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A special issue of Toxins (ISSN 2072-6651).

Deadline for manuscript submissions: closed (31 October 2021) | Viewed by 53501

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Dr. Ludger Johannes

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Cellular and Chemical Biology Unit, Institut Curie, PSL Research University, U1143 INSERM, UMR3666 CNRS, 26 rue d’Ulm, 75248 Paris CEDEX 05, France
Interests: endocytosis; intracellular trafficking; protein toxins; lectins; glycobiology; glycosphingolipids; lipid rafts; membrane compartmentalization; intracellular delivery; membrane translocation; immunotherapy; tumor targeting

Special Issue Information

Protein toxins from bacteria and plants are a serious threat to human and animal health. However, because of their intimate interactions with host cells, they have also become valuable tools to molecularly dissect cell biological functions that range from endocytosis and intracellular trafficking to cell signaling and apoptosis. Several characteristics such as ease for biochemical handling and the robustness of phenotypes are responsible for their success as cell biological tools. The in-depth understanding of their activities has, in turn, also attracted attention to their use as pharmacological tools to manipulate cellular processes that misfunction in disease situations or that can be targeted for therapeutic intervention. Cancer immunotherapy by immunotoxins or toxin subunit-based vaccines are only some of the many possibilities for using toxins in biomedical research. In this special issue of Toxins, primary research papers and review articles are assembled that address the aspects that are summarized above. The reader is thereby provided with an up-to-date perspective on some of the most recent and dynamic contributions of toxins to fundamental cell biology and applied cellular pharmacology research.

Dr. Ludger Johannes
Guest Editor

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Keywords

endocytosis
intracellular trafficking
lipid domains
apoptosis
signaling
broad spectrum inhibitors
immunotherapy

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Published Papers (11 papers)

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Editorial

Jump to: Research, Review

3 pages, 201 KiB

Open AccessEditorial

Application of Protein Toxins as Cell Biological and Pharmacological Tools

by Ludger Johannes

Toxins 2022, 14(4), 242; https://doi.org/10.3390/toxins14040242 - 28 Mar 2022

Cited by 2 | Viewed by 1861

Abstract

Protein toxins from bacteria and plants are a serious threat to human and animal health [...] Full article

(This article belongs to the Special Issue Application of Protein Toxins as Cell Biological and Pharmacological Tools)

Research

Jump to: Editorial, Review

19 pages, 5828 KiB

Open AccessArticle

The Two Sweet Sides of Janus Lectin Drive Crosslinking of Liposomes to Cancer Cells and Material Uptake

by Lina Siukstaite, Francesca Rosato, Anna Mitrovic, Peter Fritz Müller, Katharina Kraus, Simona Notova, Anne Imberty and Winfried Römer

Toxins 2021, 13(11), 792; https://doi.org/10.3390/toxins13110792 - 9 Nov 2021

Cited by 12 | Viewed by 5380

Abstract

A chimeric, bispecific Janus lectin has recently been engineered with different, rationally oriented recognition sites. It can bind simultaneously to sialylated and fucosylated glycoconjugates. Because of its multivalent architecture, this lectin reaches nanomolar avidities for sialic acid and fucose. The lectin was designed to detect hypersialylation—a dysregulation in physiological glycosylation patterns, which promotes the tumor growth and progression of several cancer types. In this study, the characteristic properties of this bispecific Janus lectin were investigated on human cells by flow cytometry and confocal microscopy in order to understand the fundamentals of its interactions. We evaluated its potential in targeted drug delivery, precisely leading to the cellular uptake of liposomal content in human epithelial cancer cells. We successfully demonstrated that Janus lectin mediates crosslinking of glyco-decorated giant unilamellar vesicles (GUVs) and H1299 lung epithelial cells. Strikingly, the Janus lectin induced the internalization of liposomal lipids and also of complete GUVs. Our findings serve as a solid proof of concept for lectin-mediated targeted drug delivery using glyco-decorated liposomes as possible drug carriers to cells of interest. The use of Janus lectin for tumor recognition certainly broadens the possibilities for engineering diverse tailor-made lectin constructs, specifically targeting extracellular structures of high significance in pathological conditions. Full article

(This article belongs to the Special Issue Application of Protein Toxins as Cell Biological and Pharmacological Tools)

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19 pages, 4759 KiB

Open AccessArticle

Engineering of Cytolethal Distending Toxin B by Its Reducing Immunogenicity and Maintaining Stability as a New Drug Candidate for Tumor Therapy; an In Silico Study

by Maryam Keshtvarz, Mahdieh Mahboobi, Marek Kieliszek, Antoni Miecznikowski, Hamid Sedighian, Milad Rezaei, Mohammad Ali Haghighi, Zahra Zareh and Ehsan Rezaei

Toxins 2021, 13(11), 785; https://doi.org/10.3390/toxins13110785 - 5 Nov 2021

Cited by 7 | Viewed by 2677

Abstract

The cytolethal distending toxin (CDT), Haemophilus ducreyi, is one of the bacterial toxins that have recently been considered for targeted therapies, especially in cancer therapies. CDT is an A-B2 exotoxin. Its catalytic subunit (CdtB) is capable of inducing DNA double strand breaks, cell cycle arrest and apoptosis in host eukaryotic cells. The sequence alignment indicates that the CdtB is structurally homologyr to phosphatases and deoxyribonucleases I (DNase I). Recently, it has been found that CdtB toxicity is mainly related to its nuclease activity. The immunogenicity of CDT can reduce its effectiveness in targeted therapies. However, the toxin can be very useful if its immunogenicity is significantly reduced. Detecting hotspot ectopic residues by computational servers and then mutating them to eliminate B-cell epitopes is a promising approach to reduce the immunogenicity of foreign protein-based therapeutics. By the mentioned method, in this study, we try to reduce the immunogenicity of the CdtB- protein sequence. This study initially screened residue of the CdtB is B-cell epitopes both linearly and conformationally. By overlapping the B-cell epitopes with the excluded conserve residues, and active and enzymatic sites, four residues were allowed to be mutated. There were two mutein options that show reduced antigenicity probability. Option one was N19F, G74I, and S161F with a VaxiJen score of 0.45 and the immune epitope database (IEDB) score of 1.80, and option two was N19F, G74I, and S161W with a VaxiJen score of 0.45 and IEDB score of 1.88. The 3D structure of the proposed sequences was evaluated and refined. The structural stability of native and mutant proteins was accessed through molecular dynamic simulation. The results showed that the mutations in the mutants caused no considerable changes in their structural stability. However, mutant 1 reveals more thermodynamic stability during the simulation. The applied approaches in this study can be used as rough guidelines for finding hot spot immunogen regions in the therapeutic proteins. Our results provide a new version of CdtB that, due to reduced immunogenicity and increased stability, can be used in toxin-based drugs such as immunotoxins. Full article

(This article belongs to the Special Issue Application of Protein Toxins as Cell Biological and Pharmacological Tools)

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20 pages, 32926 KiB

Open AccessArticle

Tamoxifen Derivatives Alter Retromer-Dependent Endosomal Tubulation and Sorting to Block Retrograde Trafficking of Shiga Toxins

by Andrey S. Selyunin, Karinel Nieves-Merced, Danyang Li, Stanton F. McHardy and Somshuvra Mukhopadhyay

Toxins 2021, 13(6), 424; https://doi.org/10.3390/toxins13060424 - 15 Jun 2021

Cited by 6 | Viewed by 2851

Abstract

Shiga toxin 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol of cells where they target ribosomes. As retrograde trafficking is essential for disease, inhibiting STx1/STx2 trafficking is therapeutically promising. Recently, we discovered that the chemotherapeutic drug tamoxifen potently inhibits the trafficking of STx1/STx2 at the critical early endosome-to-Golgi step. We further reported that the activity of tamoxifen against STx1/STx2 is independent of its selective estrogen receptor modulator (SERM) property and instead depends on its weakly basic chemical nature, which allows tamoxifen to increase endolysosomal pH and alter the recruitment of retromer to endosomes. The goal of the current work was to obtain a better understanding of the mechanism of action of tamoxifen against the more disease-relevant toxin STx2, and to differentiate between the roles of changes in endolysosomal pH and retromer function. Structure activity relationship (SAR) analyses revealed that a weakly basic amine group was essential for anti-STx2 activity. However, ability to deacidify endolysosomes was not obligatorily necessary because a tamoxifen derivative that did not increase endolysosomal pH exerted reduced, but measurable, activity. Additional assays demonstrated that protective derivatives inhibited the formation of retromer-dependent, Golgi-directed, endosomal tubules, which mediate endosome-to-Golgi transport, and the sorting of STx2 into these tubules. These results identify retromer-mediated endosomal tubulation and sorting to be fundamental processes impacted by tamoxifen; provide an explanation for the inhibitory effect of tamoxifen on STx2; and have important implications for the therapeutic use of tamoxifen, including its development for treating Shiga toxicosis. Full article

(This article belongs to the Special Issue Application of Protein Toxins as Cell Biological and Pharmacological Tools)

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25 pages, 2140 KiB

Open AccessArticle

Shiga Toxin (Stx)-Binding Glycosphingolipids of Primary Human Renal Cortical Epithelial Cells (pHRCEpiCs) and Stx-Mediated Cytotoxicity

by Johanna Detzner, Elisabeth Krojnewski, Gottfried Pohlentz, Daniel Steil, Hans-Ulrich Humpf, Alexander Mellmann, Helge Karch and Johannes Müthing

Toxins 2021, 13(2), 139; https://doi.org/10.3390/toxins13020139 - 12 Feb 2021

Cited by 7 | Viewed by 2689

Abstract

Human kidney epithelial cells are supposed to be directly involved in the pathogenesis of the hemolytic–uremic syndrome (HUS) caused by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC). The characterization of the major and minor Stx-binding glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), respectively, of primary human renal cortical epithelial cells (pHRCEpiCs) revealed GSLs with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Using detergent-resistant membranes (DRMs) and non-DRMs, Gb3Cer and Gb4Cer prevailed in the DRM fractions, suggesting their association with microdomains in the liquid-ordered membrane phase. A preference of Gb3Cer and Gb4Cer endowed with C24:0 fatty acid accompanied by minor monounsaturated C24:1-harboring counterparts was observed in DRMs, whereas the C24:1 fatty acid increased in relation to the saturated equivalents in non-DRMs. A shift of the dominant phospholipid phosphatidylcholine with saturated fatty acids in the DRM to unsaturated species in the non-DRM fractions correlated with the GSL distribution. Cytotoxicity assays gave a moderate susceptibility of pHRCEpiCs to the Stx1a and Stx2a subtypes when compared to highly sensitive Vero-B4 cells. The results indicate that presence of Stx-binding GSLs per se and preferred occurrence in microdomains do not necessarily lead to a high cellular susceptibility towards Stx. Full article

(This article belongs to the Special Issue Application of Protein Toxins as Cell Biological and Pharmacological Tools)

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Review

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20 pages, 2005 KiB

Open AccessReview

STxB as an Antigen Delivery Tool for Mucosal Vaccination

by Eric Tartour and Ludger Johannes

Toxins 2022, 14(3), 202; https://doi.org/10.3390/toxins14030202 - 10 Mar 2022

Cited by 9 | Viewed by 5019

Abstract

Immunotherapy against cancer and infectious disease holds the promise of high efficacy with minor side effects. Mucosal vaccines to protect against tumors or infections disease agents that affect the upper airways or the lung are still lacking, however. One mucosal vaccine candidate is the B-subunit of Shiga toxin, STxB. In this review, we compare STxB to other immunotherapy vectors. STxB is a non-toxic protein that binds to a glycosylated lipid, termed globotriaosylceramide (Gb3), which is preferentially expressed by dendritic cells. We review the use of STxB for the cross-presentation of tumor or viral antigens in a MHC class I-restricted manner to induce humoral immunity against these antigens in addition to polyfunctional and persistent CD4⁺ and CD8⁺ T lymphocytes capable of protecting against viral infection or tumor growth. Other literature will be summarized that documents a powerful induction of mucosal IgA and resident memory CD8⁺ T cells against mucosal tumors specifically when STxB-antigen conjugates are administered via the nasal route. It will also be pointed out how STxB-based vaccines have been shown in preclinical cancer models to synergize with other therapeutic modalities (immune checkpoint inhibitors, anti-angiogenic therapy, radiotherapy). Finally, we will discuss how molecular aspects such as low immunogenicity, cross-species conservation of Gb3 expression, and lack of toxicity contribute to the competitive positioning of STxB among the different DC targeting approaches. STxB thereby appears as an original and innovative tool for the development of mucosal vaccines in infectious diseases and cancer. Full article

(This article belongs to the Special Issue Application of Protein Toxins as Cell Biological and Pharmacological Tools)

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15 pages, 1173 KiB

Open AccessReview

Shiga Toxins as Antitumor Tools

by Aude Robert and Joëlle Wiels

Toxins 2021, 13(10), 690; https://doi.org/10.3390/toxins13100690 - 28 Sep 2021

Cited by 19 | Viewed by 6139

Abstract

Shiga toxins (Stxs), also known as Shiga-like toxins (SLT) or verotoxins (VT), constitute a family of structurally and functionally related cytotoxic proteins produced by the enteric pathogens Shigella dysenteriae type 1 and Stx-producing Escherichia coli (STEC). Infection with these bacteria causes bloody diarrhea and other pathological manifestations that can lead to HUS (hemolytic and uremic syndrome). At the cellular level, Stxs bind to the cellular receptor Gb3 and inhibit protein synthesis by removing an adenine from the 28S rRNA. This triggers multiple cellular signaling pathways, including the ribotoxic stress response (RSR), unfolded protein response (UPR), autophagy and apoptosis. Stxs cause several pathologies of major public health concern, but their specific targeting of host cells and efficient delivery to the cytosol could potentially be exploited for biomedical purposes. Moreover, high levels of expression have been reported for the Stxs receptor, Gb3/CD77, in Burkitt’s lymphoma (BL) cells and on various types of solid tumors. These properties have led to many attempts to develop Stxs as tools for biomedical applications, such as cancer treatment or imaging, and several engineered Stxs are currently being tested. We provide here an overview of these studies. Full article

(This article belongs to the Special Issue Application of Protein Toxins as Cell Biological and Pharmacological Tools)

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18 pages, 2902 KiB

Open AccessReview

Cholera Toxin as a Probe for Membrane Biology

by Anne K. Kenworthy, Stefanie S. Schmieder, Krishnan Raghunathan, Ajit Tiwari, Ting Wang, Christopher V. Kelly and Wayne I. Lencer

Toxins 2021, 13(8), 543; https://doi.org/10.3390/toxins13080543 - 3 Aug 2021

Cited by 30 | Viewed by 8686

Abstract

Cholera toxin B-subunit (CTxB) has emerged as one of the most widely utilized tools in membrane biology and biophysics. CTxB is a homopentameric stable protein that binds tightly to up to five GM1 glycosphingolipids. This provides a robust and tractable model for exploring membrane structure and its dynamics including vesicular trafficking and nanodomain assembly. Here, we review important advances in these fields enabled by use of CTxB and its lipid receptor GM1. Full article

(This article belongs to the Special Issue Application of Protein Toxins as Cell Biological and Pharmacological Tools)

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9 pages, 1503 KiB

Open AccessReview

Computational Approaches to Explore Bacterial Toxin Entry into the Host Cell

by Weria Pezeshkian, Julian C. Shillcock and John H. Ipsen

Toxins 2021, 13(7), 449; https://doi.org/10.3390/toxins13070449 - 28 Jun 2021

Cited by 4 | Viewed by 3754

Abstract

Many bacteria secrete toxic protein complexes that modify and disrupt essential processes in the infected cell that can lead to cell death. To conduct their action, these toxins often need to cross the cell membrane and reach a specific substrate inside the cell. The investigation of these protein complexes is essential not only for understanding their biological functions but also for the rational design of targeted drug delivery vehicles that must navigate across the cell membrane to deliver their therapeutic payload. Despite the immense advances in experimental techniques, the investigations of the toxin entry mechanism have remained challenging. Computer simulations are robust complementary tools that allow for the exploration of biological processes in exceptional detail. In this review, we first highlight the strength of computational methods, with a special focus on all-atom molecular dynamics, coarse-grained, and mesoscopic models, for exploring different stages of the toxin protein entry mechanism. We then summarize recent developments that are significantly advancing our understanding, notably of the glycolipid–lectin (GL-Lect) endocytosis of bacterial Shiga and cholera toxins. The methods discussed here are also applicable to the design of membrane-penetrating nanoparticles and the study of the phenomenon of protein phase separation at the surface of the membrane. Finally, we discuss other likely routes for future development. Full article

(This article belongs to the Special Issue Application of Protein Toxins as Cell Biological and Pharmacological Tools)

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21 pages, 1752 KiB

Open AccessReview

Therapeutic Uses of Bacterial Subunit Toxins

by Clifford Lingwood

Toxins 2021, 13(6), 378; https://doi.org/10.3390/toxins13060378 - 26 May 2021

Cited by 17 | Viewed by 7091

Abstract

The B subunit pentamer verotoxin (VT aka Shiga toxin-Stx) binding to its cellular glycosphingolipid (GSL) receptor, globotriaosyl ceramide (Gb₃) mediates internalization and the subsequent receptor mediated retrograde intracellular traffic of the AB5 subunit holotoxin to the endoplasmic reticulum. Subunit separation and cytosolic A subunit transit via the ER retrotranslocon as a misfolded protein mimic, then inhibits protein synthesis to kill cells, which can cause hemolytic uremic syndrome clinically. This represents one of the most studied systems of prokaryotic hijacking of eukaryotic biology. Similarly, the interaction of cholera AB5 toxin with its GSL receptor, GM1 ganglioside, is the key component of the gastrointestinal pathogenesis of cholera and follows the same retrograde transport pathway for A subunit cytosol access. Although both VT and CT are the cause of major pathology worldwide, the toxin–receptor interaction is itself being manipulated to generate new approaches to control, rather than cause, disease. This arena comprises two areas: anti neoplasia, and protein misfolding diseases. CT/CTB subunit immunomodulatory function and anti-cancer toxin immunoconjugates will not be considered here. In the verotoxin case, it is clear that Gb₃ (and VT targeting) is upregulated in many human cancers and that there is a relationship between GSL expression and cancer drug resistance. While both verotoxin and cholera toxin similarly hijack the intracellular ERAD quality control system of nascent protein folding, the more widespread cell expression of GM1 makes cholera the toxin of choice as the means to more widely utilise ERAD targeting to ameliorate genetic diseases of protein misfolding. Gb₃ is primarily expressed in human renal tissue. Glomerular endothelial cells are the primary VT target but Gb₃ is expressed in other endothelial beds, notably brain endothelial cells which can mediate the encephalopathy primarily associated with VT2-producing E. coli infection. The Gb₃ levels can be regulated by cytokines released during EHEC infection, which complicate pathogenesis. Significantly Gb₃ is upregulated in the neovasculature of many tumours, irrespective of tumour Gb₃ status. Gb₃ is markedly increased in pancreatic, ovarian, breast, testicular, renal, astrocytic, gastric, colorectal, cervical, sarcoma and meningeal cancer relative to the normal tissue. VT has been shown to be effective in mouse xenograft models of renal, astrocytoma, ovarian, colorectal, meningioma, and breast cancer. These studies are herein reviewed. Both CT and VT (and several other bacterial toxins) access the cell cytosol via cell surface ->ER transport. Once in the ER they interface with the protein folding homeostatic quality control pathway of the cell -ERAD, (ER associated degradation), which ensures that only correctly folded nascent proteins are allowed to progress to their cellular destinations. Misfolded proteins are translocated through the ER membrane and degraded by cytosolic proteosome. VT and CT A subunits have a C terminal misfolded protein mimic sequence to hijack this transporter to enter the cytosol. This interface between exogenous toxin and genetically encoded endogenous mutant misfolded proteins, provides a new therapeutic basis for the treatment of such genetic diseases, e.g., Cystic fibrosis, Gaucher disease, Krabbe disease, Fabry disease, Tay-Sachs disease and many more. Studies showing the efficacy of this approach in animal models of such diseases are presented. Full article

(This article belongs to the Special Issue Application of Protein Toxins as Cell Biological and Pharmacological Tools)

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29 pages, 41198 KiB

Open AccessReview

The Protein Toxins Ricin and Shiga Toxin as Tools to Explore Cellular Mechanisms of Internalization and Intracellular Transport

by Kirsten Sandvig, Simona Kavaliauskiene and Tore Skotland

Toxins 2021, 13(6), 377; https://doi.org/10.3390/toxins13060377 - 25 May 2021

Cited by 20 | Viewed by 4947

Abstract

Protein toxins secreted by bacteria and found in plants can be threats to human health. However, their extreme toxicity can also be exploited in different ways, e.g., to produce hybrid toxins directed against cancer cells and to study transport mechanisms in cells. Investigations during the last decades have shown how powerful these molecules are as tools in cell biological research. Here, we first present a partly historical overview, with emphasis on Shiga toxin and ricin, of how such toxins have been used to characterize processes and proteins of importance for their trafficking. In the second half of the article, we describe how one can now use toxins to investigate the role of lipid classes for intracellular transport. In recent years, it has become possible to quantify hundreds of lipid species using mass spectrometry analysis. Thus, it is also now possible to explore the importance of lipid species in intracellular transport. The detailed analyses of changes in lipids seen under conditions of inhibited toxin transport reveal previously unknown connections between syntheses of lipid classes and demonstrate the ability of cells to compensate under given conditions. Full article

(This article belongs to the Special Issue Application of Protein Toxins as Cell Biological and Pharmacological Tools)

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