Publications by authors named "Roman A Melnyk"

54 Publications

Large Clostridial Toxins: Mechanisms and Roles in Disease.

Microbiol Mol Biol Rev 2021 Jun 2:e0006421. Epub 2021 Jun 2.

Molecular Medicine Program, The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada.

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.
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http://dx.doi.org/10.1128/MMBR.00064-21DOI Listing
June 2021

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

Sci Adv 2020 12 11;6(50). Epub 2020 Dec 11.

Molecular Medicine Program, The Hospital for Sick Children, Toronto, ON, Canada.

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.
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http://dx.doi.org/10.1126/sciadv.abb0385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732195PMC
December 2020

The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis.

PLoS Pathog 2020 09 22;16(9):e1008852. Epub 2020 Sep 22.

Department of Biochemistry, University of Toronto, and Program in Molecular Medicine, The Hospital for Sick Children, Toronto, ON, Canada.

Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition.
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http://dx.doi.org/10.1371/journal.ppat.1008852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531778PMC
September 2020

Attenuated diphtheria toxin mediates siRNA delivery.

Sci Adv 2020 May 1;6(18). Epub 2020 May 1.

Department of Chemistry, University of Toronto, Toronto, ON, Canada.

Toxins efficiently deliver cargo to cells by binding to cell surface ligands, initiating endocytosis, and escaping the endolysosomal pathway into the cytoplasm. We took advantage of this delivery pathway by conjugating an attenuated diphtheria toxin to siRNA, thereby achieving gene downregulation in patient-derived glioblastoma cells. We delivered siRNA against integrin-β1 ()-a gene that promotes invasion and metastasis-and siRNA against eukaryotic translation initiation factor 3 subunit b ()-a survival gene. We demonstrated mRNA downregulation of both genes and the corresponding functional outcomes: knockdown of led to a significant inhibition of invasion, shown with an innovative 3D hydrogel model; and knockdown of resulted in significant cell death. This is the first example of diphtheria toxin being used to deliver siRNAs, and the first time a toxin-based siRNA delivery strategy has been shown to induce relevant genotypic and phenotypic effects in cancer cells.
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http://dx.doi.org/10.1126/sciadv.aaz4848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7195190PMC
May 2020

An engineered chimeric toxin that cleaves activated mutant and wild-type RAS inhibits tumor growth.

Proc Natl Acad Sci U S A 2020 07 2;117(29):16938-16948. Epub 2020 Jul 2.

Department of Microbiology and Immunology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611;

Despite nearly four decades of effort, broad inhibition of oncogenic RAS using small-molecule approaches has proven to be a major challenge. Here we describe the development of a pan-RAS biologic inhibitor composed of the RAS-RAP1-specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered chimeric toxin irreversibly cleaves and inactivates intracellular RAS at low picomolar concentrations terminating downstream signaling in receptor-bearing cells. Furthermore, we demonstrate in vivo target engagement and reduction of tumor burden in three mouse xenograft models driven by either wild-type or mutant Intracellular delivery of a potent anti-RAS biologic through a receptor-mediated mechanism represents a promising approach to developing RAS therapeutics against a broad array of cancers.
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http://dx.doi.org/10.1073/pnas.2000312117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382267PMC
July 2020

Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins.

Cell 2020 07 25;182(2):345-356.e16. Epub 2020 Jun 25.

Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; Molecular Architecture of Life Program, Canadian Institute for Advanced Research (CIFAR), Toronto, ON M5G 1M1, Canada. Electronic address:

Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.
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http://dx.doi.org/10.1016/j.cell.2020.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316060PMC
July 2020

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

Proc Natl Acad Sci U S A 2020 03 9;117(12):6792-6800. Epub 2020 Mar 9.

Molecular Medicine, Research Institute, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada;

Intestinal bile acids are known to modulate the germination and growth of Here we describe a role for intestinal bile acids in directly binding and neutralizing TcdB toxin, the primary determinant of 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 pathogenesis, these findings provide a framework for development of a mechanistic class of antitoxins.
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http://dx.doi.org/10.1073/pnas.1916965117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104382PMC
March 2020

bioPROTACs as versatile modulators of intracellular therapeutic targets including proliferating cell nuclear antigen (PCNA).

Proc Natl Acad Sci U S A 2020 03 2;117(11):5791-5800. Epub 2020 Mar 2.

Quantitative Biosciences, MSD International, Singapore 138665;

Targeted degradation approaches such as proteolysis targeting chimeras (PROTACs) offer new ways to address disease through tackling challenging targets and with greater potency, efficacy, and specificity over traditional approaches. However, identification of high-affinity ligands to serve as PROTAC starting points remains challenging. As a complementary approach, we describe a class of molecules termed biological PROTACs (bioPROTACs)-engineered intracellular proteins consisting of a target-binding domain directly fused to an E3 ubiquitin ligase. Using GFP-tagged proteins as model substrates, we show that there is considerable flexibility in both the choice of substrate binders (binding positions, scaffold-class) and the E3 ligases. We then identified a highly effective bioPROTAC against an oncology target, proliferating cell nuclear antigen (PCNA) to elicit rapid and robust PCNA degradation and associated effects on DNA synthesis and cell cycle progression. Overall, bioPROTACs are powerful tools for interrogating degradation approaches, target biology, and potentially for making therapeutic impacts.
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http://dx.doi.org/10.1073/pnas.1920251117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084165PMC
March 2020

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

Nat Commun 2020 01 23;11(1):432. Epub 2020 Jan 23.

Molecular Medicine Program, The Hospital for Sick Children Research Institute, Toronto, ON, M5G 0A4, Canada.

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.
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http://dx.doi.org/10.1038/s41467-020-14306-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978384PMC
January 2020

Drug Screen Identifies Leflunomide for Treatment of Inflammatory Bowel Disease Caused by TTC7A Deficiency.

Gastroenterology 2020 03 16;158(4):1000-1015. Epub 2019 Nov 16.

SickKids Inflammatory Bowel Disease Centre, The Hospital for Sick Children, Toronto, Ontario, Canada; Cell Biology Program, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada; Department of Pediatrics, Institute of Medical Science and Biochemistry, University of Toronto, The Hospital for Sick Children, Toronto, Ontario, Canada. Electronic address:

Background & Aims: Mutations in the tetratricopeptide repeat domain 7A gene (TTC7A) cause intestinal epithelial and immune defects. Patients can become immune deficient and develop apoptotic enterocolitis, multiple intestinal atresia, and recurrent intestinal stenosis. The intestinal disease in patients with TTC7A deficiency is severe and untreatable, and it recurs despite resection or allogeneic hematopoietic stem cell transplant. We screened drugs for those that prevent apoptosis of in cells with TTC7A deficiency and tested their effects in an animal model of the disease.

Methods: We developed a high-throughput screen to identify compounds approved by the US Food and Drug Administration that reduce activity of caspases 3 and 7 in TTC7A-knockout (TTC7A-KO) HAP1 (human haploid) cells and reduce the susceptibility to apoptosis. We validated the effects of identified agents in HeLa cells that stably express TTC7A with point mutations found in patients. Signaling pathways in cells were analyzed by immunoblots. We tested the effects of identified agents in zebrafish with disruption of ttc7a, which develop intestinal defects, and colonoids derived from biopsy samples of patients with and without mutations in TTC7A. We performed real-time imaging of intestinal peristalsis in zebrafish and histologic analyses of intestinal tissues from patients and zebrafish. Colonoids were analyzed by immunofluorescence and for ion transport.

Results: TTC7A-KO HAP1 cells have abnormal morphology and undergo apoptosis, due to increased levels of active caspases 3 and 7. We identified drugs that increased cell viability; leflunomide (used to treat patients with inflammatory conditions) reduced caspase 3 and 7 activity in cells by 96%. TTC7A-KO cells contained cleaved caspase 3 and had reduced levels of phosphorylated AKT and X-linked inhibitor of apoptosis (XIAP); incubation of these cells with leflunomide increased levels of phosphorylated AKT and XIAP and reduced levels of cleaved caspase 3. Administration of leflunomide to ttc7a zebrafish increased gut motility, reduced intestinal tract narrowing, increased intestinal cell survival, increased sizes of intestinal luminal spaces, and restored villi and goblet cell morphology. Exposure of patient-derived colonoids to leflunomide increased cell survival, polarity, and transport function.

Conclusions: In a drug screen, we identified leflunomide as an agent that reduces apoptosis and activates AKT signaling in TTC7A-KO cells. In zebrafish with disruption of ttc7a, leflunomide restores gut motility, reduces intestinal tract narrowing, and increases intestinal cell survival. This drug might be repurposed for treatment of TTC7A deficiency.
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http://dx.doi.org/10.1053/j.gastro.2019.11.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062591PMC
March 2020

Dismantling a Toxin to Disarm a Superbug.

Trends Pharmacol Sci 2019 03 23;40(3):155-156. Epub 2019 Jan 23.

Molecular Medicine, The Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

Clostridium difficile (Clostridioides difficile) is a toxin-producing, multidrug-resistant bacterium. Inhibiting the effects of toxins, which are responsible for the symptoms of disease, is viewed as a promising non-antibiotic approach to treat C. difficile infection (CDI). By inducing premature activation of toxins, Ivarsson and colleagues (Cell Chemical Biology 2018; http://doi.org/10.1016/j.chembiol.2018.10.002) uncover a clever new strategy to block toxin action.
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http://dx.doi.org/10.1016/j.tips.2019.01.007DOI Listing
March 2019

Host-targeted niclosamide inhibits C. difficile virulence and prevents disease in mice without disrupting the gut microbiota.

Nat Commun 2018 12 7;9(1):5233. Epub 2018 Dec 7.

Molecular Medicine, Hospital for Sick Children, 686 Bay St., Toronto, ON, M5G 0A4, Canada.

Clostridium difficile is the leading cause of nosocomial diarrhea and colitis in the industrialized world. Disruption of the protective gut microbiota by antibiotics enables colonization by multidrug-resistant C. difficile, which secrete up to three different protein toxins that are responsible for the gastrointestinal sequelae. Oral agents that inhibit the damage induced by toxins, without altering the gut microbiota, are urgently needed to prevent primary disease and break the cycle of antibiotic-induced disease recurrence. Here, we show that the anthelmintic drug, niclosamide, inhibits the pathogenesis of all three toxins by targeting a host process required for entry into colonocytes by each toxin. In mice infected with an epidemic strain of C. difficile, expressing all three toxins, niclosamide reduced both primary disease and recurrence, without disrupting the diversity or composition of the gut microbiota. Given its excellent safety profile, niclosamide may address an important unmet need in preventing C. difficile primary and recurrent diseases.
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http://dx.doi.org/10.1038/s41467-018-07705-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286312PMC
December 2018

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

Mol Pharm 2018 11 26;15(11):5217-5226. Epub 2018 Sep 26.

Division of Immunology and Allergy , The Hospital for Sick Children , Toronto , ON M5G 0A4 , Canada.

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.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b00735DOI Listing
November 2018

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

FEBS Lett 2018 08 16;592(16):2693-2705. Epub 2018 Aug 16.

Department of Biology, University of Waterloo, Canada.

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.
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http://dx.doi.org/10.1002/1873-3468.13208DOI Listing
August 2018

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

J Mol Biol 2018 09 7;430(18 Pt B):3190-3199. Epub 2018 Jul 7.

Molecular Medicine Program, The Hospital for Sick Children Research Institute, Toronto M5G 0A4, Ontario, Canada; Department of Biochemistry, University of Toronto, Toronto M5S 1A8, Ontario, Canada. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jmb.2018.07.001DOI Listing
September 2018

A neutralizing antibody that blocks delivery of the enzymatic cargo of toxin TcdB into host cells.

J Biol Chem 2018 01 27;293(3):941-952. Epub 2017 Nov 27.

From the Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2363,

infection is the leading cause of hospital-acquired diarrhea and is mediated by the actions of two toxins, TcdA and TcdB. The toxins perturb host cell function through a multistep process of receptor binding, endocytosis, low pH-induced pore formation, and the translocation and delivery of an N-terminal glucosyltransferase domain that inactivates host GTPases. Infection studies with isogenic strains having defined toxin deletions have established TcdB as an important target for therapeutic development. Monoclonal antibodies that neutralize TcdB function have been shown to protect against infection in animal models and reduce recurrence in humans. Here, we report the mechanism of TcdB neutralization by PA41, a humanized monoclonal antibody capable of neutralizing TcdB from a diverse array of strains. Through a combination of structural, biochemical, and cell functional studies, involving X-ray crystallography and EM, we show that PA41 recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and blocks productive translocation and delivery of the enzymatic cargo into the host cell. Our study reveals a unique mechanism of toxin neutralization by a monoclonal antibody, which involves targeting a process that is conserved across the large clostridial glucosylating toxins. The PA41 antibody described here provides a valuable tool for dissecting the mechanism of toxin pore formation and translocation across the endosomal membrane.
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http://dx.doi.org/10.1074/jbc.M117.813428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777265PMC
January 2018

Identification of novel bifunctional HIV-1 reverse transcriptase inhibitors.

J Antimicrob Chemother 2018 Jan;73(1):109-117

Department of Antiviral Research, Merck Frosst Center for Therapeutic Research, Pointe-Claire - Dorval H9R 4P8, Canada.

Objectives: The increasing prevalence of mutations in HIV-1 reverse transcriptase (RT) that confer resistance to existing NRTIs and NNRTIs underscores the need to develop RT inhibitors with novel mode-of-inhibition and distinct resistance profiles.

Methods: Biochemical assays were employed to identify inhibitors of RT activity and characterize their mode of inhibition. The antiviral activity of the inhibitors was assessed by cell-based assays using laboratory HIV-1 isolates and MT4 cells. RT variants were purified via avidin affinity columns.

Results: Compound A displayed equal or greater potency against many common NNRTI-resistant RTs (K103N and Y181C RTs) relative to WT RT. Despite possessing certain NNRTI-like properties, such as being unable to inhibit an engineered variant of RT lacking an NNRTI-binding pocket, we found that compound A was dependent on Mg2+ for binding to RT. Optimization of compound A led to more potent analogues, which retained similar activities against WT and K103N mutant viruses with submicromolar potency in a cell-based assay. One of the analogues, compound G, was crystallized in complex with RT and the structure was determined at 2.6 Å resolution. The structure indicated that compound G simultaneously interacts with the active site (Asp186), the highly conserved primer grip region (Leu234 and Trp229) and the NNRTI-binding pocket (Tyr188).

Conclusions: These findings reveal a novel class of RT bifunctional inhibitors that are not sensitive to the most common RT mutations, which can be further developed to address the deficiency of current RT inhibitors.
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http://dx.doi.org/10.1093/jac/dkx332DOI Listing
January 2018

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

J Biol Chem 2017 10 23;292(42):17290-17301. Epub 2017 Aug 23.

Molecular Medicine, The Hospital for Sick Children, Ontario M5G 0A4, Canada,

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.
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http://dx.doi.org/10.1074/jbc.M117.806687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655507PMC
October 2017

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

Crit Rev Biochem Mol Biol 2017 08 26;52(4):461-473. Epub 2017 May 26.

a Molecular Medicine Program , The Hospital for Sick Children Research Institute , Toronto , ON , Canada.

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.
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http://dx.doi.org/10.1080/10409238.2017.1325831DOI Listing
August 2017

Repurposing bacterial toxins for intracellular delivery of therapeutic proteins.

Biochem Pharmacol 2017 10 10;142:13-20. Epub 2017 Apr 10.

Molecular Medicine Program, The Hospital for Sick Children Research Institute, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bcp.2017.04.009DOI Listing
October 2017

Comment on "A small-molecule antivirulence agent for treating Clostridium difficile infection".

Sci Transl Med 2016 12;8(370):370tc2

Molecular Structure & Function, The Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada.

New insights into the mechanism of action of ebselen, a small-molecule antivirulence agent that reduces disease pathology in a mouse model of Clostridium difficile infection, suggest a different molecular target may be responsible for its efficacy.
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http://dx.doi.org/10.1126/scitranslmed.aad8926DOI Listing
December 2016

Crystal structure of Clostridium difficile toxin A.

Nat Microbiol 2016 Jan 11;1:15002. Epub 2016 Jan 11.

Chemical and Physical Biology Program, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.

Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4976693PMC
http://dx.doi.org/10.1038/nmicrobiol.2015.2DOI Listing
January 2016

Crystal structure of toxin A.

Nat Microbiol 2016;1. Epub 2016 Jan 11.

Chemical and Physical Biology Program, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA; Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA; Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37205, USA; The Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee 37212, USA.

infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics.
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http://dx.doi.org/10.1038/nmicrobiol.2015.2DOI Listing
January 2016

Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms.

Sci Adv 2016 05 20;2(5):e1501632. Epub 2016 May 20.

Program in Molecular Structure & Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.; Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

Bacterial biofilms present a significant medical challenge because they are recalcitrant to current therapeutic regimes. A key component of biofilm formation in the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharides Pel and Psl, which are involved in the formation and maintenance of the structural biofilm scaffold and protection against antimicrobials and host defenses. Given that the glycoside hydrolases PelAh and PslGh encoded in the pel and psl biosynthetic operons, respectively, are utilized for in vivo exopolysaccharide processing, we reasoned that these would provide specificity to target P. aeruginosa biofilms. Evaluating these enzymes as potential therapeutics, we demonstrate that these glycoside hydrolases selectively target and degrade the exopolysaccharide component of the biofilm matrix. PelAh and PslGh inhibit biofilm formation over a 24-hour period with a half maximal effective concentration (EC50) of 69.3 ± 1.2 and 4.1 ± 1.1 nM, respectively, and are capable of disrupting preexisting biofilms in 1 hour with EC50 of 35.7 ± 1.1 and 12.9 ± 1.1 nM, respectively. This treatment was effective against clinical and environmental P. aeruginosa isolates and reduced biofilm biomass by 58 to 94%. These noncytotoxic enzymes potentiated antibiotics because the addition of either enzyme to a sublethal concentration of colistin reduced viable bacterial counts by 2.5 orders of magnitude when used either prophylactically or on established 24-hour biofilms. In addition, PelAh was able to increase neutrophil killing by ~50%. This work illustrates the feasibility and benefits of using bacterial exopolysaccharide biosynthetic glycoside hydrolases to develop novel antibiofilm therapeutics.
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http://dx.doi.org/10.1126/sciadv.1501632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4928890PMC
May 2016

Small Molecules Take A Big Step Against Clostridium difficile.

Trends Microbiol 2015 Dec 5;23(12):746-748. Epub 2015 Nov 5.

Molecular Structure & Function, The Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

Effective treatment of Clostridium difficile infections demands a shift away from antibiotics towards toxin-neutralizing agents. Work by Bender et al., using a drug that attenuates toxin action in vivo without affecting bacterial survival, demonstrates the exciting potential of small molecules as a new modality in the fight against C. difficile.
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http://dx.doi.org/10.1016/j.tim.2015.10.009DOI Listing
December 2015

Defective mutations within the translocation domain of Clostridium difficile toxin B impair disease pathogenesis.

Pathog Dis 2016 Feb 26;74(1):ftv098. Epub 2015 Oct 26.

Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, MD 21201, USA

The Clostridium difficile toxin B is one of the main virulence factors and plays an important role in the pathogenesis of C. difficile infection (CDI). We recently revealed crucial residues in the translocation domain of TcdB for the pore formation and toxin translocation. In this study, we investigated the effects of mutating a critical site involved in pore formation, Leu-1106, to residues that differ in size and polarity (Phe, Ala, Cys, Asp). We observed a broad range of effects on TcdB function in vitro consistent with the role of this site in pore formation and translocation. We show that mice challenged systemically with a lethal dose (LD100) of the most defective mutant (L1106K) showed no symptoms of disease highlighting the importance of this residue and the translocation domain in disease pathogenesis. These findings offer insights into the structure function of the toxin translocation pore, and inform novel therapeutic strategies against CDI.
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http://dx.doi.org/10.1093/femspd/ftv098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882082PMC
February 2016

Efficient Delivery of Structurally Diverse Protein Cargo into Mammalian Cells by a Bacterial Toxin.

Mol Pharm 2015 Aug 2;12(8):2962-71. Epub 2015 Jul 2.

‡Department of Biochemistry, University of Toronto, Toronto, ON, Canada.

Platforms enabling targeted delivery of proteins into cells are needed to fully realize the potential of protein-based therapeutics with intracellular sites-of-action. Bacterial toxins are attractive systems to consider as templates for designing protein transduction systems as they naturally bind and enter specific cells with high efficiency. Here we investigated the capacity of diphtheria toxin to function as an intracellular protein delivery vector. We report that diphtheria toxin delivers an impressive array of passenger proteins spanning a range of sizes, structures, and stabilities into cells in a manner that indicates that they are "invisible" to the translocation machinery. Further, we show that α-amylase delivered into cells by a detoxified diphtheria toxin chimera digests intracellular glycogen in live cells, providing evidence that delivered cargo is folded, active, and abundant. The efficiency and versatility of diphtheria toxin over existing systems open numerous possibilities for intracellular delivery of bioactive proteins.
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http://dx.doi.org/10.1021/acs.molpharmaceut.5b00233DOI Listing
August 2015

Small molecule inhibitors of Clostridium difficile toxin B-induced cellular damage.

Chem Biol 2015 Feb 22;22(2):175-85. Epub 2015 Jan 22.

Molecular Structure & Function, The Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

Clostridium difficile causes life-threatening diarrhea through the actions of its homologous toxins TcdA and TcdB on human colonocytes. Therapeutic agents that block toxin-induced damage are urgently needed to prevent the harmful consequences of toxin action that are not addressed with current antibiotic-based treatments. Here, we developed an imaging-based phenotypic screen to identify small molecules that protected human cells from TcdB-induced cell rounding. A series of structurally diverse compounds with antitoxin activity were identified and found to act through one of a small subset of mechanisms, including direct binding and sequestration of TcdB, inhibition of endosomal maturation, and noncompetitive inhibition of the toxin glucosyltransferase activity. Distinct classes of inhibitors were used further to dissect the determinants of the toxin-mediated necrosis phenotype occurring at higher doses of toxin. These findings validate and inform novel targeting strategies for discovering small molecule agents to treat C. difficile infection.
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http://dx.doi.org/10.1016/j.chembiol.2014.12.010DOI Listing
February 2015

Derivatives of mesoxalic acid block translocation of HIV-1 reverse transcriptase.

J Biol Chem 2015 Jan 29;290(3):1474-84. Epub 2014 Oct 29.

From the Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada, the Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada, the Department of Medicine, Division of Experimental Medicine, McGill University, Quebec H3A 1A3, Canada

The pyrophosphate mimic and broad spectrum antiviral phosphonoformic acid (PFA, foscarnet) was shown to freeze the pre-translocational state of the reverse transcriptase (RT) complex of the human immunodeficiency virus type 1 (HIV-1). However, PFA lacks a specificity domain, which is seen as a major reason for toxic side effects associated with the clinical use of this drug. Here, we studied the mechanism of inhibition of HIV-1 RT by the 4-chlorophenylhydrazone of mesoxalic acid (CPHM) and demonstrate that this compound also blocks RT translocation. Hot spots for inhibition with PFA or CPHM occur at template positions with a bias toward pre-translocation. Mutations at active site residue Asp-185 compromise binding of both compounds. Moreover, divalent metal ions are required for the formation of ternary complexes with either of the two compounds. However, CPHM contains both an anchor domain that likely interacts with the catalytic metal ions and a specificity domain. Thus, although the inhibitor binding sites may partly overlap, they are not identical. The K65R mutation in HIV-1 RT, which reduces affinity to PFA, increases affinity to CPHM. Details with respect to the binding sites of the two inhibitors are provided on the basis of mutagenesis studies, structure-activity relationship analyses with newly designed CPHM derivatives, and in silico docking experiments. Together, these findings validate the pre-translocated complex of HIV-1 RT as a specific target for the development of novel classes of RT inhibitors.
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http://dx.doi.org/10.1074/jbc.M114.614305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340395PMC
January 2015
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