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13 pages, 2371 KiB

Open AccessArticle

Epsilon Toxin from Clostridium perfringens Induces the Generation of Extracellular Vesicles in HeLa Cells Overexpressing Myelin and Lymphocyte Protein

by Jonatan Dorca-Arévalo, Antonio Santana-Ruiz, Benjamín Torrejón-Escribano, Mireia Martín-Satué and Juan Blasi

Toxins 2024, 16(12), 525; https://doi.org/10.3390/toxins16120525 - 4 Dec 2024

Viewed by 967

Abstract

Epsilon toxin (ETX) from Clostridium perfringens is a pore-forming toxin (PFT) that crosses the blood–brain barrier and binds to myelin structures. In in vitro assays, ETX causes oligodendrocyte impairment, subsequently leading to demyelination. In fact, ETX has been associated with triggering multiple sclerosis. [...] Read more.

Epsilon toxin (ETX) from Clostridium perfringens is a pore-forming toxin (PFT) that crosses the blood–brain barrier and binds to myelin structures. In in vitro assays, ETX causes oligodendrocyte impairment, subsequently leading to demyelination. In fact, ETX has been associated with triggering multiple sclerosis. Myelin and lymphocyte protein (MAL) is widely considered to be the receptor for ETX as its presence is crucial for the effects of ETX on the plasma membrane of host cells that involve pore formation, resulting in cell death. To overcome the pores formed by PFTs, some host cells produce extracellular vesicles (EVs) to reduce the amount of pores inserted into the plasma membrane. The formation of EVs has not been studied for ETX in host cells. Here, we generated a highly sensitive clone from HeLa cells overexpressing the MAL-GFP protein in the plasma membrane. We observed that ETX induces the formation of EVs. Moreover, the MAL protein and ETX oligomers are found in these EVs, which are a very useful tool to decipher and study the mode of action of ETX and characterize the mechanisms involved in the binding of ETX to its receptor. Full article

(This article belongs to the Special Issue Toxin-Host Interaction of Clostridium Toxins)

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19 pages, 6415 KiB

Open AccessArticle

Proteome and Phosphoproteome Profiling Reveal the Toxic Mechanism of Clostridium perfringens Epsilon Toxin in MDCK Cells

by Nan Yue, Jing Huang, Mingxin Dong, Jiaxin Li, Shan Gao, Jing Wang, Yingshuang Wang, Dongxue Li, Xi Luo, Tingting Liu, Songyang Han, Lina Dong, Ming Chen, Jinglin Wang, Na Xu, Lin Kang and Wenwen Xin

Toxins 2024, 16(9), 394; https://doi.org/10.3390/toxins16090394 - 14 Sep 2024

Viewed by 1371

Abstract

Epsilon toxin (ETX), a potential agent of biological and toxic warfare, causes the death of many ruminants and threatens human health. It is crucial to understand the toxic mechanism of such a highly lethal and rapid course toxin. In this study, we detected [...] Read more.

Epsilon toxin (ETX), a potential agent of biological and toxic warfare, causes the death of many ruminants and threatens human health. It is crucial to understand the toxic mechanism of such a highly lethal and rapid course toxin. In this study, we detected the effects of ETX on the proteome and phosphoproteome of MDCK cells after 10 min and 30 min. A total of 44 differentially expressed proteins (DEPs) and 588 differentially phosphorylated proteins (DPPs) were screened in the 10 min group, while 73 DEPs and 489 DPPs were screened in the 30 min group. ETX-induced proteins and phosphorylated proteins were mainly located in the nucleus, cytoplasm, and mitochondria, and their enrichment pathways were related to transcription and translation, virus infection, and intercellular junction. Meanwhile, the protein–protein interaction network screened out several hub proteins, including SRSF1/2/6/7/11, SF3B1/2, NOP14/56, ANLN, GTPBP4, THOC2, and RRP1B. Almost all of these proteins were present in the spliceosome pathway, indicating that the spliceosome pathway is involved in ETX-induced cell death. Next, we used RNAi lentiviruses and inhibitors of several key proteins to verify whether these proteins play a critical role. The results confirmed that SRSF1, SF3B2, and THOC2 were the key proteins involved in the cytotoxic effect of ETX. In addition, we found that the common upstream kinase of these key proteins was SRPK1, and a reduction in the level of SRPK1 could also reduce ETX-induced cell death. This result was consistent with the phosphorylated proteomics analysis. In summary, our study demonstrated that ETX induces phosphorylation of SRSF1, SF3B2, THOC2, and SRPK1 proteins on the spliceosome pathway, which inhibits normal splicing of mRNA and leads to cell death. Full article

(This article belongs to the Special Issue Toxin-Host Interaction of Clostridium Toxins)

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18 pages, 3585 KiB

Open AccessArticle

PLGA Nanoparticle-Based Dissolving Microneedle Vaccine of Clostridium perfringens ε Toxin

by Wei Wan, Yue Li, Jing Wang, Zhiying Jin, Wenwen Xin, Lin Kang, Junhong Wang, Xiaoyang Li, Yakun Cao, Hao Yang, Jinglin Wang and Shan Gao

Toxins 2023, 15(7), 461; https://doi.org/10.3390/toxins15070461 - 19 Jul 2023

Cited by 7 | Viewed by 2720

Abstract

Epsilon toxin (ETX) is an exotoxin produced by type B and D Clostridium perfringens that causes enterotoxemia or necrotic enteritis in animals such as goats, sheep, and cattle. Vaccination is a key method in preventing such diseases. In this study, we developed a [...] Read more.

Epsilon toxin (ETX) is an exotoxin produced by type B and D Clostridium perfringens that causes enterotoxemia or necrotic enteritis in animals such as goats, sheep, and cattle. Vaccination is a key method in preventing such diseases. In this study, we developed a new type of dissolving microneedle patch (dMN) with a nanoparticle adjuvant for enhanced immune response to deliver the rETX^Y196E-C protein vaccine. We chose FDA-approved poly(lactic-co-glycolic acid) (PLGA) to prepare nanospheres as the vaccine adjuvant and introduced dimethyldioctadecylammonium bromide (DDAB) to make the surface of PLGA nanoparticles (PLGA NPs) positively charged for antigen adsorption. PLGA NPs with a diameter of 100~200 nm, a surface ZETA potential of approximately +40 mV, and good safety were successfully prepared and could effectively adsorb rETX^Y196E-C protein. Using non-toxic and antibacterial fish gelatin as the microneedle (MN) matrix, we prepared a PLGA-DDAB dMN vaccine with good mechanical properties that successfully penetrated the skin. After immunization of subcutaneous (SC) and dMN, antibody titers of the PLGA and Al adjuvant groups were similar in both two immune ways. However, in vivo neutralization experiments showed that the dMN vaccines had a better protective effect. When challenged with 100 × LD₅₀ GST-ETX, the survival rate of the MN group was 100%, while that of the SC Al group was 80%. However, a 100% protective effect was achieved in both immunization methods using PLGA NPs. In vitro neutralization experiments showed that the serum antibodies from the dMN and SC PLGA NPs groups both protect naive mice from 10 × LD₅₀ GST-ETX attack after being diluted 20 times and could also protect MDCK cells from 20 × CT₅₀ GST-ETX attack. In conclusion, the PLGA-DDAB dMN vaccine we prepared has good mechanical properties, immunogenicity, and protection, and can effectively prevent ETX poisoning. This provides a better way of delivering protein vaccines. Full article

(This article belongs to the Special Issue Toxin-Host Interaction of Clostridium Toxins)

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19 pages, 2316 KiB

Open AccessArticle

Clostridium perfringens Epsilon Toxin Binds to and Kills Primary Human Lymphocytes

by Samantha V. Shetty, Michael R. Mazzucco, Paige Winokur, Sylvia V. Haigh, Kareem Rashid Rumah, Vincent A. Fischetti, Timothy Vartanian and Jennifer R. Linden

Toxins 2023, 15(7), 423; https://doi.org/10.3390/toxins15070423 - 29 Jun 2023

Cited by 3 | Viewed by 2568

Abstract

Clostridium perfringens epsilon toxin (ETX) is the third most lethal bacterial toxin and has been suggested to be an environmental trigger of multiple sclerosis, an immune-mediated disease of the human central nervous system. However, ETX cytotoxicity on primary human cells has not been [...] Read more.

Clostridium perfringens epsilon toxin (ETX) is the third most lethal bacterial toxin and has been suggested to be an environmental trigger of multiple sclerosis, an immune-mediated disease of the human central nervous system. However, ETX cytotoxicity on primary human cells has not been investigated. In this article, we demonstrate that ETX preferentially binds to and kills human lymphocytes expressing increased levels of the myelin and lymphocyte protein MAL. Using flow cytometry, ETX binding was determined to be time and dose dependent and was highest for CD4+ cells, followed by CD8+ and then CD19+ cells. Similar results were seen with ETX-induced cytotoxicity. To determine if ETX preference for CD4+ cells was related to MAL expression, MAL gene expression was determined by RT-qPCR. CD4+ cells had the highest amount of Mal gene expression followed by CD8+ and CD19+ cells. These data indicate that primary human cells are susceptible to ETX and support the hypothesis that MAL is a main receptor for ETX. Interestingly, ETX bindings to human lymphocytes suggest that ETX may influence immune response in multiple sclerosis. Full article

(This article belongs to the Special Issue Toxin-Host Interaction of Clostridium Toxins)

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15 pages, 7662 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Domperidone Protects Cells from Intoxication with Clostridioides difficile Toxins by Inhibiting Hsp70-Assisted Membrane Translocation

by Maria Braune-Yan, Jinfang Jia, Mary Wahba, Johannes Schmid, Panagiotis Papatheodorou, Holger Barth and Katharina Ernst

Toxins 2023, 15(6), 384; https://doi.org/10.3390/toxins15060384 - 7 Jun 2023

Cited by 2 | Viewed by 2313

Abstract

Clostridioides difficile infections cause severe symptoms ranging from diarrhea to pseudomembranous colitis due to the secretion of AB-toxins, TcdA and TcdB. Both toxins are taken up into cells through receptor-mediated endocytosis, autoproteolytic processing and translocation of their enzyme domains from acidified endosomes into [...] Read more.

Clostridioides difficile infections cause severe symptoms ranging from diarrhea to pseudomembranous colitis due to the secretion of AB-toxins, TcdA and TcdB. Both toxins are taken up into cells through receptor-mediated endocytosis, autoproteolytic processing and translocation of their enzyme domains from acidified endosomes into the cytosol. The enzyme domains glucosylate small GTPases such as Rac1, thereby inhibiting processes such as actin cytoskeleton regulation. Here, we demonstrate that specific pharmacological inhibition of Hsp70 activity protected cells from TcdB intoxication. In particular, the established inhibitor VER-155008 and the antiemetic drug domperidone, which was found to be an Hsp70 inhibitor, reduced the number of cells with TcdB-induced intoxication morphology in HeLa, Vero and intestinal CaCo-2 cells. These drugs also decreased the intracellular glucosylation of Rac1 by TcdB. Domperidone did not inhibit TcdB binding to cells or enzymatic activity but did prevent membrane translocation of TcdB’s glucosyltransferase domain into the cytosol. Domperidone also protected cells from intoxication with TcdA as well as CDT toxin produced by hypervirulent strains of Clostridioides difficile. Our results reveal Hsp70 requirement as a new aspect of the cellular uptake mechanism of TcdB and identified Hsp70 as a novel drug target for potential therapeutic strategies required to combat severe Clostridioides difficile infections. Full article

(This article belongs to the Special Issue Toxin-Host Interaction of Clostridium Toxins)

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13 pages, 2011 KiB

Open AccessArticle

Genomic Insights into Virulence Factors and Multi-Drug Resistance in Clostridium perfringens IRMC2505A

by Reem AlJindan, Doaa M. AlEraky, Maha Farhat, Noor B. Almandil, Sayed AbdulAzeez and Jesu Francis Borgio

Toxins 2023, 15(6), 359; https://doi.org/10.3390/toxins15060359 - 25 May 2023

Cited by 8 | Viewed by 3416

Abstract

Clostridium perfringens is a spore-forming, Gram-positive anaerobic pathogen that causes several disorders in humans and animals. A multidrug-resistant Clostridium strain was isolated from the fecal sample of a patient who was clinically suspected of gastrointestinal infection and had a recent history of antibiotic [...] Read more.

Clostridium perfringens is a spore-forming, Gram-positive anaerobic pathogen that causes several disorders in humans and animals. A multidrug-resistant Clostridium strain was isolated from the fecal sample of a patient who was clinically suspected of gastrointestinal infection and had a recent history of antibiotic exposure and diarrhea. The strain was identified by 16s rRNA sequencing as Clostridium perfringens. The strain’s pathogenesis was analyzed through its complete genome, specifically antimicrobial resistance-related genes. The Clostridium perfringens IRMC2505A genome contains 19 (Alr, Ddl, dxr, EF-G, EF-Tu, folA, Dfr, folP, gyrA, gyrB, Iso-tRNA, kasA, MurA, rho, rpoB, rpoC, S10p, and S12p) antibiotic-susceptible genetic species according to the k-mer-based detection of antimicrobial resistance genes. Genome mapping using CARD and VFDB databases revealed significant (p-value = 1 × 10⁻²⁶) genes with aligned reads against antibiotic-resistant genes or virulence factors, including phospholipase C, perfringolysin O, collagenase, hyaluronidase, alpha-clostripain, exo-alpha-sialidase, and sialidase activity. In conclusion, this is the first report on C. perfringens from Saudi Arabia that conducted whole genome sequencing of IRMC2505A and confirmed the strain as an MDR bacterium with several virulence factors. Developing control strategies requires a detailed understanding of the epidemiology of C. perfringens, its virulence factors, and regional antimicrobial resistance patterns. Full article

(This article belongs to the Special Issue Toxin-Host Interaction of Clostridium Toxins)

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Review

Jump to: Research

14 pages, 2539 KiB

Open AccessReview

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

by Richard W. Titball

Toxins 2024, 16(4), 180; https://doi.org/10.3390/toxins16040180 - 7 Apr 2024

Cited by 1 | Viewed by 2391

Abstract

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, [...] Read more.

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. Full article

(This article belongs to the Special Issue Toxin-Host Interaction of Clostridium Toxins)

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27 pages, 3347 KiB

Open AccessEditor’s ChoiceReview

Recent Developments in Vaccine Design: From Live Vaccines to Recombinant Toxin Vaccines

by Sonal Gupta and Sabine Pellett

Toxins 2023, 15(9), 563; https://doi.org/10.3390/toxins15090563 - 8 Sep 2023

Cited by 17 | Viewed by 7374

Abstract

Vaccines are one of the most effective strategies to prevent pathogen-induced illness in humans. The earliest vaccines were based on live inoculations with low doses of live or related pathogens, which carried a relatively high risk of developing the disease they were meant [...] Read more.

Vaccines are one of the most effective strategies to prevent pathogen-induced illness in humans. The earliest vaccines were based on live inoculations with low doses of live or related pathogens, which carried a relatively high risk of developing the disease they were meant to prevent. The introduction of attenuated and killed pathogens as vaccines dramatically reduced these risks; however, attenuated live vaccines still carry a risk of reversion to a pathogenic strain capable of causing disease. This risk is completely eliminated with recombinant protein or subunit vaccines, which are atoxic and non-infectious. However, these vaccines require adjuvants and often significant optimization to induce robust T-cell responses and long-lasting immune memory. Some pathogens produce protein toxins that cause or contribute to disease. To protect against the effects of such toxins, chemically inactivated toxoid vaccines have been found to be effective. Toxoid vaccines are successfully used today at a global scale to protect against tetanus and diphtheria. Recent developments for toxoid vaccines are investigating the possibilities of utilizing recombinant protein toxins mutated to eliminate biologic activity instead of chemically inactivated toxins. Finally, one of the most contemporary approaches toward vaccine design utilizes messenger RNA (mRNA) as a vaccine candidate. This approach was used globally to protect against coronavirus disease during the COVID-19 pandemic that began in 2019, due to its advantages of quick production and scale-up, and effectiveness in eliciting a neutralizing antibody response. Nonetheless, mRNA vaccines require specialized storage and transport conditions, posing challenges for low- and middle-income countries. Among multiple available technologies for vaccine design and formulation, which technology is most appropriate? This review focuses on the considerable developments that have been made in utilizing diverse vaccine technologies with a focus on vaccines targeting bacterial toxins. We describe how advancements in vaccine technology, combined with a deeper understanding of pathogen–host interactions, offer exciting and promising avenues for the development of new and improved vaccines. Full article

(This article belongs to the Special Issue Toxin-Host Interaction of Clostridium Toxins)

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