Structures of distant diphtheria toxin homologs reveal functional determinants of an evolutionarily conserved toxin scaffold.

Diphtheria toxin (DT) is the archetype for bacterial exotoxins implicated in human diseases and has played a central role in defining the field of toxinology since its discovery in 1888. Despite being one of the most extensively characterized bacterial toxins, the origins and evolutionary adaptation of DT to human hosts remain unknown. Here, we determined the first high-resolution structures of DT homologs outside of the Corynebacterium genus. DT homologs from Streptomyces albireticuli (17% identity to DT) and Seinonella peptonophila (20% identity to DT), despite showing no toxicity toward human cells, display significant structural similarities to DT sharing both the overall Y-shaped architecture of DT as well as the individual folds of each domain. Through a systematic investigation of individual domains, we show that the functional determinants of host range extend beyond an inability to bind cellular receptors; major differences in pH-induced pore-formation and cytosolic release further dictate the delivery of toxic catalytic moieties into cells, thus providing multiple mechanisms for a conserved structural fold to adapt to different hosts. Our work provides structural insights into the expanding DT family of toxins, and highlights key transitions required for host adaptation.

Translocation expands the scope of the large clostridial toxin family.

Large clostridial toxins (LCTs) are a family of six homologous disease-causing proteins characterised by their large size (>200 kDa) and conserved multidomain architectures. Using their central translocation and receptor-binding domain (T domain), LCTs bind host cell receptors and translocate their upstream glycosyltransferase and cysteine protease domain across the endosomal membrane and into the cytosol. The recent discovery of hundreds of LCT-like T domains in diverse genomic contexts and domain architectures from bacteria other than clostridia has provided significant new insights into the enigmatic process of LCT translocation, but also has put the definition of what constitutes an LCT into question. In this opinion article, we discuss how these findings have expanded our understanding of LCT translocation and reshaped the scope of the LCT family.

Large Clostridial Toxins: Mechanisms and Roles in Disease.

Large clostridial toxins (LCTs) are a family of bacterial exotoxins that infiltrate and destroy target cells. Members of the LCT family include Clostridioides difficile toxins TcdA and TcdB, Paeniclostridium sordellii toxins TcsL and TcsH, Clostridium novyi toxin TcnA, and Clostridium perfringens toxin TpeL. Since the 19th century, LCT-secreting bacteria have been isolated from the blood, organs, and wounds of diseased individuals, and LCTs have been implicated as the primary virulence factors in a variety of infections, including C. difficile infection and some cases of wound-associated gas gangrene. Clostridia express and secrete LCTs in response to various physiological signals. LCTs invade host cells by binding specific cell surface receptors, ultimately leading to internalization into acidified vesicles. Acidic pH promotes conformational changes within LCTs, which culminates in translocation of the N-terminal glycosyltransferase and cysteine protease domain across the endosomal membrane and into the cytosol, leading first to cytopathic effects and later to cytotoxic effects. The focus of this review is on the role of LCTs in infection and disease, the mechanism of LCT intoxication, with emphasis on recent structural work and toxin subtyping analysis, and the genomic discovery and characterization of LCT homologues. We provide a comprehensive review of these topics and offer our perspective on emerging questions and future research directions for this enigmatic family of toxins.

Intestinal bile acids directly modulate the structure and function of C. difficile TcdB toxin.

Intestinal bile acids are known to modulate the germination and growth of Clostridioides difficile Here we describe a role for intestinal bile acids in directly binding and neutralizing TcdB toxin, the primary determinant of C. difficile disease. We show that individual primary and secondary bile acids reversibly bind and inhibit TcdB to varying degrees through a mechanism that requires the combined oligopeptide repeats region to which no function has previously been ascribed. We find that bile acids induce TcdB into a compact "balled up" conformation that is no longer able to bind cell surface receptors. Lastly, through a high-throughput screen designed to identify bile acid mimetics we uncovered nonsteroidal small molecule scaffolds that bind and inhibit TcdB through a bile acid-like mechanism. In addition to suggesting a role for bile acids in C. difficile pathogenesis, these findings provide a framework for development of a mechanistic class of C. difficile antitoxins.

The C. difficile toxin B membrane translocation machinery is an evolutionarily conserved protein delivery apparatus.

Large Clostridial Toxins (LCTs) are a family of six homologous protein toxins that are implicated in severe disease. LCTs infiltrate host cells using a translocation domain (LCT-T) that contains both cell-surface receptor binding sites and a membrane translocation apparatus. Despite much effort, LCT translocation remains poorly understood. Here we report the identification of 1104 LCT-T homologs, with 769 proteins from bacteria outside of clostridia. Sequences are widely distributed in pathogenic and host-associated species, in a variety of contexts and architectures. Consistent with these homologs being functional toxins, we show that a distant LCT-T homolog from Serratia marcescens acts as a pH-dependent translocase to deliver its effector into host cells. Based on evolutionary footprinting of LCT-T homologs, we further define an evolutionarily conserved translocase region that we show is an autonomous translocase capable of delivering heterologous cargo into host cells. Our work uncovers a broad class of translocating toxins and provides insights into LCT translocation.

Direct Detection of Membrane-Inserting Fragments Defines the Translocation Pores of a Family of Pathogenic Toxins.

Large clostridial toxins (LCTs) are a family of homologous proteins toxins that are directly responsible for the symptoms associated with a number of clostridial infections that cause disease in humans and in other animals. LCTs damage tissues by delivering a glucosyltransferase domain, which inactivates small GTPases, across the endosomal membrane and into the cytosol of target cells. Elucidating the mechanism of translocation for LCTs has been hampered by difficulties associated with identifying marginally hydrophobic segments that insert into the bounding membrane to form the translocation pore. Here, we directly measured the membrane-insertion partitioning propensity for segments spanning the putative pore-forming region using a translocon-mediated insertion assay and synthetic peptides. We identified membrane-inserting segments, as well as a conserved and functionally important negatively charged residue that requires protonation for efficient membrane insertion. We provide a model of the LCT pore, which provides insights into translocation for this enigmatic family of α-helical translocases.

Structural and Mechanical Roles for the C-Terminal Nonrepetitive Domain Become Apparent in Recombinant Spider Aciniform Silk.

Spider aciniform (or wrapping) silk is the toughest of the seven types of spider silks/glue due to a combination of high elasticity and strength. Like most spider silk proteins (spidroins), aciniform spidroin (AcSp1) has a large core repetitive domain flanked by relatively short N- and C-terminal nonrepetitive domains (the NTD and CTD, respectively). The major ampullate silk protein (MaSp) CTD has been shown to control protein solubility and fiber formation, but the aciniform CTD function remains unknown. Here, we compare fiber mechanical properties, solution-state structuring, and fibrous state secondary structural composition, and orientation relative to native aciniform silk for two AcSp1 repeat units with or without fused AcSp1- and MaSp-derived CTDs alongside three AcSp1 repeat units without a CTD. The native AcSp1 CTD uniquely modulated fiber mechanical properties, relative to all other constructs, directly correlating to a native-like structural transformation and alignment.

Clostridium difficile toxins A and B: Receptors, pores, and translocation into cells.

The most potent toxins secreted by pathogenic bacteria contain enzymatic moieties that must reach the cytosol of target cells to exert their full toxicity. Toxins such as anthrax, diphtheria, and botulinum toxin all use three well-defined functional domains to intoxicate cells: a receptor-binding moiety that triggers endocytosis into acidified vesicles by binding to a specific host-cell receptor, a translocation domain that forms pores across the endosomal membrane in response to acidic pH, and an enzyme that translocates through these pores to catalytically inactivate an essential host cytosolic substrate. The homologous toxins A (TcdA) and Toxin B (TcdB) secreted by Clostridium difficile are large enzyme-containing toxins that for many years have eluded characterization. The cell-surface receptors for these toxins, the non-classical nature of the pores that they form in membranes, and mechanism of translocation have remained undefined, exacerbated, in part, by the lack of any structural information for the central ∼1000 amino acid translocation domain. Recent advances in the identification of receptors for TcdB, high-resolution structural information for the translocation domain, and a model for the pore have begun to shed light on the mode-of-action of these toxins. Here, we will review TcdA/TcdB uptake and entry into mammalian cells, with focus on receptor binding, endocytosis, pore formation, and translocation. We will highlight how these toxins diverge from classical models of translocating toxins, and offer our perspective on key unanswered questions for TcdA/TcdB binding and entry into mammalian cells.

Tracking Transitions in Spider Wrapping Silk Conformation and Dynamics by (19)F Nuclear Magnetic Resonance Spectroscopy.

Aciniform silk protein (AcSp1) is the primary component of wrapping silk, the toughest of the spider silks because of a combination of high tensile strength and extensibility. Argiope trifasciata AcSp1 contains a core repetitive domain with at least 14 homogeneous 200-amino acid units ("W" units). Upon fibrillogenesis, AcSp1 converts from an α-helix-rich soluble state to a mixed α-helical/ß-sheet conformation. Solution-state nuclear magnetic resonance (NMR) spectroscopy allowed demonstration of variable local stability within the W unit, but comprehensive characterization was confounded by spectral overlap, which was exacerbated by decreased chemical shift dispersion upon denaturation. Here, (19)F NMR spectroscopy, in the context of a single W unit (W1), is applied to track changes in structure and dynamics. Four strategic positions in the W unit were mutated to tryptophan and biosynthetically labeled with 5-fluorotryptophan (5F-Trp). Simulated annealing-based structure calculations implied that these substitutions should be tolerated, while circular dichroism (CD) spectroscopy and (1)H-(15)N chemical shift displacements indicated minimal structural perturbation in W1 mutants. Fiber formation by W2 concatemers containing 5F-Trp substitutions in both W units demonstrated retention of functionality, a somewhat surprising finding in light of sequence conservation between species. Each 5F-Trp-labeled W1 exhibited a unique (19)F chemical shift, line width, longitudinal relaxation time constant (T1), and solvent isotope shift. Perturbation to (19)F chemical shift and nuclear spin relaxation parameters reflected changes in the conformation and dynamics at each 5F-Trp site upon addition of urea and dodecylphosphocholine (DPC). (19)F NMR spectroscopy allowed unambiguous localized tracking throughout titration with each perturbant, demonstrating distinct behavior for each perturbant not previously revealed by heteronuclear NMR experiments.

Spider wrapping silk fibre architecture arising from its modular soluble protein precursor.

Spiders store spidroins in their silk glands as high concentration aqueous solutions, spinning these dopes into fibres with outstanding mechanical properties. Aciniform (or wrapping) silk is the toughest spider silk and is devoid of the short amino acid sequence motifs characteristic of the other spidroins. Using solution-state NMR spectroscopy, we demonstrate that the 200 amino acid Argiope trifasciata AcSp1 repeat unit contrasts with previously characterized spidroins, adopting a globular 5-helix bundle flanked by intrinsically disordered N- and C-terminal tails. Split-intein-mediated segmental NMR-active isotope-enrichment allowed unambiguous demonstration of modular and malleable "beads-on-a-string" concatemeric behaviour. Concatemers form fibres upon manual drawing with silk-like morphology and mechanical properties, alongside secondary structuring and orientation consistent with native AcSp1 fibres. AcSp1 structural stability varies locally, with the fifth helix denaturing most readily. The structural transition of aciniform spidroin from a mostly α-helical dope to a mixed α-helix/ß-sheet-containing fibre can be directly related to spidroin architecture and stability.

Nanoparticle self-assembly by a highly stable recombinant spider wrapping silk protein subunit.

Artificial spider silk proteins may form fibers with exceptional strength and elasticity. Wrapping silk, or aciniform silk, is the toughest of the spider silks, and has a very different protein composition than other spider silks. Here, we present the characterization of an aciniform protein (AcSp1) subunit named W1, consisting of one AcSp1 199 residue repeat unit from Argiope trifasciata. The structural integrity of recombinant W1 is demonstrated in a variety of buffer conditions and time points. Furthermore, we show that W1 has a high thermal stability with reversible denaturation at ∼71°C and forms self-assembled nanoparticle in near-physiological conditions. W1 therefore represents a highly stable and structurally robust module for protein-based nanoparticle formation.