Evolutionary analyses of the gasdermin family suggest conserved roles in infection response despite loss of pore-forming functionality

Background Gasdermins are ancient (>500million-years-ago) proteins, constituting a family of pore-forming proteins that allow the release of intracellular content including proinflammatory cytokines. Despite their importance in the immune response, and although gasdermin and gasdermin-like genes have been identified across a wide range of animal and non-animal species, there is limited information about the evolutionary history of the gasdermin family, and their functional roles after infection. In this study, we assess the lytic functions of different gasdermins across Metazoa species, and use a mouse model of sepsis to evaluate the expression of the different gasdermins during infection. Results We show that the majority of gasdermin family members from distantly related animal clades are pore-forming, in line with the function of the ancestral proto-gasdermin and gasdermin-like proteins of Bacteria. We demonstrate the first expansion of this family occurred through a duplication of the ancestral gasdermin gene which formed gasdermin E and pejvakin prior to the divergence of cartilaginous fish and bony fish ~475 mya. We show that pejvakin from cartilaginous fish and mammals lost the pore-forming functionality and thus its role in cell lysis. We describe that the pore-forming gasdermin A formed ~320 mya as a duplication of gasdermin E prior to the divergence of the Sauropsida clade (the ancestral lineage of reptiles, turtles, and birds) and the Synapsid clade (the ancestral lineage of mammals). We then demonstrate that the gasdermin A gene duplicated to form the rest of the gasdermin family including gasdermins B, C, and D: pore-forming proteins that present a high variation of the exons in the linker sequence, which in turn allows for diverse activation pathways. Finally, we describe expression of murine gasdermin family members in different tissues in a mouse sepsis model, indicating function during infection response. Conclusions In this study we explored the evolutionary history of the gasdermin proteins in animals and demonstrated that the pore-formation functionality has been conserved from the ancient proto-gasdermin protein. We also showed that one gasdermin family member, pejvakin, lost its pore-forming functionality, but that all gasdermin family members, including pejvakin, likely retained a role in inflammation and the physiological response to infection.

Clostridium perfringens Beta2 toxin forms highly cation-selective channels in lipid bilayers

Clostridium perfringens is a potent producer of a variety of toxins. Well studied from these are five toxins (alpha, Beta (CPB), epsilon, iota and CPE) that are produced by seven toxinotype strains (A–G) of C. perfringens. Besides these toxins, C. perfringens produces also another toxin that causes necrotizing enterocolitis in piglets. This toxin termed consensus Beta2 toxin (cCPB2) has a molecular mass of 27,620 Da and shows only little homology to CPB and no one to the other toxins of C. perfringens. Its primary action on cells remained unknown to date. cCPB2 was heterogeneously expressed as fusion protein with GST in Escherichia coli and purified to homogeneity. Although cCPB2 does not exhibit the typical structure of beta-stranded pore-forming proteins and contains no indication for the presence of amphipathic alpha-helices we could demonstrate that cCPB2 is a pore-forming component with an extremely high activity in lipid bilayers. The channels have a single-channel conductance of about 700 pS in 1 M KCl and are highly cation-selective as judged from selectivity measurements in the presence of salt gradients. The high cation selectivity is caused by the presence of net negative charges in or near the channel that allowed an estimate of the channel size being about 1.4 nm wide. Our measurements suggest that the primary effect of cCPB2 is the formation of cation-selective channels followed by necrotic enteritis in humans and animals. We searched in databases for homologs of cCPB2 and constructed a cladogram representing the phylogenetic relationship to the next relatives of cCPB2.

Molecular and structural aspects of gasdermin family pores and insights into gasdermin-elicited programmed cell death

Pyroptosis is a highly inflammatory and lytic type of programmed cell death (PCD) commenced by inflammasomes, which sense perturbations in the cytosolic environment. Recently, several ground-breaking studies have linked a family of pore-forming proteins known as gasdermins (GSDMs) to pyroptosis. The human genome encodes six GSDM proteins which have a characteristic feature of forming pores in the plasma membrane resulting in the disruption of cellular homeostasis and subsequent induction of cell death. GSDMs have an N-terminal cytotoxic domain and an auto-inhibitory C-terminal domain linked together through a flexible hinge region whose proteolytic cleavage by various enzymes releases the N-terminal fragment that can insert itself into the inner leaflet of the plasma membrane by binding to acidic lipids leading to pore formation. Emerging studies have disclosed the involvement of GSDMs in various modalities of PCD highlighting their role in diverse cellular and pathological processes. Recently, the cryo-EM structures of the GSDMA3 and GSDMD pores were resolved which have provided valuable insights into the pore formation process of GSDMs. Here, we discuss the current knowledge regarding the role of GSDMs in PCD, structural and molecular aspects of autoinhibition, and pore formation mechanism followed by a summary of functional consequences of gasdermin-induced membrane permeabilization.

Role of Gasdermins in the Biogenesis of Apoptotic Cell–Derived Exosomes

The gasdermins are a family of pore-forming proteins that has recently been suggested to play a central role in the pyroptosis and the release of inflammatory cytokines. Here, we describe the novel roles of gasdermins in the biogenesis of apoptotic cell–derived exosomes. In apoptotic cells, GADMA, GSDMC, GSDMD, and GSDME increased the release of ApoExos, and both their full-length and cleaved forms were localized in the exosomal membrane. GSDMB and DFNB59, on the other hand, negatively affected the release of ApoExos. The caspase-mediated cleavage of gasdermins, especially GSDME, is suggested to increase Ca2+ influx to cytosol through endosomal pores and thus increase the biogenesis of ApoExos. In addition, the GSDME-mediated biogenesis of ApoExos depended on the ESCRT-III complex and endosomal recruitment of Ca2+-dependent proteins: annexins A2 and A7, the PEF domain family proteins sorcin and grancalcin, and the Bro1 domain protein HD-PTP. Therefore, we propose that the biogenesis of ApoExos begins when gasdermin-mediated endosomal pores increase cytosolic Ca2+, continues through the recruitment of annexin-sorcin/grancalcin-HD-PTP, and is completed when the ESCRT-III complex synthesizes intraluminal vesicles in the multivesicular bodies of dying cells. Finally, we found that GSDME-bearing tumors released ApoExos to induce inflammatory responses in the in vivo 4T1 orthotropic model of breast cancer. The data presented in this study indicate that the switch from apoptosis to pyroptosis could drive the transfer of mass signals to nearby or distant living cells and tissues by way of extracellular vesicles, and that gasdermins play critical roles in that process.

The Curious Case of Earthworms and COVID-19

Earthworms have been used for centuries in traditional medicine and are used globally as an ecotoxicological standard test species. Studies of the earthworm Eisenia fetida have shown that exposure to nanomaterials activates a primary corona-response, which is covering the nanomaterial with native proteins, the same response as to biological invaders such as a virus. We outline that the earthworm Eisenia fetida is possibly immune to COVID-19 (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2), and we describe the likely mechanisms of highly receptor-specific pore-forming proteins (PFPs). A non-toxic version of this protein is available, and we hypothesize that it is possible to use the earthworm’s PFPs based anti-viral mechanism as a therapeutic model for human SARS-CoV-2 and other corona viruses. The proteins can be used as a drug, for example, delivered with a nanoparticle in a similar way to the current COVID-19 vaccines. Obviously, careful consideration should be given to the potential risk of toxicity elicited by lysenin for in vivo usage. We aim to share this view to activate its exploration by the wider scientific community while promoting a potential therapeutic development.

Physiological Functions of CRAC Channels

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ signaling pathway that is evolutionarily conserved across eukaryotes. SOCE is triggered physiologically when the endoplasmic reticulum (ER) Ca2+ stores are emptied through activation of inositol-1,4,5-trisphosphate receptors. SOCE is mediated by the Ca2+ release-activated Ca2+ (CRAC) channels, which are highly Ca2+ selective. Upon store depletion, the ER Ca2+-sensing STIM proteins aggregate and gain extended conformations spanning the ER-plasma membrane junctional space to bind and activate Orai, the pore-forming proteins of hexameric CRAC channels. In recent years, studies on STIM and Orai tissue-specific knockout mice and gain- and loss-of-function mutations in humans have shed light on the physiological functions of SOCE in various tissues. Here, we describe recent findings on the composition of native CRAC channels and their physiological functions in immune, muscle, secretory, and neuronal systems to draw lessons from transgenic mice and human diseases caused by altered CRAC channel activity. Expected final online publication date for the Annual Review of Physiology, Volume 84 is February 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Pore Forming Protein Induced Biomembrane Reorganization and Dynamics: A Focused Review

Pore forming proteins are a broad class of pathogenic proteins secreted by organisms as virulence factors due to their ability to form pores on the target cell membrane. Bacterial pore forming toxins (PFTs) belong to a subclass of pore forming proteins widely implicated in bacterial infections. Although the action of PFTs on target cells have been widely investigated, the underlying membrane response of lipids during membrane binding and pore formation has received less attention. With the advent of superresolution microscopy as well as the ability to carry out molecular dynamics (MD) simulations of the large protein membrane assemblies, novel microscopic insights on the pore forming mechanism have emerged over the last decade. In this review, we focus primarily on results collated in our laboratory which probe dynamic lipid reorganization induced in the plasma membrane during various stages of pore formation by two archetypal bacterial PFTs, cytolysin A (ClyA), an α-toxin and listeriolysin O (LLO), a β-toxin. The extent of lipid perturbation is dependent on both the secondary structure of the membrane inserted motifs of pore complex as well as the topological variations of the pore complex. Using confocal and superresolution stimulated emission depletion (STED) fluorescence correlation spectroscopy (FCS) and MD simulations, lipid diffusion, cholesterol reorganization and deviations from Brownian diffusion are correlated with the oligomeric state of the membrane bound protein as well as the underlying membrane composition. Deviations from free diffusion are typically observed at length scales below ∼130 nm to reveal the presence of local dynamical heterogeneities that emerge at the nanoscale—driven in part by preferential protein binding to cholesterol and domains present in the lipid membrane. Interrogating the lipid dynamics at the nanoscale allows us further differentiate between binding and pore formation of β- and α-PFTs to specific domains in the membrane. The molecular insights gained from the intricate coupling that occurs between proteins and membrane lipids and receptors during pore formation are expected to improve our understanding of the virulent action of PFTs.

Decoding assembly of alpha-helical transmembrane pores through intermediate states

Membrane-active pore-forming alpha-helical peptides and proteins are well known for their dynamic assembly mechanism and it has been critical to delineate the pore-forming structures in the membrane. Previously, attempts have been made to elucidate their assembly mechanism and there is a large gap due to complex pathways by which these membrane-active pores impart their effect. Here we demonstrate the multi-step structural assembly pathway of alpha-helical peptide pores formed by a 37 amino-acid synthetic peptide, pPorU based on the natural porin from Corynebacterium urealyticum using single-channel electrical recordings. More specifically, we report detectable intermediates states during membrane insertion and pore formation of pPorU. The fully assembled pore is functional and exhibited unusually large stable conductance and voltage-dependent gating, generally applicable to a range of pore-forming proteins. Furthermore, we used rationally designed mutants to understand the role of specific amino acids in the assembly of these peptide pores. Mutant peptides that differ from wild-type peptides produced noisy, unstable intermediate states and low conductance pores, demonstrating sequence specificity in the pore-formation process supported by molecular dynamics simulations. We suggest that our study contributes to understanding the mechanism of action of alpha-helical pores and antimicrobial peptides and should be of broad interest to bioengineers to build peptide-based nanopore sensors.