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.

Exploiting the diphtheria toxin internalization receptor enhances delivery of proteins to lysosomes for enzyme replacement therapy.

Enzyme replacement therapy, in which a functional copy of an enzyme is injected either systemically or directly into the brain of affected individuals, has proven to be an effective strategy for treating certain lysosomal storage diseases. The inefficient uptake of recombinant enzymes via the mannose-6-phosphate receptor, however, prohibits the broad utility of replacement therapy. Here, to improve the efficiency and efficacy of lysosomal enzyme uptake, we exploited the strategy used by diphtheria toxin to enter into the endolysosomal network of cells by creating a chimera between the receptor-binding fragment of diphtheria toxin and the lysosomal hydrolase TPP1. We show that chimeric TPP1 binds with high affinity to target cells and is efficiently delivered into lysosomes. Further, we show superior uptake of chimeric TPP1 over TPP1 alone in brain tissue following intracerebroventricular injection in mice lacking TPP1, demonstrating the potential of this strategy for enhancing lysosomal storage disease therapy.

Intracellular Delivery of Human Purine Nucleoside Phosphorylase by Engineered Diphtheria Toxin Rescues Function in Target Cells.

Despite a wealth of potential applications inside target cells, protein-based therapeutics are largely limited to extracellular targets due to the inability of proteins to readily cross biological membranes and enter the cytosol. Bacterial toxins, which deliver a cytotoxic enzyme into cells as part of their intoxication mechanism, hold great potential as platforms for delivering therapeutic protein cargo into cells. Diphtheria toxin (DT) has been shown to be capable of delivering an array of model proteins of varying sizes, structures, and stabilities into mammalian cells as amino-terminal fusions. Here, seeking to expand the utility of DT as a delivery vector, we asked whether an active human enzyme, purine nucleoside phosphorylase (PNP), could be delivered by DT into cells to rescue PNP deficiency. Using a series of biochemical and cellular readouts, we demonstrate that PNP is efficiently delivered into target cells in a receptor- and translocation-dependent manner. In patient-derived PNP-deficient lymphocytes and pluripotent stem cell-differentiated neurons, we show that human PNP is efficiently translocated into target cells by DT, where it is able to restore intracellular hypoxanthine levels. Further, through replacement of the native receptor-binding moiety of DT with single-chain variable fragments that were selected to bind mouse HBEGF, we show that PNP can be retargeted into mouse splenocytes from PNP-deficient mice, resulting in restoration of the proliferative capacity of T-cells. These findings highlight the versatility of the DT delivery platform and provide an attractive approach for the delivery of PNP as well as other cytosolic enzymes implicated in disease.

Identification of a diphtheria toxin-like gene family beyond the Corynebacterium genus.

Diphtheria toxin (DT), produced by Corynebacterium diphtheria, is the causative agent of diphtheria and one of the most potent protein toxins known; however, it has an unclear evolutionary history. Here, we report the discovery of a DT-like gene family in several bacterial lineages outside of Corynebacterium, including Austwickia and Streptomyces. These DT-like genes form sister lineages in the DT phylogeny and conserve key DT features including catalytic and translocation motifs, but possess divergent receptor-binding domains. DT-like genes are not associated with corynephage, but have undergone lateral transfer through a separate mechanism. The discovery of the first non-Corynebacterium homologs of DT sheds light on its evolutionary origin and highlights novelties that may have resulted in the emergence of DT targeting humans.

Functional defects in Clostridium difficile TcdB toxin uptake identify CSPG4 receptor-binding determinants.

Clostridium difficile is a major nosocomial pathogen that produces two exotoxins, TcdA and TcdB, with TcdB thought to be the primary determinant in human disease. TcdA and TcdB are large, multidomain proteins, each harboring a cytotoxic glucosyltransferase domain that is delivered into the cytosol from endosomes via a translocation domain after receptor-mediated endocytosis of toxins from the cell surface. Although there are currently no known host cell receptors for TcdA, three cell-surface receptors for TcdB have been identified: CSPG4, NECTIN3, and FZD1/2/7. The sites on TcdB that mediate binding to each receptor are not defined. Furthermore, it is not known whether the combined repetitive oligopeptide (CROP) domain is involved in or required for receptor binding. Here, in a screen designed to identify sites in TcdB that are essential for target cell intoxication, we identified a region at the junction of the translocation and the CROP domains that is implicated in CSPG4 binding. Using a series of C-terminal truncations, we show that the CSPG4-binding site on TcdB extends into the CROP domain, requiring three short repeats for binding and for full toxicity on CSPG4-expressing cells. Consistent with the location of the CSPG4-binding site on TcdB, we show that the anti-TcdB antibody bezlotoxumab, which binds partially within the first three short repeats, prevents CSPG4 binding to TcdB. In addition to establishing the binding region for CSPG4, this work ascribes for the first time a role in TcdB CROPs in receptor binding and further clarifies the relative roles of host receptors in TcdB pathogenesis.

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.

Repurposing bacterial toxins for intracellular delivery of therapeutic proteins.

Despite enormous efforts, achieving efficacious levels of proteins inside mammalian cells remains one of the greatest challenges in biologics-based drug discovery and development. The inability of proteins to readily cross biological membranes precludes access to the wealth of intracellular targets and applications that lie within mammalian cells. Existing methods of delivery commonly suffer from an inability to target specific cells and tissues, poor endosomal escape, and limited in vivo efficacy. The aim of the present commentary is to highlight the potential of certain classes of bacterial toxins, which naturally deliver a large protein into the cytosolic compartment of target cells after binding a host cell-surface receptor with high affinity, as robust protein delivery platforms. We review the progress made in recent years toward demonstrating the utility of these systems at delivering a wide variety of protein cargo, with special attention paid to three distinct toxin-based platforms. We contend that with recent advances in protein deimmunization strategies, bacterial toxins are poised to introduce biologics into the inner sanctum of cells and treat a wealth of heretofore untreatable diseases with a new generation of therapeutics.

The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions.

UNLABELLED: FimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels-and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)-by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the "FimV C-terminal domain," which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV861-919, containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV689, a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions. IMPORTANCE: FimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs.

Mechanism for accurate, protein-assisted DNA annealing by Deinococcus radiodurans DdrB.

Accurate pairing of DNA strands is essential for repair of DNA double-strand breaks (DSBs). How cells achieve accurate annealing when large regions of single-strand DNA are unpaired has remained unclear despite many efforts focused on understanding proteins, which mediate this process. Here we report the crystal structure of a single-strand annealing protein [DdrB (DNA damage response B)] in complex with a partially annealed DNA intermediate to 2.2 Å. This structure and supporting biochemical data reveal a mechanism for accurate annealing involving DdrB-mediated proofreading of strand complementarity. DdrB promotes high-fidelity annealing by constraining specific bases from unauthorized association and only releases annealed duplex when bound strands are fully complementary. To our knowledge, this mechanism provides the first understanding for how cells achieve accurate, protein-assisted strand annealing under biological conditions that would otherwise favor misannealing.

Crystal structure of the DdrB/ssDNA complex from Deinococcus radiodurans reveals a DNA binding surface involving higher-order oligomeric states.

The ability of Deinococcus radiodurans to recover from extensive DNA damage is due in part to its ability to efficiently repair its genome, even following severe fragmentation by hundreds of double-strand breaks. The single-strand annealing pathway plays an important role early during the recovery process, making use of a protein, DdrB, shown to greatly stimulate ssDNA annealing. Here, we report the structure of DdrB bound to ssDNA to 2.3 Å. Pentameric DdrB was found to assemble into higher-order structures that coat ssDNA. To gain further mechanistic insight into the protein's function, a number of point mutants were generated altering both DNA binding and higher order oligomerization. This work not only identifies higher-order DdrB associations but also suggests the presence of an extended DNA binding surface running along the 'top' surface of a DdrB pentamer and continuing down between two individual subunits of the ring structure. Together this work sheds new insight into possible mechanisms for DdrB function in which higher-order assemblies of DdrB pentamers assist in the pairing of complementary ssDNA using an extended DNA binding surface.