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Aegerolysins are remarkable proteins. They are distributed over the tree of life, being relatively widespread in bacteria and fungi, but also present in some insects, plants, protozoa, and viruses. Despite their abundance in cells of certain developmental stages and their presence in secretomes, only a few aegerolysins have been studied in detail. Their function, in particular, is intriguing. Here, we summarize previously published findings on the distribution, molecular interactions, and function of these versatile aegerolysins. They have very diverse protein sequences but a common fold. The machine learning approach of the AlphaFold2 algorithm, which incorporates physical and biological knowledge of protein structures and multisequence alignments, provides us new insights into the aegerolysins and their pore-forming partners, complemented by additional genomic support. We hypothesize that aegerolysins are involved in the mechanisms of competitive exclusion in the niche. [ABSTRACT FROM AUTHOR]

Gamma delta (GD) T cells are an unconventional T cell type present in both the epidermis and the dermis of human skin. They are critical to regulating skin inflammation, wound healing, and anti-microbial defense. Similar to CD8+ cytotoxic T cells expressing an alpha beta (AB) TCR, GD T cells have cytolytic capabilities. They play an important role in elimination of cutaneous tumors and virally infected cells and have also been implicated in pathogenicity of several autoimmune diseases. T cell cytotoxicity is associated with the expression of the pore forming protein Perforin. Perforin is an innate immune protein containing a membrane attack complex perforin-like (MACPF) domain and functions by forming pores in the membranes of target cells, which allow granzymes and reactive oxygen species to enter the cells and destroy them. Perforin-2, encoded by the gene MPEG1, is a newly discovered member of this protein family that is critical for clearance of intracellular bacteria. Cutaneous GD T cells express both Perforin and Perforin-2, but many questions remain regarding the role that these proteins play in GD T cell mediated cytotoxicity against tumors and bacterial pathogens. Here, we review what is known about Perforin expression by skin GD T cells and the mechanisms that contribute to Perforin activation.

Gamma delta (GD) T cells are an unconventional T cell type present in both the epidermis and the dermis of human skin. They are critical to regulating skin inflammation, wound healing, and anti-microbial defense. Similar to CD8+ cytotoxic T cells expressing an alpha beta (AB) TCR, GD T cells have cytolytic capabilities. They play an important role in elimination of cutaneous tumors and virally infected cells and have also been implicated in pathogenicity of several autoimmune diseases. T cell cytotoxicity is associated with the expression of the pore forming protein Perforin. Perforin is an innate immune protein containing a membrane attack complex perforin-like (MACPF) domain and functions by forming pores in the membranes of target cells, which allow granzymes and reactive oxygen species to enter the cells and destroy them. Perforin-2, encoded by the gene MPEG1 , is a newly discovered member of this protein family that is critical for clearance of intracellular bacteria. Cutaneous GD T cells express both Perforin and Perforin-2, but many questions remain regarding the role that these proteins play in GD T cell mediated cytotoxicity against tumors and bacterial pathogens. Here, we review what is known about Perforin expression by skin GD T cells and the mechanisms that contribute to Perforin activation. [ABSTRACT FROM AUTHOR]

A hybrid membrane system based on 1-octadecanethiol (ODT) and 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC) has been investigated in respect to the incorporation of the transport component C2IIa of Clostridium botulinum C2 toxin into such bilayers. Such model systems are particularly interesting surrogates for studying fundamental transport phenomena across cell membranes. Here, we present structural and electrochemical studies at a model ODT-POPC hybrid membrane system established at a solid gold support substrate before and after the incorporation of pores. Pore incorporation was investigated via macroscopic electrochemical characterization using cyclic voltammetry (CV), and microscopic studies using atomic force microscopy (AFM) and AFM-scanning electrochemical microscopy (SECM) recording current-distance curves at the supported lipid membranes (SLMs) before and after the incorporation of pores. Recently developed AFM-SECM probes with integrated conical platinum composite electrodes were used for these spatially resolved studies. Finally, control experiments blocking the pores were performed using chloroquine (CQ), which is a known inhibitor for C2IIa proteins. The number of C2IIa pores incorporated in an ODT-POPC hybrid membrane was determined as 6.6*10 4 pores/mm 2 based on the single pore conductance model. Blocking with chloroquine has significantly reduced the number of active pores to 2.6*10 4 pores/mm 2 . To the best of our knowledge, the associated visualization via AFM was accomplished for the first time. [ABSTRACT FROM AUTHOR]

Abstract: The various steps that perforin (PFN), a critical mediator of innate immune response, undertakes to form a transmembrane pore remains poorly understood. We have used surface plasmon resonance (SPR) to dissect mechanism of pore formation. The membrane association of PFN was calcium dependent irrespective of pH. However, PFN does not permeabilize large or giant unilamellar vesicles (GUV) at pH 5.5 even though the monomers bind to the membranes in the presence of calcium. It was possible to activate adsorbed PFN and to induce membrane permeabilization by simply raising pH to a physiological level (pH 7.4). These results were independently confirmed on GUV and Jurkat cells. The conformational state of PFN at either pH was further assessed with monoclonal antibodies Pf-80 and Pf-344. Pf-344 maps to a linear epitope within region 373–388 of epidermal growth factor (EGF)-like domain while the Pf-80 appears to recognize a conformational epitope. Pf-344 interacts with the EGF-like domain after PFN monomers undergo pore formation, the site recognized by Pf-80 is only accessible at acidic but not neutral pH. Thus, the Pf-80 mAb likely interacts with a region of the monomer that participates in oligomerization prior to insertion of the monomer into the lipid bilayer and thus may have therapeutic utility against PFN-mediated immunopathology. [ABSTRACT FROM AUTHOR]

[Display omitted] Organisms have evolved mechanisms in which cellular membranes can both be targeted and punctured thereby killing the targeted cell. One such mechanism involves the deployment of pore forming proteins (PFPs) which function by oligomerizing on cell membranes and inserting a physical pore spanning the membrane. This pore can lead to cell death by either causing osmotic flux or allowing the delivery of a secondary toxin. Pore forming proteins can be broadly classified into different families depending on the structure of the final pore; either α-PFPs using channels made from α -helices or β-PFPs using channels made from β-barrels. There are many different β-PFPs and an emerging superfamily is the aerolysin-ETX/MTX-2 superfamily. A comparison between the members of this superfamily reveals the pore forming domain is a common module yet the receptor binding region is highly variable. These structural and architectural variations lead to differences in the target recognition and determine the site of activity. Closer investigation of the topology of the family also suggests that the Toxin_10 family of PFPs could be considered as part of the aerolysin-ETX/MTX-2 superfamily. Comparatively, far less is known about how Toxin_10 proteins assemble into the final pore structure than aerolysin-ETX/MTX-2 proteins. This review aims to collate the pore forming protein members and bridge the structural similarities between the aerolysin-ETX/MTX-2 superfamily and the insecticidal Toxin_10 subfamily. [ABSTRACT FROM AUTHOR]

Over the last 25 years, the biology of membrane proteins, including the PFPs-membranes interactions is seeking attention for the development of successful drug molecules against a number of infectious diseases. Pore forming toxins (PFTs), the largest family of PFPs are considered as a group of virulence factors produced in a large number of pathogenic systems which include streptococcus, pneumonia, Staphylococcus aureus, E. coli, Mycobacterium tuberculosis, group A and B streptococci, Corynebacterium diphtheria and many more. PFTs are generally utilized by the disease causing pathogens to disrupt the host first line of defense i.e. host cell membranes through pore formation strategy. Although, pore formation is the principal mode of action of the PFTs but they can have additional adverse effects on the hosts including immune evasion. Recently, structural investigation of different PFTs have imparted the molecular mechanistic insights into how PFTs get transformed from its inactive state to active toxic state. On the basis of their structural entity, PFTs have been classified in different types and their mode of actions alters in terms of pore formation and corresponding cellular toxicity. Although pathogen genome analysis can identify the probable PFTs depending upon their structural diversity, there are so many PFTs which utilize the local environmental conditions to generate their pore forming ability using a novel strategy which is known as "conformational switch" of a protein. This conformational switch is considered as characteristics of the phase shifting proteins which were often utilized by many pathogenic systems to protect them from the invaders through allosteric communication between distant regions of the protein. In this chapter, we discuss the structure function relationships of PFTs and how activity of PFTs varies with the change in the environmental conditions has been explored. Finally, we demonstrate these structural insights to develop therapeutic potential to treat the infections caused by multidrug resistant pathogens., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Pore forming proteins (PFPs) undergo dramatic conformational changes to punch holes in the target membrane. These PFPs have the ability to self-assemble, by way of oligomerization, and have the capacity to transform from a water soluble state (commonly referred to as fluid phase) to a membrane adhered form. Accordingly, PFPs are metastable, that is they are inert until the right conditions cause the release of potential energy stored in the conformational fold leading to a vast structural rearrangement into a membrane-inserted oligomeric form. However, the metastable state of PFPs poses a problem of leading to aggregation and precipitation in conditions typically required for structural biology techniques. Here, we discuss the protein chemistry of the MACPF protein complement component 9 (C9). C9 is part of a larger complex assembly known as the membrane attack complex (MAC) that has been studied extensively for its ability to form pores in bacteria. An unusual artifact of human C9 is the ability to form a soluble oligomeric state of the channel portion of the MAC, called polyC9. PolyC9 formation does not require the presence of membranes or other complement factors. It is only in recent years that structural studies of the MAC have become successful owing to improved recombinant DNA expression systems and the improvement of high-resolution techniques (both X-ray crystallography and single particle cryo-EM). We discuss the expression and purification of recombinant C9, crystallization of the soluble monomeric form of C9 and the preparation of the oligomeric polyC9., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Pore forming proteins are released as water-soluble monomers that form-mostly oligomeric-pores in target membranes. Our understanding of such pore formation relies in part on the direct visualization of their assemblies on and in the membrane. Here, we discuss the application of atomic force microscopy (AFM) to visualize and understand membrane pore formation, illustrated specifically by studies of proteins of the MACPF/CDC superfamily on supported lipid bilayers. Besides detailed protocols, we also point out common imaging artefacts and strategies to avoid them, and briefly outline how AFM can be effectively used in conjunction with other methods., (Copyright © 2021 Elsevier Inc. All rights reserved.)