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Special Issue "Novel Aspects of Bacterial AB5-Toxins"

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A special issue of Toxins (ISSN 2072-6651). This special issue belongs to the section "Bacterial Toxins".

Deadline for manuscript submissions: closed (31 January 2022) | Viewed by 10063

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Prof. Dr. Holger Barth

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Institut für Pharmakologie und Toxikologie, Universitätsklinikum Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany
Interests: actin; bacterial protein toxins; macrophages; rho-gtpases; cellular uptake and intracellular membrane transport of bacterial toxins; interaction of bacterial toxins with host cell chaperones; bacterial toxins as molecular trojan horses for drug delivery
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Prof. Dr. Herbert Schmidt

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Institute of Food Science and Biotechnologie, Department of Food Microbiology and -hygiene, University of Hohenheim, Garbenstrasse 28, 70599 Stuttgart, Germany

Special Issue Information

Dear Colleagues,

Bacterial AB5 toxins consist of a highly specialized group of composite proteins, which have been implicated in serious human diseases. Current important members of the bacterial AB5 toxin family are cholera toxin (Ctx) of Vibrio cholerae, pertussis toxin (Ptx) of Bordetella pertussis, Shiga toxin (Stx), subtilase cytotoxin (SubAB), and heat-labile Enterotoxin (Lt) of pathogenic Escherichia coli. More recently, other bacterial AB5 toxins have been described, such as ArtAB of Salmonella enterica serovar Typhi and Ecx of E. coli. AB5 toxins share a similar hexameric architecture, consisting of an enzymatic subunit (A) and five receptor-binding B subunits that form a pentamer. Whereas the enzymatic A subunits mediate the toxic effects in their eukaryotic target cells, the B subunits guide the holotoxin to the target cells by specific receptor binding and finally deliver the A subunits into the cytosol where they interact with their substrate molecules. Binding, uptake, and intracellular transport of AB5 toxins follow similar paths but are toxin-specific, as described in the literature.

During recent years, interesting new aspects of AB5 toxins concerning subunit structures, assembly, binding, uptake, and the AB5 paradigm have been revealed by several authors. Moreover, attractive novel approaches to specifically inhibit toxin uptake and thereby cell intoxication were reported. The aim of this Special Issue is to present novel aspects of AB5 toxins to get more detailed insights into the structure and function of this important group of protein exotoxins.

Prof. Dr. Holger Barth
Prof. Dr. Herbert Schmidt
Guest Editors

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Keywords

AB5 toxins
Shiga toxin
subtilase cytotoxin
Pertussistoxin
Choleratoxin
subunit assembly
binding
translocation
function
inhibitors

Published Papers (5 papers)

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Research

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Open AccessArticle

Cell-Free Systems Enable the Production of AB₅ Toxins for Diagnostic Applications

Franziska Ramm

Lena Jack

Danny Kaser

Jeffrey L. Schloßhauer

Anne Zemella

and

Stefan Kubick

Toxins 2022, 14(4), 233; https://doi.org/10.3390/toxins14040233 - 23 Mar 2022

Cited by 3 | Viewed by 1741

Abstract

Cell-free protein synthesis (CFPS) represents a versatile key technology for the production of toxic proteins. As a cell lysate, rather than viable cells, is used, the toxic effects on the host organism can be circumvented. The open nature of cell-free systems allows for the addition of supplements affecting protein concentration and folding. Here, we present the cell-free synthesis and functional characterization of two AB₅ toxins, namely the cholera toxin (Ctx) and the heat-labile enterotoxin (LT), using two eukaryotic cell-free systems based on Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cells. Through an iterative optimization procedure, the synthesis of the individual AB₅ toxins was established, and the formation of multimeric structures could be shown by autoradiography. A functional analysis was performed using cell-based assays, thereby demonstrating that the LT complex induced the characteristic cell elongation of target cells after 24 h. The LT complex induced cell death at higher concentrations, starting at an initial concentration of 5 nM. The initial toxic effects of the Ctx multimer could already be detected at 4 nM. The detection and characterization of such AB₅ toxins is of utmost importance, and the monitoring of intracellular trafficking facilitates the further identification of the mechanism of action of these toxins. We showed that the B-subunit of LT (LTB) could be fluorescently labeled using an LTB-Strep fusion protein, which is a proof-of-concept for future Trojan horse applications. Further, we performed a mutational analysis of the CtxA subunit as its template was modified, and an amber stop codon was inserted into CtxA’s active site. Subsequently, a non-canonical amino acid was site-specifically incorporated using bio-orthogonal systems. Finally, a fluorescently labeled CtxA protein was produced using copper-catalyzed click reactions as well as a Staudinger ligation. As expected, the modified Ctx multimer no longer induced toxic effects. In our study, we showed that CFPS could be used to study the active centers of toxins by inserting mutations. Additionally, this methodology can be applied for the design of Trojan horses and targeted toxins, as well as enabling the intracellular trafficking of toxins as a prerequisite for the analysis of the toxin’s mechanism of action. Full article

(This article belongs to the Special Issue Novel Aspects of Bacterial AB5-Toxins)

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Open AccessArticle

Combined Action of Shiga Toxin Type 2 and Subtilase Cytotoxin in the Pathogenesis of Hemolytic Uremic Syndrome

and

Toxins 2021, 13(8), 536; https://doi.org/10.3390/toxins13080536 - 29 Jul 2021

Cited by 4 | Viewed by 1349

Abstract

Shiga toxin-producing E. coli (STEC) produces Stx1 and/or Stx2, and Subtilase cytotoxin (SubAB). Since these toxins may be present simultaneously during STEC infections, the purpose of this work was to study the co-action of Stx2 and SubAB. Stx2 + SubAB was assayed in vitro on monocultures and cocultures of human glomerular endothelial cells (HGEC) with a human proximal tubular epithelial cell line (HK-2) and in vivo in mice after weaning. The effects in vitro of both toxins, co-incubated and individually, were similar, showing that Stx2 and SubAB contribute similarly to renal cell damage. However, in vivo, co-injection of toxins lethal doses reduced the survival time of mice by 24 h and mice also suffered a strong decrease in the body weight associated with a lowered food intake. Co-injected mice also exhibited more severe histological renal alterations and a worsening in renal function that was not as evident in mice treated with each toxin separately. Furthermore, co-treatment induced numerous erythrocyte morphological alterations and an increase of free hemoglobin. This work shows, for the first time, the in vivo effects of Stx2 and SubAB acting together and provides valuable information about their contribution to the damage caused in STEC infections. Full article

(This article belongs to the Special Issue Novel Aspects of Bacterial AB5-Toxins)

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Open AccessArticle

Cytotoxic Effects of Recombinant StxA2-His in the Absence of Its Corresponding B-Subunit

and

Toxins 2021, 13(5), 307; https://doi.org/10.3390/toxins13050307 - 26 Apr 2021

Cited by 1 | Viewed by 2120

Abstract

AB₅ protein toxins are produced by certain bacterial pathogens and are composed of an enzymatically active A-subunit and a B-subunit pentamer, the latter being responsible for cell receptor recognition, cellular uptake, and transport of the A-subunit into the cytosol of eukaryotic target cells. Two members of the AB₅ toxin family were described in Shiga toxin-producing Escherichia coli (STEC), namely Shiga toxin (Stx) and subtilase cytotoxin (SubAB). The functional paradigm of AB toxins includes the B-subunit being mandatory for the uptake of the toxin into its target cells. Recent studies have shown that this paradigm cannot be maintained for SubAB, since SubA alone was demonstrated to intoxicate human epithelial cells in vitro. In the current study, we raised the hypothesis that this may also be true for the A-subunit of the most clinically relevant Stx-variant, Stx2a. After separate expression and purification, the recombinant Stx2a subunits StxA2a-His and StxB2a-His were applied either alone or in combination in a 1:5 molar ratio to Vero B4, HeLa, and HCT-116 cells. For all cell lines, a cytotoxic effect of StxA2a-His alone was detected. Competition experiments with Stx and SubAB subunits in combination revealed that the intoxication of StxA2a-His was reduced by addition of SubB1-His. This study showed that the enzymatic subunit StxA2a alone was active on different cells and might therefore play a yet unknown role in STEC disease development. Full article

(This article belongs to the Special Issue Novel Aspects of Bacterial AB5-Toxins)

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Review

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Open AccessEditor’s ChoiceReview

Novel Strategies to Inhibit Pertussis Toxin

Katharina Ernst

Toxins 2022, 14(3), 187; https://doi.org/10.3390/toxins14030187 - 03 Mar 2022

Viewed by 3016

Abstract

Pertussis, also known as whooping cough, is a respiratory disease caused by infection with Bordetella pertussis, which releases several virulence factors, including the AB-type pertussis toxin (PT). The characteristic symptom is severe, long-lasting paroxysmal coughing. Especially in newborns and infants, pertussis symptoms, such as leukocytosis, can become life-threatening. Despite an available vaccination, increasing case numbers have been reported worldwide, including Western countries such as Germany and the USA. Antibiotic treatment is available and important to prevent further transmission. However, antibiotics only reduce symptoms if administered in early stages, which rarely occurs due to a late diagnosis. Thus, no causative treatments against symptoms of whooping cough are currently available. The AB-type protein toxin PT is a main virulence factor and consists of a binding subunit that facilitates transport of an enzyme subunit into the cytosol of target cells. There, the enzyme subunit ADP-ribosylates inhibitory α-subunits of G-protein coupled receptors resulting in disturbed cAMP signaling. As an important virulence factor associated with severe symptoms, such as leukocytosis, and poor outcomes, PT represents an attractive drug target to develop novel therapeutic strategies. In this review, chaperone inhibitors, human peptides, small molecule inhibitors, and humanized antibodies are discussed as novel strategies to inhibit PT. Full article

(This article belongs to the Special Issue Novel Aspects of Bacterial AB5-Toxins)

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Open AccessReview

Novel Aspects of the SubA Subunit of the Subtilase Cytotoxin

Katharina Sessler

Herbert Schmidt

and

Holger Barth

Toxins 2022, 14(2), 156; https://doi.org/10.3390/toxins14020156 - 21 Feb 2022

Viewed by 1000

Abstract

The subtilase cytotoxin (SubAB) belongs to the family of AB₅ toxins and is produced together with Shiga toxin (Stx) by certain Stx-producing E. coli strains (STEC). For most AB-type toxins, it is assumed that cytotoxic effects can only be induced by a complete holotoxin complex consisting of SubA and SubB. However, it has been shown for SubAB that the enzymatically active subunit SubA, without its transport and binding domain SubB, induces cell death in different eukaryotic cell lines. Interestingly, the molecular structure of SubA resembles that of the SubAB complex. SubA alone is capable of binding to cells and then being taken up autonomously. Once inside the host cell, SubA is transported, similar to the SubAB holotoxin, via a retrograde transport into the endoplasmatic reticulum (ER). In the ER, it exhibits its enzymatic activity by cleaving the chaperone BiP/GRP78 and thereby triggering cell death. Therefore, the existence of toxic single SubA subunits that have not found a B-pentamer for holotoxin assembly might improve the pathogenic potential of subtilase-producing strains. Moreover, from a pharmacological aspect, SubA might be an interesting molecule for the targeted transport of therapeutic molecules into the ER, in order to investigate and specifically modulate processes in the context of ER stress-associated diseases. Since recent studies on bacterial AB₅ toxins contributed mainly to the understanding of the biology of AB-type holotoxins, this mini-review specifically focus on that recently observed single A-effect of the subtilase cytotoxin and addresses whether a fundamental shift of the traditional AB₅ paradigm might be required. Full article

(This article belongs to the Special Issue Novel Aspects of Bacterial AB5-Toxins)

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