Special Issue "RTX Toxins"

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

Deadline for manuscript submissions: closed (30 September 2019).

Special Issue Editor

Prof. Roland Benz
E-Mail Website
Guest Editor
Department of Life Sciences and Chemistry, Jacobs-University Bremen, Campusring 1, 28759 Bremen, Germany

Special Issue Information

Dear Colleagues,

RTX toxins (Repeats in ToXin) represent a steadily increasing family of gram-negative bacterial proteins containing functionally important glycine-rich and aspartate-containing nonapeptide repeats of the consensus sequence G-G-X-G-(N/D)-D-X-(L/I/F)-X (where X can be any amino acid). The repeats are able to bind calcium ions with high affinity to form a special structure that is important for target cell recognition. RTX proteins comprise many different functional categories. The most well-known of these is the RTX cytolysin family, where HlyA of uropathogenic Escherichia coli may represent the classical example of a pore-forming RTX toxin. Besides the cytolysins, the RTX proteins also contain subfamilies of many diverse functions. These subfamilies consist of RTX adhesins; enzymatic toxins; bacteriocins; surface layer proteins; and hydrolytic enzymes, such as proteases and lipases. More than 1000 RTX proteins are known to date. Common to all RTX proteins is their secretion via a type-one secretion system (T1SS) across the bacterial inner and outer membranes. This system is composed of different proteins that are encoded on the rtx-operon in transcriptional order. It comprises an inner membrane ATPase, a linker protein to combine inner and outer membrane components and an outer membrane pore. The rtx-operon may also code for an acyltransferase that modifies together with a bacterial acyl carrier protein the RTX toxins. The secreted RTX proteins have approximately a 60 amino acid-long secretion signal at the C-terminus.

This Special Issue will focus on the function of RTX toxins of different subfamilies. This comprises the cytolysin subfamily, which represents severe pathogenicity factors for diseases in humans or animals and other key virulence factor such as the adenylate cyclase toxin (Act, CyaA) of the whooping cough agent Bordetella pertussis. The large RTX protein family of the adhesins will also be a topic of this Special Issue together with biofilm associated RTX proteins.

Prof. Roland Benz
Guest Editor

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Keywords

  • RTX toxin
  • RTX metalloprotease and lipase
  • MARTX toxin
  • cytolysin
  • virulence factors
  • protein folding
  • Ca2+ activation
  • type 1 secretion
  • ACT

Published Papers (10 papers)

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Research

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Open AccessArticle
Calcium-Induced Activity and Folding of a Repeat in Toxin Lipase from Antarctic Pseudomonas fluorescens Strain AMS8
Toxins 2020, 12(1), 27; https://doi.org/10.3390/toxins12010027 - 01 Jan 2020
Abstract
It is hypothesized that the Ca2+ ions were involved in the activity, folding and stabilization of many protein structures. Many of these proteins contain repeat in toxin (RTX) motifs. AMS8 lipase from Antarctic Pseudomonas fluorescens strain AMS8 was found to have three [...] Read more.
It is hypothesized that the Ca2+ ions were involved in the activity, folding and stabilization of many protein structures. Many of these proteins contain repeat in toxin (RTX) motifs. AMS8 lipase from Antarctic Pseudomonas fluorescens strain AMS8 was found to have three RTX motifs. So, this research aimed to examine the influence of Ca2+ ion towards the activity and folding of AMS8 lipase through various biophysical characterizations. The results showed that CaCl2 increased lipase activity. The far-UV circular dichroism (CD) and Fourier-transform infrared (FTIR) analysis suggested that the secondary structure content was improved with the addition of CaCl2. Fluorescence spectroscopy analysis showed that the presence of CaCl2 increased protein folding and compactness. Dynamic light scattering (DLS) analysis suggested that AMS8 lipase became aggregated at a high concentration of CaCl2.The binding constant (Kd) value from the isothermal titration calorimetry (ITC) analysis proved that the Ca2+ ion was tightly bound to the AMS8 lipase. In conclusion, Ca2+ ions play crucial roles in the activity and folding of the AMS8 lipase. Calcium binding to RTX nonapeptide repeats sequences will induced the formation and folding of the RTX parallel β-roll motif repeat structure. Full article
(This article belongs to the Special Issue RTX Toxins)
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Open AccessArticle
Channel Formation by LktA of Mannheimia (Pasteurella) haemolytica in Lipid Bilayer Membranes and Comparison of Channel Properties with Other RTX-Cytolysins
Toxins 2019, 11(10), 604; https://doi.org/10.3390/toxins11100604 - 17 Oct 2019
Abstract
Cytolysin LktA is one of the major pathogenicity factors of Mannheimia haemolytica (formerly Pasteurella haemolytica) that is the cause of pasteurellosis, also known as shipping fever pneumonia, causing substantial loss of sheep and cattle during transport. LktA belongs to the family of [...] Read more.
Cytolysin LktA is one of the major pathogenicity factors of Mannheimia haemolytica (formerly Pasteurella haemolytica) that is the cause of pasteurellosis, also known as shipping fever pneumonia, causing substantial loss of sheep and cattle during transport. LktA belongs to the family of RTX-toxins (Repeats in ToXins) that are produced as pathogenicity factors by a variety of Gram-negative bacteria. Sublytic concentrations of LktA cause inflammatory responses of ovine leukocytes. Higher concentrations result in formation of transmembrane channels in target cells that may cause cell lysis and apoptosis. In this study we investigated channel formation by LktA in artificial lipid bilayer membranes made of different lipids. LktA purified from culture supernatants by polyethylene glycol 4000 precipitation and lyophilization had to be activated to frequently form channels by solution in 6 M urea. The LktA channels had a single-channel conductance of about 60 pS in 0.1 M KCl, which is about one tenth of the conductance of most RTX-toxins with the exception of adenylate cyclase toxin of Bordetella pertussis. The LktA channels are highly cation-selective caused by negative net charges. The theoretical treatment of the conductance of LktA as a function of the bulk aqueous concentration allowed a rough estimate of the channel diameter, which is around 1.5 nm. The size of the LktA channel is discussed with respect to channels formed by other RTX-toxins. We present here the first investigation of LktA in a reconstituted system. Full article
(This article belongs to the Special Issue RTX Toxins)
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Open AccessArticle
Distinct Spatiotemporal Distribution of Bacterial Toxin-Produced Cellular cAMP Differentially Inhibits Opsonophagocytic Signaling
Toxins 2019, 11(6), 362; https://doi.org/10.3390/toxins11060362 - 20 Jun 2019
Abstract
Myeloid phagocytes have evolved to rapidly recognize invading pathogens and clear them through opsonophagocytic killing. The adenylate cyclase toxin (CyaA) of Bordetella pertussis and the edema toxin (ET) of Bacillus anthracis are both calmodulin-activated toxins with adenylyl cyclase activity that invade host cells [...] Read more.
Myeloid phagocytes have evolved to rapidly recognize invading pathogens and clear them through opsonophagocytic killing. The adenylate cyclase toxin (CyaA) of Bordetella pertussis and the edema toxin (ET) of Bacillus anthracis are both calmodulin-activated toxins with adenylyl cyclase activity that invade host cells and massively increase the cellular concentrations of a key second messenger molecule, 3’,5’-cyclic adenosine monophosphate (cAMP). However, the two toxins differ in the kinetics and mode of cell entry and generate different cAMP concentration gradients within the cell. While CyaA rapidly penetrates cells directly across their plasma membrane, the cellular entry of ET depends on receptor-mediated endocytosis and translocation of the enzymatic subunit across the endosomal membrane. We show that CyaA-generated membrane-proximal cAMP gradient strongly inhibits the activation and phosphorylation of Syk, Vav, and Pyk2, thus inhibiting opsonophagocytosis. By contrast, at similar overall cellular cAMP levels, the ET-generated perinuclear cAMP gradient poorly inhibits the activation and phosphorylation of these signaling proteins. Hence, differences in spatiotemporal distribution of cAMP produced by the two adenylyl cyclase toxins differentially affect the opsonophagocytic signaling in myeloid phagocytes. Full article
(This article belongs to the Special Issue RTX Toxins)
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Open AccessArticle
Rapid Purification of Endotoxin-Free RTX Toxins
Toxins 2019, 11(6), 336; https://doi.org/10.3390/toxins11060336 - 12 Jun 2019
Abstract
Cytolytic leukotoxins of the repeat in toxin (RTX) family are large proteins excreted by gram-negative bacterial pathogens through the type 1 secretion system (T1SS). Due to low yields and poor stability in cultures of the original pathogens, it is useful to purify recombinant [...] Read more.
Cytolytic leukotoxins of the repeat in toxin (RTX) family are large proteins excreted by gram-negative bacterial pathogens through the type 1 secretion system (T1SS). Due to low yields and poor stability in cultures of the original pathogens, it is useful to purify recombinant fatty-acylated RTX cytolysins from inclusion bodies produced in E. coli. Such preparations are, however, typically contaminated by high amounts of E. coli lipopolysaccharide (LPS or endotoxin). We report a simple procedure for purification of large amounts of biologically active and endotoxin-free RTX toxins. It is based on the common feature of RTX cytolysins that are T1SS-excreted as unfolded polypeptides and fold into a biologically active toxin only upon binding of calcium ions outside of the bacterial cell. Mimicking this process, the RTX proteins are solubilized from inclusion bodies with buffered 8 M urea, bound onto a suitable chromatographic medium under denaturing conditions and the contaminating LPS is removed through extensive on-column washes with buffers containing 6 to 8 M urea and 1% Triton X-100 or Triton X-114. Extensive on-column rinsing with 8 M urea buffer removes residual detergent and the eluted highly active RTX protein preparations then contain only trace amounts of LPS. The procedure is exemplified using four prototypic RTX cytolysins, the Bordetella pertussis CyaA and the hemolysins of Escherichia coli (HlyA), Kingella kingae (RtxA), and Actinobacillus pleuropneumoniae (ApxIA). Full article
(This article belongs to the Special Issue RTX Toxins)
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Review

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Open AccessReview
RTX Toxins Ambush Immunity’s First Cellular Responders
Toxins 2019, 11(12), 720; https://doi.org/10.3390/toxins11120720 - 10 Dec 2019
Abstract
The repeats-in-toxin (RTX) family represents a unique class of bacterial exoproteins. The first family members described were toxins from Gram-negative bacterial pathogens; however, additional members included exoproteins with diverse functions. Our review focuses on well-characterized RTX family toxins from Aggregatibacter actinomycetemcomitans (LtxA), Mannheimia [...] Read more.
The repeats-in-toxin (RTX) family represents a unique class of bacterial exoproteins. The first family members described were toxins from Gram-negative bacterial pathogens; however, additional members included exoproteins with diverse functions. Our review focuses on well-characterized RTX family toxins from Aggregatibacter actinomycetemcomitans (LtxA), Mannheimia haemolytica (LktA), Bordetella pertussis (CyaA), uropathogenic Escherichia coli (HlyA), and Actinobacillus pleuropneumoniae (ApxIIIA), as well as the studies that have honed in on a single host cell receptor for RTX toxin interactions, the β2 integrins. The β2 integrin family is composed of heterodimeric members with four unique alpha subunits and a single beta subunit. β2 integrins are only found on leukocytes, including neutrophils and monocytes, the first responders to inflammation following bacterial infection. The LtxA, LktA, HlyA, and ApxIIIA toxins target the shared beta subunit, thereby targeting all types of leukocytes. Specific β2 integrin family domains are required for the RTX toxin’s cytotoxic activity and are summarized here. Research examining the domains of the RTX toxins required for cytotoxic and hemolytic activity is also summarized. RTX toxins attack and kill phagocytic immune cells expressing a single integrin family, providing an obvious advantage to the pathogen. The critical question that remains, can the specificity of the RTX-β2 integrin interaction be therapeutically targeted? Full article
(This article belongs to the Special Issue RTX Toxins)
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Open AccessReview
RTX Toxins of Animal Pathogens and Their Role as Antigens in Vaccines and Diagnostics
Toxins 2019, 11(12), 719; https://doi.org/10.3390/toxins11120719 - 10 Dec 2019
Abstract
Exotoxins play a central role in the pathologies caused by most major bacterial animal pathogens. The large variety of vertebrate and invertebrate hosts in the animal kingdom is reflected by a large variety of bacterial pathogens and toxins. The group of repeats in [...] Read more.
Exotoxins play a central role in the pathologies caused by most major bacterial animal pathogens. The large variety of vertebrate and invertebrate hosts in the animal kingdom is reflected by a large variety of bacterial pathogens and toxins. The group of repeats in the structural toxin (RTX) toxins is particularly abundant among bacterial pathogens of animals. Many of these toxins are described as hemolysins due to their capacity to lyse erythrocytes in vitro. Hemolysis by RTX toxins is due to the formation of cation-selective pores in the cell membrane and serves as an important marker for virulence in bacterial diagnostics. However, their physiologic relevant targets are leukocytes expressing β2 integrins, which act as specific receptors for RTX toxins. For various RTX toxins, the binding to the CD18 moiety of β2 integrins has been shown to be host specific, reflecting the molecular basis of the host range of RTX toxins expressed by bacterial pathogens. Due to the key role of RTX toxins in the pathogenesis of many bacteria, antibodies directed against specific RTX toxins protect against disease, hence, making RTX toxins valuable targets in vaccine research and development. Due to their specificity, several structural genes encoding for RTX toxins have proven to be essential in modern diagnostic applications in veterinary medicine. Full article
(This article belongs to the Special Issue RTX Toxins)
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Open AccessReview
Structure–Function Relationships of the Repeat Domains of RTX Toxins
Toxins 2019, 11(11), 657; https://doi.org/10.3390/toxins11110657 - 12 Nov 2019
Abstract
RTX proteins are a large family of polypeptides of mainly Gram-negative origin that are secreted into the extracellular medium by a type I secretion system featuring a non-cleavable C-terminal secretion signal, which is preceded by a variable number of nine-residue tandem repeats. The [...] Read more.
RTX proteins are a large family of polypeptides of mainly Gram-negative origin that are secreted into the extracellular medium by a type I secretion system featuring a non-cleavable C-terminal secretion signal, which is preceded by a variable number of nine-residue tandem repeats. The three-dimensional structure forms a parallel β-roll, where β-strands of two parallel sheets are connected by calcium-binding linkers in such a way that a right-handed spiral is built. The Ca2+ ions are an integral part of the structure, which cannot form without them. The structural determinants of this unique architecture will be reviewed with its conservations and variations together with the implication for secretion and folding of these proteins. The general purpose of the RTX domains appears to act as an internal chaperone that keeps the polypeptide unfolded in the calcium-deprived cytosol and triggers folding in the calcium-rich extracellular medium. A rather recent addition to the structural biology of the RTX toxin is a variant occurring in a large RTX adhesin, where this non-canonical β-roll binds to ice and diatoms. Full article
(This article belongs to the Special Issue RTX Toxins)
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Open AccessReview
Genetics, Toxicity, and Distribution of Enterohemorrhagic Escherichia coli Hemolysin
Toxins 2019, 11(9), 502; https://doi.org/10.3390/toxins11090502 - 29 Aug 2019
Abstract
The ability to produce enterohemolysin is regarded as a potential virulence factor for enterohemorrhagic Escherichia coli (EHEC) and is frequently associated with severe human diseases such as hemorrhagic colitis (HC) and the hemolytic uremic syndrome (HUS). The responsible toxin, which has also been [...] Read more.
The ability to produce enterohemolysin is regarded as a potential virulence factor for enterohemorrhagic Escherichia coli (EHEC) and is frequently associated with severe human diseases such as hemorrhagic colitis (HC) and the hemolytic uremic syndrome (HUS). The responsible toxin, which has also been termed EHEC-hemolysin (EHEC-Hly, syn. Ehx), belongs to the Repeats in Toxin (RTX)-family of pore-forming cytolysins and is characterized by the formation of incomplete turbid lysis zones on blood agar plates containing defibrinated sheep erythrocytes. Besides the expression of Shiga toxins (Stx) and the locus of enterocyte effacement (LEE), EHEC-Hly is a commonly used marker for the detection of potential pathogenic E. coli strains, although its exact role in pathogenesis is not completely understood. Based on the current knowledge of EHEC-Hly, this review describes the influence of various regulator proteins, explains the different mechanisms leading to damage of target cells, discusses the diagnostic role, and gives an insight of the prevalence and genetic evolution of the toxin. Full article
(This article belongs to the Special Issue RTX Toxins)
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Open AccessReview
Aggregatibacter actinomycetemcomitans Leukotoxin (LtxA; Leukothera®): Mechanisms of Action and Therapeutic Applications
Toxins 2019, 11(9), 489; https://doi.org/10.3390/toxins11090489 - 26 Aug 2019
Cited by 3
Abstract
Aggregatibacter actinomycetemcomitans is an oral pathogen that produces the RTX toxin, leukotoxin (LtxA; Leukothera®). A. actinomycetemcomitans is strongly associated with the development of localized aggressive periodontitis. LtxA acts as a virulence factor for A. actinomycetemcomitans to subvert the host immune response [...] Read more.
Aggregatibacter actinomycetemcomitans is an oral pathogen that produces the RTX toxin, leukotoxin (LtxA; Leukothera®). A. actinomycetemcomitans is strongly associated with the development of localized aggressive periodontitis. LtxA acts as a virulence factor for A. actinomycetemcomitans to subvert the host immune response by binding to the β2 integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) on white blood cells (WBCs), causing cell death. In this paper, we reviewed the state of knowledge on LtxA interaction with WBCs and the subsequent mechanisms of induced cell death. Finally, we touched on the potential therapeutic applications of LtxA (trade name Leukothera®) toxin therapy for the treatment of hematological malignancies and immune-mediated diseases. Full article
(This article belongs to the Special Issue RTX Toxins)
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Other

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Open AccessBrief Report
The Adenylate Cyclase (CyaA) Toxin from Bordetella pertussis Has No Detectable Phospholipase A (PLA) Activity In Vitro
Toxins 2019, 11(2), 111; https://doi.org/10.3390/toxins11020111 - 13 Feb 2019
Abstract
The adenylate cyclase (CyaA) toxin produced in Bordetella pertussis is the causative agent of whooping cough. CyaA exhibits the remarkable capacity to translocate its N-terminal adenyl cyclase domain (ACD) directly across the plasma membrane into the cytosol of eukaryotic cells. Once translocated, calmodulin [...] Read more.
The adenylate cyclase (CyaA) toxin produced in Bordetella pertussis is the causative agent of whooping cough. CyaA exhibits the remarkable capacity to translocate its N-terminal adenyl cyclase domain (ACD) directly across the plasma membrane into the cytosol of eukaryotic cells. Once translocated, calmodulin binds and activates ACD, leading to a burst of cAMP that intoxicates the target cell. Previously, Gonzalez-Bullon et al. reported that CyaA exhibits a phospholipase A activity that could destabilize the membrane to facilitate ACD membrane translocation. However, Bumba and collaborators lately reported that they could not replicate these results. To clarify this controversy, we assayed the putative PLA activity of two CyaA samples purified in two different laboratories by using two distinct fluorescent probes reporting either PLA2 or both PLA1 and PLA2 activities, as well as in various experimental conditions (i.e., neutral or negatively charged membranes in different buffers.) However, we could not detect any PLA activity in these CyaA batches. Thus, our data independently confirm that CyaA does not possess any PLA activity. Full article
(This article belongs to the Special Issue RTX Toxins)
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