Tyrosine-modifying glycosylation by Yersinia effectors.

Mono-O-glycosylation of target proteins by bacterial toxins or effector proteins is a well-known mechanism by which bacteria interfere with essential functions of host cells. The respective glycosyltransferases are important virulence factors such as the Clostridioides difficile toxins A and B. Here, we describe two glycosyltransferases of Yersinia species that have a high sequence identity: YeGT from the zoonotic pathogen Yersinia enterocolitica and YkGT from the murine pathogen Yersinia kristensenii. We show that both modify Rho family proteins by attachment of GlcNAc at tyrosine residues (Tyr-34 in RhoA). Notably, the enzymes differed in their target protein specificity. While YeGT modified RhoA, B, and C, YkGT possessed a broader substrate spectrum and glycosylated not only Rho but also Rac and Cdc42 subfamily proteins. Mutagenesis studies indicated that residue 177 is important for this broader target spectrum. We determined the crystal structure of YeGT shortened by 16 residues N terminally (sYeGT) in the ligand-free state and bound to UDP, the product of substrate hydrolysis. The structure assigns sYeGT to the GT-A family. It shares high structural similarity to glycosyltransferase domains from toxins. We also demonstrated that the 16 most N-terminal residues of YeGT and YkGT are important for the mediated translocation into the host cell using the pore-forming protective antigen of anthrax toxin. Mediated introduction into HeLa cells or ectopic expression of YeGT and YkGT caused morphological changes and redistribution of the actin cytoskeleton. The data suggest that YeGT and YkGT are likely bacterial effectors belonging to the family of tyrosine glycosylating bacterial glycosyltransferases.

An Updated View on the Cellular Uptake and Mode-of-Action of Clostridioides difficile Toxins.

Research on the human gut pathogen Clostridioides (C.) difficile and its toxins continues to attract much attention as a consequence of the threat to human health posed by hypervirulent strains. Toxin A (TcdA) and Toxin B (TcdB) are the two major virulence determinants of C. difficile. Both are single-chain proteins with a similar multidomain architecture. Certain hypervirulent C. difficile strains also produce a third toxin, namely binary toxin CDT (C. difficile transferase). C. difficile toxins are the causative agents of C. difficile-associated diseases (CDADs), such as antibiotics-associated diarrhea and pseudomembranous colitis. For that reason, considerable efforts have been expended to unravel their molecular mode-of-action and the cellular mechanisms responsible for their uptake. Many of these studies have been conducted in European laboratories. Here, we provide an update on our previous review (Papatheodorou et al. Adv Exp Med Biol, 2018) on important advances in C. difficile toxins research.

Antibodies Directed Against GalNAc- and GlcNAc-O-Tyrosine Posttranslational Modifications - a New Tool for Glycoproteomic Detection.

In the last decade, it was discovered that protein mucin-type O-glycosylation and O-GlcNAcylation modify Tyr residues besides the well explored Thr and Ser amino acids. Several glycoproteomic studies have identified α-GalNAc-O-Tyr modifications, and studies propose that ß-GlcNAc-O-Tyr also exists as a new group of posttranslational modifications (PTMs). Specific bacterial toxins have further been identified to modify host GTPases with α-GlcNAc-O-Tyr to promote bacterial virulence. Despite being identified on numerous proteins, the biological roles, biosynthesis and expression of GalNAc- and GlcNAc-O-Tyr modifications are poorly understood. A major obstacle is the lack of tools to specifically detect and identify proteins containing these modifications. With this in mind, we prepared vaccine constructs and raised antibodies to enable selective detection of proteins carrying these new PTMs. The obtained polyclonal antibody sera were evaluated using ELISA and glycopeptide microarrays and were found to be highly selective for GlcNAc- and GalNAc-O-Tyr glycopeptides over the corresponding Ser- and Thr-modifications. For microarray analysis, synthetic GlcNAc- and GalNAc-O-Tyr Fmoc-amino acids were prepared and applied in Fmoc-SPPS to obtain an extensive O-glycopeptide library. After affinity purification, the antibodies were applied in western blot analysis and showed specific detection of α-GlcNAc-O-Tyr modified RhoA GTPase.

Clostridioides difficile Binary Toxin Is Recognized by the Toll-Like Receptor 2/6 Heterodimer to Induce a Nuclear Factor-κB Response.

Clostridioides difficile infection (CDI) represents a significant burden on the health care system, one that is exacerbated by the emergence of binary toxin (CDT)-producing hypervirulent C. difficile strains. Previous work from our laboratory has shown that Toll-like receptor 2 (TLR2) recognizes CDT to induce inflammation. Here we explore the interactions of CDT with TLR2 and the impact on host immunity during CDI. We found that the TLR2/6 heterodimer, not TLR2/1, is responsible for CDT recognition, and that gene pathways including nuclear factor-κB and MAPK downstream of TLR2/6 are upregulated in mice with intact TLR2/6 signaling during CDI.

Cryo-EM structure of the fully-loaded asymmetric anthrax lethal toxin in its heptameric pre-pore state.

Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA), that catalyzes the unfolding of its cytotoxic substrates lethal factor (LF) and edema factor (EF), followed by translocation into the host cell. Substrate recruitment to the heptameric PA pre-pore and subsequent translocation, however, are not well understood. Here, we report three high-resolution cryo-EM structures of the fully-loaded anthrax lethal toxin in its heptameric pre-pore state, which differ in the position and conformation of LFs. The structures reveal that three LFs interact with the heptameric PA and upon binding change their conformation to form a continuous chain of head-to-tail interactions. As a result of the underlying symmetry mismatch, one LF binding site in PA remains unoccupied. Whereas one LF directly interacts with a part of PA called α-clamp, the others do not interact with this region, indicating an intermediate state between toxin assembly and translocation. Interestingly, the interaction of the N-terminal domain with the α-clamp correlates with a higher flexibility in the C-terminal domain of the protein. Based on our data, we propose a model for toxin assembly, in which the relative position of the N-terminal α-helices in the three LFs determines which factor is translocated first.

Clostridium difficile Toxin Biology.

Clostridium difficile is the cause of antibiotics-associated diarrhea and pseudomembranous colitis. The pathogen produces three protein toxins: C. difficile toxins A (TcdA) and B (TcdB), and C. difficile transferase toxin (CDT). The single-chain toxins TcdA and TcdB are the main virulence factors. They bind to cell membrane receptors and are internalized. The N-terminal glucosyltransferase and autoprotease domains of the toxins translocate from low-pH endosomes into the cytosol. After activation by inositol hexakisphosphate (InsP6), the autoprotease cleaves and releases the glucosyltransferase domain into the cytosol, where GTP-binding proteins of the Rho/Ras family are mono-O-glucosylated and, thereby, inactivated. Inactivation of Rho proteins disturbs the organization of the cytoskeleton and affects multiple Rho-dependent cellular processes, including loss of epithelial barrier functions, induction of apoptosis, and inflammation. CDT, the third C. difficile toxin, is a binary actin-ADP-ribosylating toxin that causes depolymerization of actin, thereby inducing formation of the microtubule-based protrusions. Recent progress in understanding of the toxins' actions include insights into the toxin structures, their interaction with host cells, and functional consequences of their actions.

Involvement of N-glycans in binding of Photorhabdus luminescens Tc toxin.

Photorhabdus luminescens Tc toxins are large tripartite ABC-type toxin complexes, composed of TcA, TcB and TcC proteins. Tc toxins are widespread and have shown a tropism for a variety of targets including insect, mammalian and human cells. However, their receptors and the specific mechanisms of uptake into target cells remain unknown. Here, we show that the TcA protein TcdA1 interacts with N-glycans, particularly Lewis X/Y antigens. This is confirmed using N-acetylglucosamine transferase I (Mgat1 gene product)-deficient Chinese hamster ovary (CHO) Lec1 cells, which are highly resistant to intoxication by the Tc toxin complex most likely due to the absence of complex N-glycans. Restoring Mgat1 gene activity, and hence complex N-glycan biosynthesis, recapitulated the sensitivity of these cells to the toxin. Exogenous addition of Lewis X trisaccharide partially inhibits intoxication in wild-type cells. Additionally, sialic acid also largely reduced binding of the Tc toxin. Moreover, proteolytic activation of TcdA1 alters glycan-binding and uptake into target cells. The data suggest that TcdA1-binding is most likely multivalent, and carbohydrates probably work cooperatively to facilitate binding and intoxication.

Defined stereoisomers of 2″-amino NAD+ and their activity against human sirtuins and a bacterial (ADP-ribosyl) transferase.

Nicotinamide adenine dinucleotide (NAD+) is an important biomolecule with essential roles at the intersection of energy metabolism, epigenetic regulation and cell signalling. Synthetic analogues of NAD+ are therefore of great interest as chemical tools for medicinal chemistry, chemical biology and drug discovery. Herein, we report the chemical synthesis and full analytical characterisation of three stereoisomers of 2″-amino NAD+, and their biochemical evaluation against two classes of NAD+-consuming enzymes: the human sirtuins 1-3, and the bacterial toxin TccC3. To rationalise the observed activities, molecular docking experiments were carried out with SIRT1 and SIRT2, which identified the correct orientation of the pyrophosphate linkage as a major determinant for activity in this series. These results, together with results from stability tests and a conformational analysis, allow, for the first time, a side-by-side comparison of the chemical and biochemical features, and analytical properties, of different 2″-amino NAD+ stereoisomers. Our findings provide insight into the recognition of co-substrate analogues by sirtuins, and will greatly facilitate the application of these important NAD+ analogues as chemical tool compounds for mechanistic studies with these as well as other NAD+-dependent enyzmes.

Human peptide α-defensin-1 interferes with Clostridioides difficile toxins TcdA, TcdB, and CDT.

The human pathogenic bacterium Clostridioides difficile produces two exotoxins TcdA and TcdB, which inactivate Rho GTPases thereby causing C. difficile-associated diseases (CDAD) including life-threatening pseudomembranous colitis. Hypervirulent strains produce additionally the binary actin ADP-ribosylating toxin CDT. These strains are hallmarked by more severe forms of CDAD and increased frequency and severity. Once in the cytosol, the toxins act as enzymes resulting in the typical clinical symptoms. Therefore, targeting and inactivation of the released toxins are of peculiar interest. Prompted by earlier findings that human α-defensin-1 neutralizes TcdB, we investigated the effects of the defensin on all three C. difficile toxins. Inhibition of TcdA, TcdB, and CDT was demonstrated by analyzing toxin-induced changes in cell morphology, substrate modification, and decrease in transepithelial electrical resistance. Application of α-defensin-1 protected cells and human intestinal organoids from the cytotoxic effects of TcdA, TcdB, CDT, and their combination which is attributed to a direct interaction between the toxins and α-defensin-1. In mice, the application of α-defensin-1 reduced the TcdA-induced damage of intestinal loops in vivo. In conclusion, human α-defensin-1 is a specific and potent inhibitor of the C. difficile toxins and a promising agent to develop novel therapeutic options against C. difficile infections.

The chaperonin TRiC/CCT is essential for the action of bacterial glycosylating protein toxins like Clostridium difficile toxins A and B.

Various bacterial protein toxins, including Clostridium difficile toxins A (TcdA) and B (TcdB), attack intracellular target proteins of host cells by glucosylation. After receptor binding and endocytosis, the toxins are translocated into the cytosol, where they modify target proteins (e.g., Rho proteins). Here we report that the activity of translocated glucosylating toxins depends on the chaperonin TRiC/CCT. The chaperonin subunits CCT4/5 directly interact with the toxins and enhance the refolding and restoration of the glucosyltransferase activities of toxins after heat treatment. Knockdown of CCT5 by siRNA and HSF1A, an inhibitor of TRiC/CCT, blocks the cytotoxic effects of TcdA and TcdB. In contrast, HSP90, which is involved in the translocation and uptake of ADP ribosylating toxins, is not involved in uptake of the glucosylating toxins. We show that the actions of numerous glycosylating toxins from various toxin types and different species depend on TRiC/CCT. Our data indicate that the TRiC/CCT chaperonin system is specifically involved in toxin uptake and essential for the action of various glucosylating protein toxins acting intracellularly on target proteins.

A cysteine protease-like domain enhances the cytotoxic effects of the Photorhabdus asymbiotica toxin PaTox.

The nematode mutualistic bacterium Photorhabdus asymbiotica produces a large virulence-associated multifunctional protein toxin named PaTox. A glycosyltransferase domain and a deamidase domain of this large toxin function as effectors that specifically target host Rho GTPases and heterotrimeric G proteins, respectively. Modification of these intracellular regulators results in toxicity toward insects and mammalian cells. In this study, we identified a cysteine protease-like domain spanning PaTox residues 1844-2114 (PaToxP), upstream of these two effector domains and characterized by three conserved amino acid residues (Cys-1865, His-1955, and Asp-1975). We determined the crystal structure of the PaToxP C1865A variant by native single-wavelength anomalous diffraction of sulfur atoms (sulfur-SAD). At 2.0 Å resolution, this structure revealed a catalytic site typical for papain-like cysteine proteases, comprising a catalytic triad, oxyanion hole, and typical secondary structural elements. The PaToxP structure had highest similarity to that of the AvrPphB protease from Pseudomonas syringae classified as a C58-protease. Furthermore, we observed that PaToxP shares structural homology also with non-C58-cysteine proteases, deubiquitinases, and deamidases. Upon delivery into insect larvae, PaToxP alone without full-length PaTox had no toxic effects. Yet, PaToxP expression in mammalian cells was toxic and enhanced the apoptotic phenotype induced by PaTox in HeLa cells. We propose that PaToxP is a C58-like cysteine protease module that is essential for full PaTox activity.

The Legionella effector LtpM is a new type of phosphoinositide-activated glucosyltransferase.

Legionella pneumophila causes Legionnaires' disease, a severe form of pneumonia. L. pneumophila translocates more than 300 effectors into host cells via its Dot/Icm (Defective in organelle trafficking/Intracellular multiplication) type IV secretion system to enable its replication in target cells. Here, we studied the effector LtpM, which is encoded in a recombination hot spot in L. pneumophila Paris. We show that a C-terminal phosphoinositol 3-phosphate (PI3P)-binding domain, also found in otherwise unrelated effectors, targets LtpM to the Legionella-containing vacuole and to early and late endosomes. LtpM expression in yeast caused cytotoxicity. Sequence comparison and structural homology modeling of the N-terminal domain of LtpM uncovered a remote similarity to the glycosyltransferase (GT) toxin PaTox from the bacterium Photorhabdus asymbiotica; however, instead of the canonical DxD motif of GT-A type glycosyltransferases, essential for enzyme activity and divalent cation coordination, we found that a DxN motif is present in LtpM. Using UDP-glucose as sugar donor, we show that purified LtpM nevertheless exhibits glucohydrolase and autoglucosylation activity in vitro and demonstrate that PI3P binding activates LtpM's glucosyltransferase activity toward protein substrates. Substitution of the aspartate or the asparagine in the DxN motif abolished the activity of LtpM. Moreover, whereas all glycosyltransferase toxins and effectors identified so far depend on the presence of divalent cations, LtpM is active in their absence. Proteins containing LtpM-like GT domains are encoded in the genomes of other L. pneumophila isolates and species, suggesting that LtpM is the first member of a novel family of glycosyltransferase effectors employed to subvert hosts.

Salmonella Typhimurium effector SseI inhibits chemotaxis and increases host cell survival by deamidation of heterotrimeric Gi proteins.

Salmonella enterica serotype Typhimurium (S. Typhimurium) is one of the most frequent causes of food-borne illness in humans and usually associated with acute self-limiting gastroenteritis. However, in immunocompromised patients, the pathogen can disseminate and lead to severe systemic diseases. S. Typhimurium are facultative intracellular bacteria. For uptake and intracellular life, Salmonella translocate numerous effector proteins into host cells using two type-III secretion systems (T3SS), which are encoded within Salmonella pathogenicity islands 1 (SPI-1) and 2 (SPI-2). While SPI-1 effectors mainly promote initial invasion, SPI-2 effectors control intracellular survival and proliferation. Here, we elucidate the mode of action of Salmonella SPI-2 effector SseI, which is involved in control of systemic dissemination of S. Typhimurium. SseI deamidates a specific glutamine residue of heterotrimeric G proteins of the Gαi family, resulting in persistent activation of the G protein. Gi activation inhibits cAMP production and stimulates PI3-kinase γ by Gαi-released Gßγ subunits, resulting in activation of survival pathways by phosphorylation of Akt and mTOR. Moreover, SseI-induced deamidation leads to non-polarized activation of Gαi and, thereby, to loss of directed migration of dendritic cells.

Cytotoxicity of Clostridium difficile toxins A and B requires an active and functional SREBP-2 pathway.

Clostridium difficile is associated with antibiotic-associated diarrhea and pseudomembranous colitis in humans. Its 2 major toxins, toxins A and B, enter host cells and inactivate GTPases of the Ras homologue/rat sarcoma family by glucosylation. Pore formation of the toxins in the endosomal membrane enables the translocation of their glucosyltransferase domain into the cytosol, and membrane cholesterol is crucial for this process. Here, we asked whether the activity of the sterol regulatory element-binding protein 2 (SREBP-2) pathway, which regulates the cholesterol content in membranes, affects the susceptibility of target cells toward toxins A and B. We show that the SREBP-2 pathway is crucial for the intoxication process of toxins A and B by using pharmacological inhibitors (PF-429242, 25-hydroxycholesterol) and cells that are specifically deficient in SREBP-2 pathway signaling. SREBP-2 pathway inhibition disturbed the cholesterol-dependent pore formation of toxin B in cellular membranes. Preincubation with the cholesterol-lowering drug simvastatin protected cells from toxin B intoxication. Inhibition of the SREBP-2 pathway was without effect when the enzyme portion of toxin B was introduced into target cells via the cell delivery property of anthrax protective antigen. Taken together, these findings allowed us to identify the SREBP-2 pathway as a suitable target for the development of antitoxin therapeutics against C. difficile toxins A and B.-Papatheodorou, P., Song, S., López-Ureña, D., Witte, A., Marques, F., Ost, G. S., Schorch, B., Chaves-Olarte, E., Aktories, K. Cytotoxicity of Clostridium difficile toxins A and B requires an active and functional SREBP-2 pathway.

Revisiting an old antibiotic: bacitracin neutralizes binary bacterial toxins and protects cells from intoxication.

The antibiotic bacitracin (Bac) inhibits cell wall synthesis of gram-positive bacteria. Here, we discovered a totally different activity of Bac: the neutralization of bacterial exotoxins. Bac prevented intoxication of mammalian cells with the binary enterotoxins Clostridium botulinum C2, C. perfringens ι, C. difficile transferase (CDT), and Bacillus anthracis lethal toxin. The transport (B) subunits of these toxins deliver their respective enzyme (A) subunits into cells. Following endocytosis, the B subunits form pores in membranes of endosomes, which mediate translocation of the A subunits into the cytosol. Bac inhibited formation of such B pores in lipid bilayers in vitro and in living cells, thereby preventing translocation of the A subunit into the cytosol. Bac preserved the epithelial integrity of toxin-treated CaCo-2 monolayers, a model for the human gut epithelium. In conclusion, Bac should be discussed as a therapeutic option against infections with medically relevant toxin-producing bacteria, including C. difficile and B. anthracis, because it inhibits bacterial growth and neutralizes the secreted toxins.-Schnell, L., Felix, I., Müller, B., Sadi, M., von Bank, F., Papatheodorou, P., Popoff, M. R., Aktories, K., Waltenberger, E., Benz, R., Weichbrodt, C., Fauler, M., Frick, M., Barth, H. Revisiting an old antibiotic: bacitracin neutralizes binary bacterial toxins and protects cells from intoxication.

Photorhabdus luminescens Tc toxin is inhibited by the protease inhibitor MG132 and activated by protease cleavage resulting in increased binding to target cells.

Photorhabdus luminescens Tc toxins consist of the cell-binding component TcA, the linker component TcB, and the enzyme component TcC. TccC3, a specific isoform of TcC, ADP-ribosylates actin and causes redistribution of the actin cytoskeleton. TccC5, another isoform of TcC, ADP-ribosylates and activates Rho proteins. Here, we report that the proteasome inhibitor MG132 blocks the intoxication of cells by Tc toxin. The inhibitory effect of MG132 was not observed, when the ADP-ribosyltransferase domain of the TcC component was introduced into target cells by protective antigen, which is the binding and delivery component of anthrax toxin. Additionally, MG132 affected neither pore formation by TcA in artificial membranes nor binding of the toxin to cells. Furthermore, the in vitro ADP-ribosylation of actin by the enzyme domain of TccC3 was not affected by MG132. Similar to MG132, several calpain inhibitors blocked the action of the Tc toxin. Proteolytic cleavage of the binding component TcA induced by P. luminescens protease PrtA1 or by collagenase largely increased the toxicity of the Tc toxin. MG132 exhibited no inhibitory effect on the cleaved TcA component. Moreover, binding of TcA to target cells was largely increased after cleavage. The data indicate that Tc toxin is activated by proteolytic processing of the TcA component, resulting in increased receptor binding. Toxin processing is probably inhibited by MG132.

Mechanism of Tc toxin action revealed in molecular detail.

Tripartite Tc toxin complexes of bacterial pathogens perforate the host membrane and translocate toxic enzymes into the host cell, including in humans. The underlying mechanism is complex but poorly understood. Here we report the first, to our knowledge, high-resolution structures of a TcA subunit in its prepore and pore state and of a complete 1.7 megadalton Tc complex. The structures reveal that, in addition to a translocation channel, TcA forms four receptor-binding sites and a neuraminidase-like region, which are important for its host specificity. pH-induced opening of the shell releases an entropic spring that drives the injection of the TcA channel into the membrane. Binding of TcB/TcC to TcA opens a gate formed by a six-bladed ß-propeller and results in a continuous protein translocation channel, whose architecture and properties suggest a novel mode of protein unfolding and translocation. Our results allow us to understand key steps of infections involving Tc toxins at the molecular level.

A syringe-like injection mechanism in Photorhabdus luminescens toxins.

Photorhabdus luminescens is an insect pathogenic bacterium that is symbiotic with entomopathogenic nematodes. On invasion of insect larvae, P. luminescens is released from the nematodes and kills the insect through the action of a variety of virulence factors including large tripartite ABC-type toxin complexes (Tcs). Tcs are typically composed of TcA, TcB and TcC proteins and are biologically active only when complete. Functioning as ADP-ribosyltransferases, TcC proteins were identified as the actual functional components that induce actin-clustering, defects in phagocytosis and cell death. However, little is known about the translocation of TcC into the cell by the TcA and TcB components. Here we show that TcA in P. luminescens (TcdA1) forms a transmembrane pore and report its structure in the prepore and pore state determined by cryoelectron microscopy. We find that the TcdA1 prepore assembles as a pentamer forming an α-helical, vuvuzela-shaped channel less than 1.5 nanometres in diameter surrounded by a large outer shell. Membrane insertion is triggered not only at low pH as expected, but also at high pH, explaining Tc action directly through the midgut of insects. Comparisons with structures of the TcdA1 pore inserted into a membrane and in complex with TcdB2 and TccC3 reveal large conformational changes during membrane insertion, suggesting a novel syringe-like mechanism of protein translocation. Our results demonstrate how ABC-type toxin complexes bridge a membrane to insert their lethal components into the cytoplasm of the host cell. We believe that the proposed mechanism is characteristic of the whole ABC-type toxin family. This explanation of toxin translocation is a step towards understanding the host-pathogen interaction and the complex life cycle of P. luminescens and other pathogens, including human pathogenic bacteria, and serves as a strong foundation for the development of biopesticides.