation, we compared the hydrogen–deuterium exchange pattern between lyseninWT and its mutant lyseninV88C/Y131C. This mutation arrests lysenin oligomers in the prepore state at the membrane surface and does not affect the structural dynamics of the water-soluble form of lysenin. In contrast, membrane-bound lyseninV88C/Y131C exhibited increased structural stabilization, especially within the twisted β-sheet of the N-terminal domain. We demonstrated that the structural stabilization of the lysenin prepore started at the site of lysenin's initial interaction with the lipid membrane and was transmitted to the twisted β-sheet of the N-terminal domain, and that lyseninV88C/Y131C was arrested in this conformation. In lyseninWT, stabilization of these regions drove the conformational changes necessary for pore formation. [ABSTRACT FROM AUTHOR]