Pore-forming Toxin Research Articles

The Molecular Architecture and Mode of Action of Clostridium perfringens ε-Toxin

Clostridium perfringens ε-toxin has long been associated with a severe enterotoxaemia of livestock animals, and more recently, was proposed to play a role in the etiology of multiple sclerosis in humans. The remarkable potency of the toxin has intrigued researchers for many decades, who suggested that this indicated an enzymatic mode of action. Recently, there have been major breakthroughs by finding that it is a pore-forming toxin which shows exquisite specificity for cells bearing the myelin and lymphocyte protein (MAL) receptor. This review details the molecular structures of the toxin, the evidence which identifies MAL as the receptor and the possible roles of other cell membrane components in toxin binding. The information on structure and mode of action has allowed the functions of individual amino acids to be investigated and has led to the creation of mutants with reduced toxicity that could serve as vaccines. In spite of this progress, there are still a number of key questions around the mode of action of the toxin which need to be further investigated.

Dry and liquid formulations of IBT-V02, a novel multi-component toxoid vaccine, are effective against Staphylococcus aureus isolates from low-to-middle income countries.

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTIs) in the U.S. as well as more serious invasive diseases, including bacteremia, sepsis, endocarditis, surgical site infections, osteomyelitis, and pneumonia. These infections are exacerbated by the emergence of antibiotic-resistant clinical isolates such as methicillin-resistant S. aureus (MRSA), highlighting the need for alternatives to antibiotics to treat bacterial infections. We have previously developed a multi-component toxoid vaccine (IBT-V02) in a liquid formulation with efficacy against multiple strains of Staphylococcus aureus prevalent in the industrialized world. However, liquid vaccine formulations are not compatible with the paucity of cold chain storage infrastructure in many low-to-middle income countries (LMICs). Furthermore, whether our IBT-V02 vaccine formulations are protective against S. aureus isolates from LMICs is unknown. To overcome these limitations, we developed lyophilized and spray freeze-dried formulations of IBT-V02 vaccine and demonstrated that both formulations had comparable biophysical attributes as the liquid formulation, including similar levels of toxin neutralizing antibodies and protective efficacy against MRSA infections in murine and rabbit models. To enhance the relevancy of our findings, we then performed a multi-dimensional screen of 83 S. aureus clinical isolates from LMICs (e.g., Democratic Republic of Congo, Palestine, and Cambodia) to rationally down-select strains to test in our in vivo models based on broad expression of IBT-V02 targets (i.e., pore-forming toxins and superantigens). IBT-V02 polyclonal antisera effectively neutralized toxins produced by the S. aureus clinical isolates from LMICs. Notably, the lyophilized IBT-V02 formulation exhibited significant in vivo efficacy in various preclinical infection models against the S. aureus clinical isolates from LMICs, which was comparable to our liquid formulation. Collectively, our findings suggested that lyophilization is an effective alternative to liquid vaccine formulations of our IBT-V02 vaccine against S. aureus infections, which has important implications for protection from S. aureus isolates from LMICs.

PRMT-7/PRMT7 activates HLH-30/TFEB to guard plasma membrane integrity compromised by bacterial pore-forming toxins

ABSTRACT Bacterial pore-forming toxins (PFTs) that disrupt host plasma membrane integrity (PMI) significantly contribute to the virulence of various pathogens. However, how host cells protect PMI in response to PFT perforation in vivo remains obscure. Previously, we demonstrated that the HLH-30/TFEB-dependent intrinsic cellular defense (INCED) is elicited by PFT to maintain PMI in Caenorhabditis elegans intestinal epithelium. Yet, the molecular mechanism for the full activation of HLH-30/TFEB by PFT remains elusive. Here, we reveal that PRMT-7 (protein arginine methyltransferase-7) is indispensable to the nuclear transactivation of HLH-30 elicited by PFTs. We demonstrate that PRMT-7 participates in the methylation of HLH-30 on its RAG complex binding domain to facilitate its nuclear localization and activation. Moreover, we showed that PRMT7 is evolutionarily conserved to regulate TFEB cellular localization and repair plasma damage caused by PFTs in human intestinal cells. Together, our observations not only unveil a novel PRMT-7/PRMT7-dependent post-translational regulation of HLH-30/TFEB but also shed insight on the evolutionarily conserved mechanism of the INCED against PFT in metazoans.

Gasdermin D promotes influenza virus-induced mortality through neutrophil amplification of inflammation

Influenza virus activates cellular inflammasome pathways, which can be both beneficial and detrimental to infection outcomes. Here, we investigate the function of the inflammasome-activated, pore-forming protein gasdermin D (GSDMD) during infection. Ablation of GSDMD in knockout (KO) mice (Gsdmd−/−) significantly attenuates influenza virus-induced weight loss, lung dysfunction, lung histopathology, and mortality compared with wild type (WT) mice, despite similar viral loads. Infected Gsdmd−/− mice exhibit decreased inflammatory gene signatures shown by lung transcriptomics. Among these, diminished neutrophil gene activation signatures are corroborated by decreased detection of neutrophil elastase and myeloperoxidase in KO mouse lungs. Indeed, directly infected neutrophils are observed in vivo and infection of neutrophils in vitro induces release of DNA and tissue-damaging enzymes that is largely dependent on GSDMD. Neutrophil depletion in infected WT mice recapitulates the reductions in mortality, lung inflammation, and lung dysfunction observed in Gsdmd−/− animals, while depletion does not have additive protective effects in Gsdmd−/− mice. These findings implicate a function for GSDMD in promoting lung neutrophil responses that amplify influenza virus-induced inflammation and pathogenesis. Targeting the GSDMD/neutrophil axis may provide a therapeutic avenue for treating severe influenza.

Migraine and gasdermin D: a new perspective on the inflammatory basis of migraine.

Migraine is a common and disabling primary headache disorder and inflammation is a proposed factor in the complex ethiology of the disease. Gasdermin D (GSDMD) is a membrane pore-forming protein acting through the caspase system. End result is cell death caused by leakage of intracellular components to extracellular space which also results in inflammation. Stemming from this knowledge, the potential role of GSDMD in migraine was investigated in this prospective study. This prospective study was conducted between September 2022 to April 2023. 47 patients with migraine were designated as the patient group, whereas 47 healthy volunteers were designated as the control group. Serum GSDMD levels of both groups were compared, with an additional comparison between migraine patients during symptom-free and attack periods. Migraine related characteristics of the patients were also included in the study. Median GSDMD levels of the patient and control group did not reveal a significant difference. Nausea, vomiting and severity of headache were found to be correlated with GSDMD levels in migraine patients. Patients with nausea revealed a higher GSDMD level compared to patients without nausea during both symptom-free and attack periods (p = 0.021 and p = 0.01, respectively). Nausea was correlated to higher GSDMD levels in the patient population during symptom-free period (p = 0.030). The severity of pain was positively correlated with GSDMD levels during the attack period (p < 0.001). Gasdermin family and GSDMD in particular are promising prospects for therapy in a wide spectrum of disorders. Gasdermin proteins are candidates to be the focus for future studies both related to pathogenesis and drug therapy in migraine and varying inflammatory-driven clinical pictures.

Abstract 725: Listeria-inspired phagosome escape drives STING responses to CD47 blockade

Abstract Purpose: Through phagocytosis, antigen-presenting cells (APCs) engulf and neutralize malignant cells. ​However, engulfed tumor cells are destroyed in phagolysosomes leaving few peptides to enter cytosol, where cross-presentation of antigens and immune activation occurs. Listeria (L.) monocytogenes escape phagolysosomes by secreting a pore-forming protein Listeriolysin O (LLO). Our breakthrough centers on harnessing LLO to create LLO-CD47, a novel protein-antibody conjugate that fuses the myeloid checkpoint anti-CD47 to LLO. By promoting tumor cell-phagocytosis and the release of tumor peptides and DNA from phagolysosomes, we hypothesize that LLO-CD47 enhances the immune functionalities of APCs. Procedures: Anti-CD47 was conjugated to LLO using a water-soluble SPDP crosslinker and purified by affinity chromatography. Transmission electron microscopy (TEM) was used to visualize the integrity of macrophage phagolysosomes following treatment. C57BL/6 mouse bone marrow-derived macrophages (BMDMs) were used to study M2-to-Ml polarization, tumor cell phagocytosis, STING activation, and antigen presentation. Mice bearing orthotopically implanted spontaneously metastatic murine triple-negative 4T1 tumors and E0771 tumors received intratumoral (IT) or intraperitoneal (IP) injection. CD8+ T cells or tumor-associated macrophages (TAMs) were depleted from tumor-bearing mice using an anti-CD8 antibody or anti-CSF-1R antibody prior to treatment. Results: 1. LLO-CD47 skews BMDMs from M2-to-M1 inflammatory phenotypes and enhances the phagocytosis of E0771 tumor cells. 2. BMDMs visualized by TEM show breaches in phagosome membranes following LLO-CD47, but not anti-CD47, treatment. 3. LLO-CD47 increases levels of phosphorylated STING, IFN, and TNFa in BMDMs in vitro and TAMs in vivo, relative to anti-CD47. 4. IT delivery of LLO-CD47 inhibits the growth and metastasis of orthotopically implanted murine breast tumors relative to anti-CD47. Harvested tumors show increased M1 polarized macrophages and CD8+ T cells, which express high levels of PD-1. 5. Co-administration of IP anti-PD-1 and LLO-CD47 delays the progression of breast tumor metastasis and prolongs animal survival relative to animals treated with anti-PD-1 and anti-CD47. 6. The elimination of CD8+ T cells or TAMs abrogates the antitumor effect of LLO-CD47. 7. IP administration of LLO-CD47 showed acceptable toxicity with stable body weight gain and no long-term hematological, renal, or hepatic toxicities. Conclusion: Conjugation of LLO to anti-CD47 enhances macrophage STING activation, antigen presentation, and tumor cell phagocytosis. Our work represents the first antibody-drug conjugate specifically designed to enhance immune activation against cancer.​ Citation Format: Benjamin R. Schrank, Yifan Wang, Abin Antony, Wen Jiang. Listeria-inspired phagosome escape drives STING responses to CD47 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 725.

Large conductance voltage-and calcium-activated K+ (BK) channel in health and disease.

Large Conductance Voltage- and Calcium-activated K+ (BK) channels are transmembrane pore-forming proteins that regulate cell excitability and are also expressed in non-excitable cells. They play a role in regulating vascular tone, neuronal excitability, neurotransmitter release, and muscle contraction. Dysfunction of the BK channel can lead to arterial hypertension, hearing disorders, epilepsy, and ataxia. Here, we provide an overview of BK channel functioning and the implications of its abnormal functioning in various diseases. Understanding the function of BK channels is crucial for comprehending the mechanisms involved in regulating vital physiological processes, both in normal and pathological conditions, controlled by BK. This understanding may lead to the development of therapeutic interventions to address BK channelopathies.

A Comparative Analysis of the Venom System between Two Morphotypes of the Sea Anemone Actinia equina.

The current study investigates the venom-delivery system of green and red morphotypes of the sea anemone Actinia equina to disclose its potential as a source of bioactive compounds. We compared the two morphotypes using electron and optical microscopy, proteomics, and toxicity assessment on zebrafish embryos. Specialized venom-injecting cells (nematocysts) are equally distributed and found in the tentacles of both varieties. Proteomics revealed proteins of interest in both red and green Actinia, yielding the three most abundant Gene Ontology (GO) terms related to the biological processes "proteolysis", "hemolysis in another organism" and "lipid catabolic process". Neurotoxins and cytolytic toxins similar to known cnidarian toxins like PsTX-60A and AvTX-60A, for instance, were identified in both types. Extracts from green and red anemones were toxic to zebrafish embryos, with green anemone venom appearing to be more potent. The findings highlight the presence of proteinaceous toxins in A. equina and the potential for different varieties to possess distinct bioactive compounds. Notably, pore-forming toxins are suggested for molecular probes and immunotoxins, making them valuable assets for potential biotechnological and biomedical purposes.

New insights into Gasdermin D pore formation.

Gasdermin D (GSDMD) is a pore-forming protein that perforates the plasma membrane (PM) during pyroptosis, a pro-inflammatory form of cell death, to induce the unconventional secretion of inflammatory cytokines and, ultimately, cell lysis. GSDMD is activated by protease-mediated cleavage of its active N-terminal domain from the autoinhibitory C-terminal domain. Inflammatory caspase-1, -4/5 are the main activators of GSDMD via either the canonical or non-canonical pathways of inflammasome activation, but under certain stimuli, caspase-8 and other proteases can also activate GSDMD. Activated GSDMD can oligomerize and assemble into various nanostructures of different sizes and shapes that perforate cellular membranes, suggesting plasticity in pore formation. Although the exact mechanism of pore formation has not yet been deciphered, cysteine residues are emerging as crucial modulators of the oligomerization process. GSDMD pores and thus the outcome of pyroptosis can be modulated by various regulatory mechanisms. These include availability of activated GSDMD at the PM, control of the number of GSDMD pores by PM repair mechanisms, modulation of the lipid environment and post-translational modifications. Here, we review the latest findings on the mechanisms that induce GSDMD to form membrane pores and how they can be tightly regulated for cell content release and cell fate modulation.

Non-viral Gene Therapy for Melanoma Using Lysenin from Eisenia Foetida.

Earthworms, long utilized in traditional medicine, serve as a source of inspiration for modern therapeutics. Lysenin, a defensive factor in the coelom fluid of the earthworm Eisenia fetida, has multiple bioactivities. However, the inherent toxicity of Lysenin as a pore-forming protein (PFP) restricts its application in therapy. Here, a gene therapy strategy based on Lysenin for cancer treatment is presented. The formulation consists of polymeric nanoparticles complexed with the plasmid encoding Lysenin. After transfection in vitro, melanoma cells can express Lysenin, resulting in necrosis, autophagy, and immunogenic cell death. The secretory signal peptide alters the intracellular distribution of the expressed product of Lysenin, thereby potentiating its anticancer efficacy. The intratumor injection of Lysenin gene formulation can efficiently kill the transfected melanoma cells and activate the antitumor immune response. Notably, no obvious systemic toxicity is observed during the treatment. Non-viralgene therapy based on Lysenin derived from Eisenia foetida exhibits potential in cancer therapy, which can inspire future cancer therapeutics.

Erythronecroptosis: an overview of necroptosis or programmed necrosis in red blood cells.

Necroptosis is considered a programmed necrosis that requires receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), and pore-forming mixed lineage kinase domain-like protein (MLKL) to trigger a regulated cell membrane lysis. Membrane rupture in necroptosis has been shown to fuel innate immune response due to release of damage-associated molecular patterns (DAMPs). Recently published studies indicate that mature erythrocytes can undergo necroptosis as well. In this review, we provide an outline of multiple cell death modes occurring in erythrocytes, discuss possible immunological aspects of diverse erythrocyte cell deaths, summarize available evidence related to the ability of erythrocytes to undergo necroptosis, outline key involved molecular mechanisms, and discuss the potential implication of erythrocyte necroptosis in the physiology and pathophysiology. Furthermore, we aim to highlight the interplay between necroptosis and eryptosis signaling in erythrocytes, emphasizing specific characteristics of these pathways distinct from their counterparts in nucleated cells. Thus, our review provides a comprehensive summary of the current knowledge of necroptosis in erythrocytes. To reflect critical differences between necroptosis of nucleated cells and necroptosis of erythrocytes, we suggest a term erythronecroptosis for necroptosis of enucleated cells.

Abstract 1191 TAK1 inhibition translocates pore-forming proteins, MLKL and gasdermins into mitochondria to generate reactive oxygen species

Abstract 1191 TAK1 inhibition translocates pore-forming proteins, MLKL and gasdermins into mitochondria to generate reactive oxygen species

NINJ1 mediates inflammatory cell death, PANoptosis, and lethality during infection conditions and heat stress

Innate immunity provides the first line of defense through multiple mechanisms, including pyrogen production and cell death. While elevated body temperature during infection is beneficial to clear pathogens, heat stress (HS) can lead to inflammation and pathology. Links between pathogen exposure, HS, cytokine release, and inflammation have been observed, but fundamental innate immune mechanisms driving pathology during pathogen exposure and HS remain unclear. Here, we use multiple genetic approaches to elucidate innate immune pathways in infection or LPS and HS models. Our results show that bacteria and LPS robustly increase inflammatory cell death during HS that is dependent on caspase-1, caspase-11, caspase-8, and RIPK3 through the PANoptosis pathway. Caspase-7 also contributes to PANoptosis in this context. Furthermore, NINJ1 is an important executioner of this cell death to release inflammatory molecules, independent of other pore-forming executioner proteins, gasdermin D, gasdermin E, and MLKL. In an in vivo HS model, mortality is reduced by deleting NINJ1 and fully rescued by deleting key PANoptosis molecules. Our findings suggest that therapeutic strategies blocking NINJ1 or its upstream regulators to prevent PANoptosis may reduce the release of inflammatory mediators and benefit patients.

Two Toxoplasma gondii putative pore-forming proteins, GRA47 and GRA72, influence small molecule permeability of the parasitophorous vacuole.

Toxoplasma gondii is a parasite that poses significant health risks to those with impaired immunity. It replicates inside host cells shielded by the PVM, which controls nutrient and waste exchange with the host. GRA72, previously identified as essential in the absence of the GRA17 nutrient channel, is implicated in forming an alternative nutrient channel. Here we found that GRA47 associates with GRA72 and is also important for the PVM's permeability to small molecules. Removal of GRA47 leads to distorted vacuoles and impairs small molecule transport across the PVM, resembling the effects of GRA17 and GRA72 deletions. Structural models suggest GRA47 and GRA72 form distinct pore structures, with a pore-lining histidine critical to their function. Toxoplasma strains lacking GRA47 or those with a histidine mutation have impaired growth and reduced virulence in mice, highlighting these proteins as potential targets for new treatments against toxoplasmosis.

Estimates of differential toxin expression governing heterogeneous intracellular lifespans of Streptococcus pneumoniae.

Following invasion of the host cell, pore-forming toxins secreted by pathogens compromise vacuole integrity and expose the microbe to diverse intracellular defence mechanisms. However, the quantitative correlation between toxin expression levels and consequent pore dynamics, fostering the intracellular life of pathogens, remains largely unexplored. In this study, using Streptococcus pneumoniae and its secreted pore-forming toxin pneumolysin (Ply) as a model system, we explored various facets of host-pathogen interactions in the host cytosol. Using time-lapse fluorescence imaging, we monitored pore formation dynamics and lifespans of different pneumococcal subpopulations inside host cells. Based on experimental histograms of various event timescales such as pore formation time, vacuolar death or cytosolic escape time and total degradation time, we developed a mathematical model based on first-passage processes that could correlate the event timescales to intravacuolar toxin accumulation. This allowed us to estimate Ply production rate, burst size and threshold Ply quantities that trigger these outcomes. Collectively, we present a general method that illustrates a correlation between toxin expression levels and pore dynamics, dictating intracellular lifespans of pathogens.

A cholesterol-binding bacterial toxin provides a strategy for identifying a specific Scap inhibitor that blocks lipid synthesis in animal cells

Lipid synthesis is regulated by the actions of Scap, a polytopic membrane protein that binds cholesterol in membranes of the endoplasmic reticulum (ER). When ER cholesterol levels are low, Scap activates SREBPs, transcription factors that upregulate genes for synthesis of cholesterol, fatty acids, and triglycerides. When ER cholesterol levels rise, the sterol binds to Scap, triggering conformational changes that prevent activation of SREBPs and halting synthesis of lipids. To achieve a molecular understanding of how cholesterol regulates the Scap/SREBP machine and to identify therapeutics for dysregulated lipid metabolism, cholesterol-mimetic compounds that specifically bind and inhibit Scap are needed. To accomplish this goal, we focused on Anthrolysin O (ALO), a pore-forming bacterial toxin that binds cholesterol with a specificity and sensitivity that is uncannily similar to Scap. We reasoned that a small molecule that would bind and inhibit ALO might also inhibit Scap. High-throughput screening of a ~300,000-compound library for ALO-binding unearthed one molecule, termed UT-59, which binds to Scap's cholesterol-binding site. Upon binding, UT-59 triggers the same conformation changes in Scap as those induced by cholesterol and blocks activation of SREBPs and lipogenesis in cultured cells. UT-59 also inhibits SREBP activation in the mouse liver. Unlike five previously reported inhibitors of SREBP activation, UT-59 is the only one that acts specifically by binding to Scap's cholesterol-binding site. Our approach to identify specific Scap inhibitors such as UT-59 holds great promise in developing therapeutic leads for human diseases stemming from elevated SREBP activation, such as fatty liver and certain cancers.

An aptamer-triggered hybridization chain reaction strategy for ultra-sensitive biological nanopore detection of aflatoxin B1

Biological nanopore refers to a type of pore-forming membrane proteins, providing a unique platform for single molecule detection of DNA, RNA, peptide, etc. The direct detection of small molecules, especially of food chemical contaminants by biological nanopore, remains challenging due to their fast translocation dynamics, trace amount, and the interference from the complex food matrix. The effective recognition and signal conversion methods need to be developed. Herein, we employ an aptamer-triggered hybridization chain reaction (AtHCR) strategy for the detection of a mycotoxin, aflatoxin B1 (AFB1) by an α-hemolysin nanopore. In the absence of AFB1, the formed dsDNA polymers in HCR show a low frequency of long-time blockage signals. The presence of AFB1 can inhibit the hybridization chain reaction through the interaction between AFB1 and its aptamer, so the remaining hairpins can easily block the nanopore and induce frequent long-time blockage signals. The results show that the event frequency of long-blockage has a linear relationship with the AFB1 concentration in the range of 6.6 × 10−1–6.6 × 102 pmol/L. The recognition and amplification effect of AtHCR allows nanopore to detect AFB1 at a detection limit of 0.54 pmol/L. Furthermore, the developed AtHCR strategy can help biological nanopore to detect AFB1 in corn sample with satisfactory accuracy. The recognition and amplification effect achieved by coupling AtHCR to biological nanopore offers an enzyme-free single-molecule technology for the rapid detection of food chemical contaminants.

Cryo-EM assists in capturing all the intermediate steps of pore formation by bacterial pore-forming toxins on the plasma membrane

Cryo-EM assists in capturing all the intermediate steps of pore formation by bacterial pore-forming toxins on the plasma membrane

25-Hydroxycholesterol prevents the lytic activity of the pore-forming toxin Cytolysin A

25-Hydroxycholesterol prevents the lytic activity of the pore-forming toxin Cytolysin A

Klebicin E, a pore-forming bacteriocin of Klebsiella pneumoniae, exploits the porin OmpC and the Ton system for translocation

Bacteriocins, which have narrow-spectrum activity and limited adverse effects, are promising alternatives to antibiotics. In this study, we identified klebicin E (KlebE), a small bacteriocin derived from Klebsiella pneumoniae. KlebE exhibited strong efficacy against multidrug-resistant K. pneumoniae isolates and conferred a significant growth advantage to the producing strain during intraspecies competition. A giant unilamellar vesicle leakage assay demonstrated the unique membrane permeabilization effect of KlebE, suggesting it is a pore-forming toxin. In addition to a colicin A-like C-terminal domain, KlebE also has a disordered N-terminal domain and a globular central domain. Pulldown assays and soft agar overlay experiments revealed the essential role of the outer membrane porin OmpC and the Ton system in KlebE recognition and cytotoxicity. Strong binding between KlebE and both OmpC and TonB was observed. The TonB-box, a crucial component of the toxin-TonB interaction, was identified as the 7-amino acid sequence (E3ETLTVV9) located in the N-terminal region. Further studies showed that a region near the bottom of the central domain of KlebE plays a primary role in recognizing OmpC, with eight residues surrounding this region identified as essential for KlebE toxicity. Finally, based on the discrepancies in OmpC sequences between the KlebE-resistant and sensitive strains, it was found that the 91st residue of OmpC, an aspartic acid residue, is a key determinant of KlebE toxicity. The identification and characterization of this toxin will facilitate the development of bacteriocin-based therapies targeting multidrug-resistant K. pneumoniae infections.