Editorial

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Open AccessEditorial

Cellular Entry of Binary and Pore-Forming Bacterial Toxins

by Alexey S. Ladokhin

Toxins 2018, 10(1), 11; https://doi.org/10.3390/toxins10010011 - 26 Dec 2017

Cited by 4 | Viewed by 2896

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(This article belongs to the Special Issue Cellular Entry of Binary and Pore-Forming Bacterial Toxins)

Research

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Open AccessFeature PaperArticle

Interaction of Cholesterol with Perfringolysin O: What Have We Learned from Functional Analysis?

by Sergey N. Savinov and Alejandro P. Heuck

Toxins 2017, 9(12), 381; https://doi.org/10.3390/toxins9120381 - 23 Nov 2017

Cited by 27 | Viewed by 8276

Abstract

Cholesterol-dependent cytolysins (CDCs) constitute a family of pore-forming toxins secreted by Gram-positive bacteria. These toxins form transmembrane pores by inserting a large β-barrel into cholesterol-containing membranes. Cholesterol is absolutely required for pore-formation. For most CDCs, binding to cholesterol triggers conformational changes that lead [...] Read more.

Cholesterol-dependent cytolysins (CDCs) constitute a family of pore-forming toxins secreted by Gram-positive bacteria. These toxins form transmembrane pores by inserting a large β-barrel into cholesterol-containing membranes. Cholesterol is absolutely required for pore-formation. For most CDCs, binding to cholesterol triggers conformational changes that lead to oligomerization and end in pore-formation. Perfringolysin O (PFO), secreted by Clostridium perfringens, is the prototype for the CDCs. The molecular mechanisms by which cholesterol regulates the cytolytic activity of the CDCs are not fully understood. In particular, the location of the binding site for cholesterol has remained elusive. We have summarized here the current body of knowledge on the CDCs-cholesterol interaction, with focus on PFO. We have employed sterols in aqueous solution to identify structural elements in the cholesterol molecule that are critical for its interaction with PFO. In the absence of high-resolution structural information, site-directed mutagenesis data combined with binding studies performed with different sterols, and molecular modeling are beginning to shed light on this interaction. Full article

(This article belongs to the Special Issue Cellular Entry of Binary and Pore-Forming Bacterial Toxins)

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Open AccessFeature PaperArticle

Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain

by Alexey S. Ladokhin, Mauricio Vargas-Uribe, Mykola V. Rodnin, Chiranjib Ghatak and Onkar Sharma

Toxins 2017, 9(10), 299; https://doi.org/10.3390/toxins9100299 - 22 Sep 2017

Cited by 12 | Viewed by 6185

Abstract

Cellular entry of diphtheria toxin is a multistage process involving receptor targeting, endocytosis, and translocation of the catalytic domain across the endosomal membrane into the cytosol. The latter is ensured by the translocation (T) domain of the toxin, capable of undergoing conformational refolding [...] Read more.

Cellular entry of diphtheria toxin is a multistage process involving receptor targeting, endocytosis, and translocation of the catalytic domain across the endosomal membrane into the cytosol. The latter is ensured by the translocation (T) domain of the toxin, capable of undergoing conformational refolding and membrane insertion in response to the acidification of the endosomal environment. While numerous now classical studies have demonstrated the formation of an ion-conducting conformation—the Open-Channel State (OCS)—as the final step of the refolding pathway, it remains unclear whether this channel constitutes an in vivo translocation pathway or is a byproduct of the translocation. To address this question, we measure functional activity of known OCS-blocking mutants with H-to-Q replacements of C-terminal histidines of the T-domain. We also test the ability of these mutants to translocate their own N-terminus across lipid bilayers of model vesicles. The results of both experiments indicate that translocation activity does not correlate with previously published OCS activity. Finally, we determined the topology of TH5 helix in membrane-inserted T-domain using W281 fluorescence and its depth-dependent quenching by brominated lipids. Our results indicate that while TH5 becomes a transbilayer helix in a wild-type protein, it fails to insert in the case of the OCS-blocking mutant H322Q. We conclude that the formation of the OCS is not necessary for the functional translocation by the T-domain, at least in the histidine-replacement mutants, suggesting that the OCS is unlikely to constitute a translocation pathway for the cellular entry of diphtheria toxin in vivo. Full article

(This article belongs to the Special Issue Cellular Entry of Binary and Pore-Forming Bacterial Toxins)

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Open AccessArticle

Asymmetric Cryo-EM Structure of Anthrax Toxin Protective Antigen Pore with Lethal Factor N-Terminal Domain

by Alexandra J. Machen, Narahari Akkaladevi, Caleb Trecazzi, Pierce T. O’Neil, Srayanta Mukherjee, Yifei Qi, Rebecca Dillard, Wonpil Im, Edward P. Gogol, Tommi A. White and Mark T. Fisher

Toxins 2017, 9(10), 298; https://doi.org/10.3390/toxins9100298 - 22 Sep 2017

Cited by 10 | Viewed by 10003

Abstract

The anthrax lethal toxin consists of protective antigen (PA) and lethal factor (LF). Understanding both the PA pore formation and LF translocation through the PA pore is crucial to mitigating and perhaps preventing anthrax disease. To better understand the interactions of the LF-PA [...] Read more.

The anthrax lethal toxin consists of protective antigen (PA) and lethal factor (LF). Understanding both the PA pore formation and LF translocation through the PA pore is crucial to mitigating and perhaps preventing anthrax disease. To better understand the interactions of the LF-PA engagement complex, the structure of the LF_N-bound PA pore solubilized by a lipid nanodisc was examined using cryo-EM. CryoSPARC was used to rapidly sort particle populations of a heterogeneous sample preparation without imposing symmetry, resulting in a refined 17 Å PA pore structure with 3 LF_N bound. At pH 7.5, the contributions from the three unstructured LF_N lysine-rich tail regions do not occlude the Phe clamp opening. The open Phe clamp suggests that, in this translocation-compromised pH environment, the lysine-rich tails remain flexible and do not interact with the pore lumen region. Full article

(This article belongs to the Special Issue Cellular Entry of Binary and Pore-Forming Bacterial Toxins)

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Open AccessArticle

Evidence for Complex Formation of the Bacillus cereus Haemolysin BL Components in Solution

by Franziska Tausch, Richard Dietrich, Kristina Schauer, Robert Janowski, Dierk Niessing, Erwin Märtlbauer and Nadja Jessberger

Toxins 2017, 9(9), 288; https://doi.org/10.3390/toxins9090288 - 16 Sep 2017

Cited by 24 | Viewed by 6176

Abstract

Haemolysin BL is an important virulence factor regarding the diarrheal type of food poisoning caused by Bacillus cereus. However, the pathogenic importance of this three-component enterotoxin is difficult to access, as nearly all natural B. cereus culture supernatants additionally contain the highly [...] Read more.

Haemolysin BL is an important virulence factor regarding the diarrheal type of food poisoning caused by Bacillus cereus. However, the pathogenic importance of this three-component enterotoxin is difficult to access, as nearly all natural B. cereus culture supernatants additionally contain the highly cytotoxic Nhe, the second three-component toxin involved in the aetiology of B. cereus-induced food-borne diseases. To better address the toxic properties of the Hbl complex, a system for overexpression and purification of functional, cytotoxic, recombinant (r)Hbl components L₂, L₁ and B from E. coli was established and an nheABC deletion mutant was constructed from B. cereus reference strain F837/76. Furthermore, 35 hybridoma cell lines producing monoclonal antibodies (mAbs) against Hbl L₂, L₁ and B were generated. While mAbs 1H9 and 1D8 neutralized Hbl toxicity and thus, represent important tools for future investigations of the mode-of-action of Hbl on the target cell surface, mAb 1D7, in contrast, even enhanced Hbl toxicity by supporting the binding of Hbl B to the cell surface. By using the specific mAbs in Dot blots, indirect and hybrid sandwich enzyme immuno assays (EIAs), complex formation between Hbl L₁ and B, as well as L₁ and L₂ in solution could be shown for the first time. Surface plasmon resonance experiments with the rHbl components confirmed these results with K_D values of 4.7 × 10⁻⁷ M and 1.5 × 10⁻⁷ M, respectively. These findings together with the newly created tools lay the foundation for the detailed elucidation of the molecular mode-of-action of the highly complex three-component Hbl toxin. Full article

(This article belongs to the Special Issue Cellular Entry of Binary and Pore-Forming Bacterial Toxins)

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Open AccessArticle

Listeriolysin O Regulates the Expression of Optineurin, an Autophagy Adaptor That Inhibits the Growth of Listeria monocytogenes

by Madhu Puri, Luigi La Pietra, Mobarak Abu Mraheil, Rudolf Lucas, Trinad Chakraborty and Helena Pillich

Toxins 2017, 9(9), 273; https://doi.org/10.3390/toxins9090273 - 05 Sep 2017

Cited by 17 | Viewed by 8130

Abstract

Autophagy, a well-established defense mechanism, enables the elimination of intracellular pathogens including Listeria monocytogenes. Host cell recognition results in ubiquitination of L. monocytogenes and interaction with autophagy adaptors p62/SQSTM1 and NDP52, which target bacteria to autophagosomes by binding to microtubule-associated protein [...] Read more.

Autophagy, a well-established defense mechanism, enables the elimination of intracellular pathogens including Listeria monocytogenes. Host cell recognition results in ubiquitination of L. monocytogenes and interaction with autophagy adaptors p62/SQSTM1 and NDP52, which target bacteria to autophagosomes by binding to microtubule-associated protein 1 light chain 3 (LC3). Although studies have indicated that L. monocytogenes induces autophagy, the significance of this process in the infectious cycle and the mechanisms involved remain poorly understood. Here, we examined the role of the autophagy adaptor optineurin (OPTN), the phosphorylation of which by the TANK binding kinase 1 (TBK1) enhances its affinity for LC3 and promotes autophagosomal degradation, during L. monocytogenes infection. In LC3- and OPTN-depleted host cells, intracellular replicating L. monocytogenes increased, an effect not seen with a mutant lacking the pore-forming toxin listeriolysin O (LLO). LLO induced the production of OPTN. In host cells expressing an inactive TBK1, bacterial replication was also inhibited. Our studies have uncovered an OPTN-dependent pathway in which L. monocytogenes uses LLO to restrict bacterial growth. Hence, manipulation of autophagy by L. monocytogenes, either through induction or evasion, represents a key event in its intracellular life style and could lead to either cytosolic growth or persistence in intracellular vacuolar structures. Full article

(This article belongs to the Special Issue Cellular Entry of Binary and Pore-Forming Bacterial Toxins)

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Open AccessArticle

The Vip3Ag4 Insecticidal Protoxin from Bacillus thuringiensis Adopts A Tetrameric Configuration That Is Maintained on Proteolysis

by Leopoldo Palma, David J. Scott, Gemma Harris, Salah-Ud Din, Thomas L. Williams, Oliver J. Roberts, Mark T. Young, Primitivo Caballero and Colin Berry

Toxins 2017, 9(5), 165; https://doi.org/10.3390/toxins9050165 - 14 May 2017

Cited by 35 | Viewed by 6028

Abstract

The Vip3 proteins produced during vegetative growth by strains of the bacterium Bacillus thuringiensis show insecticidal activity against lepidopteran insects with a mechanism of action that may involve pore formation and apoptosis. These proteins are promising supplements to our arsenal of insecticidal proteins, [...] Read more.

The Vip3 proteins produced during vegetative growth by strains of the bacterium Bacillus thuringiensis show insecticidal activity against lepidopteran insects with a mechanism of action that may involve pore formation and apoptosis. These proteins are promising supplements to our arsenal of insecticidal proteins, but the molecular details of their activity are not understood. As a first step in the structural characterisation of these proteins, we have analysed their secondary structure and resolved the surface topology of a tetrameric complex of the Vip3Ag4 protein by transmission electron microscopy. Sites sensitive to proteolysis by trypsin are identified and the trypsin-cleaved protein appears to retain a similar structure as an octomeric complex comprising four copies each of the ~65 kDa and ~21 kDa products of proteolysis. This processed form of the toxin may represent the active toxin. The quality and monodispersity of the protein produced in this study make Vip3Ag4 a candidate for more detailed structural analysis using cryo-electron microscopy. Full article

(This article belongs to the Special Issue Cellular Entry of Binary and Pore-Forming Bacterial Toxins)

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Review

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Open AccessReview

Structural Features of Apicomplexan Pore-Forming Proteins and Their Roles in Parasite Cell Traversal and Egress

by Alfredo J. Guerra and Vern B. Carruthers

Toxins 2017, 9(9), 265; https://doi.org/10.3390/toxins9090265 - 29 Aug 2017

Cited by 11 | Viewed by 7198

Abstract

Apicomplexan parasites cause diseases, including malaria and toxoplasmosis, in a range of hosts, including humans. These intracellular parasites utilize pore-forming proteins that disrupt host cell membranes to either traverse host cells while migrating through tissues or egress from the parasite-containing vacuole after replication. [...] Read more.

Apicomplexan parasites cause diseases, including malaria and toxoplasmosis, in a range of hosts, including humans. These intracellular parasites utilize pore-forming proteins that disrupt host cell membranes to either traverse host cells while migrating through tissues or egress from the parasite-containing vacuole after replication. This review highlights recent insight gained from the newly available three-dimensional structures of several known or putative apicomplexan pore-forming proteins that contribute to cell traversal or egress. These new structural advances suggest that parasite pore-forming proteins use distinct mechanisms to disrupt host cell membranes at multiple steps in parasite life cycles. How proteolytic processing, secretion, environment, and the accessibility of lipid receptors regulate the membranolytic activities of such proteins is also discussed. Full article

(This article belongs to the Special Issue Cellular Entry of Binary and Pore-Forming Bacterial Toxins)

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Open AccessReview

Cellular Entry of Clostridium perfringens Iota-Toxin and Clostridium botulinum C2 Toxin

by Masaya Takehara, Teruhisa Takagishi, Soshi Seike, Masataka Oda, Yoshihiko Sakaguchi, Junzo Hisatsune, Sadayuki Ochi, Keiko Kobayashi and Masahiro Nagahama

Toxins 2017, 9(8), 247; https://doi.org/10.3390/toxins9080247 - 11 Aug 2017

Cited by 21 | Viewed by 7239

Abstract

Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin are composed of two non-linked proteins, one being the enzymatic component and the other being the binding/translocation component. These latter components recognize specific receptors and oligomerize in plasma membrane lipid-rafts, mediating the uptake of the [...] Read more.

Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin are composed of two non-linked proteins, one being the enzymatic component and the other being the binding/translocation component. These latter components recognize specific receptors and oligomerize in plasma membrane lipid-rafts, mediating the uptake of the enzymatic component into the cytosol. Enzymatic components induce actin cytoskeleton disorganization through the ADP-ribosylation of actin and are responsible for cell rounding and death. This review focuses upon the recent advances in cellular internalization of clostridial binary toxins. Full article

(This article belongs to the Special Issue Cellular Entry of Binary and Pore-Forming Bacterial Toxins)

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Other

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643 KiB

Open AccessPerspective

The Unexpected Tuners: Are LncRNAs Regulating Host Translation during Infections?

by Primoz Knap, Toma Tebaldi, Francesca Di Leva, Marta Biagioli, Mauro Dalla Serra and Gabriella Viero

Toxins 2017, 9(11), 357; https://doi.org/10.3390/toxins9110357 - 03 Nov 2017

Cited by 5 | Viewed by 4663

Abstract

Pathogenic bacteria produce powerful virulent factors, such as pore-forming toxins, that promote their survival and cause serious damage to the host. Host cells reply to membrane stresses and ionic imbalance by modifying gene expression at the epigenetic, transcriptional and translational level, to recover [...] Read more.

Pathogenic bacteria produce powerful virulent factors, such as pore-forming toxins, that promote their survival and cause serious damage to the host. Host cells reply to membrane stresses and ionic imbalance by modifying gene expression at the epigenetic, transcriptional and translational level, to recover from the toxin attack. The fact that the majority of the human transcriptome encodes for non-coding RNAs (ncRNAs) raises the question: do host cells deploy non-coding transcripts to rapidly control the most energy-consuming process in cells—i.e., host translation—to counteract the infection? Here, we discuss the intriguing possibility that membrane-damaging toxins induce, in the host, the expression of toxin-specific long non-coding RNAs (lncRNAs), which act as sponges for other molecules, encoding small peptides or binding target mRNAs to depress their translation efficiency. Unravelling the function of host-produced lncRNAs upon bacterial infection or membrane damage requires an improved understanding of host lncRNA expression patterns, their association with polysomes and their function during this stress. This field of investigation holds a unique opportunity to reveal unpredicted scenarios and novel approaches to counteract antibiotic-resistant infections. Full article

(This article belongs to the Special Issue Cellular Entry of Binary and Pore-Forming Bacterial Toxins)

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