Structural Determinants of Clostridium difficile Toxin A Glucosyltransferase Activity

Pruitt, Rory; Chumbler, Nicole M.; Rutherford, Stacey A.; Farrow, Melissa A.; Friedman, David; Spiller, Benjamin W.; Lacy, D. Borden

doi:10.1074/jbc.m111.298414

Cited by 74 publications

(112 citation statements)

References 51 publications

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“…However, it is still possible that Y219 in NleB1 interacts with UDP-GlcNAc by hydrophobic stacking, as observed for numerous aromatic side chain-containing amino acids (39,54,55). Alternatively, Y219 in NleB1 may interact with UDP-GlcNAc via hydrogen bonding, as the equivalent tyrosine in C. difficile toxin A (Y283) is positioned in close proximity to two carbonyls of the ribose ring and a water molecule (56,57 …”

Section: Discussionmentioning

confidence: 99%

Mutagenesis and Functional Analysis of the Bacterial Arginine Glycosyltransferase Effector NleB1 from Enteropathogenic Escherichia coli

et al. 2016

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Enteropathogenic Escherichia coli (EPEC) interferes with host cell signaling by injecting virulence effector proteins into enterocytes via a type III secretion system (T3SS). NleB1 is a novel T3SS glycosyltransferase effector from EPEC that transfers a single Enteropathogenic Escherichia coli (EPEC) is an extracellular diarrheagenic pathogen of children that utilizes a type III secretion system (T3SS) to inject virulence effector proteins directly into host intestinal cells. These effectors subvert host cell function and are encoded by genes either within the locus of enterocyte effacement (LEE) pathogenicity island (1) or outside the LEE (non-Lee-encoded [Nle] effectors) (2). A number of non-LEEencoded effectors that disrupt host cell signaling have been characterized in recent years (3, 4). For example, both NleC and NleE inhibit NF-B signaling and, consequently, block the production of inflammatory cytokines, such as interleukin-8 (5-7). NleB1 was recently characterized to be a glycosyltransferase effector that modifies a conserved arginine residue in the Fas-associated death domain protein (FADD) as well as the death domain-containing proteins TRADD and RIPK1, thereby blocking host death receptor signaling and apoptosis (8, 9). Modification of FADD prevents its recruitment to the Fas receptor and the subsequent recruitment and activation of caspase-8 (8, 9).Glycosyltransferases are enzymes that catalyze the transfer of a sugar moiety from an activated sugar donor substrate containing a phosphate-leaving group, such as nucleoside diphosphate sugars, to a specific acceptor substrate (10, 11). Cytosolic glycosylation is a vital molecular mechanism by which various bacterial toxins and effector proteins subvert eukaryotic host cell function (12). For example, Clostridium difficile toxins A and B modify small Rho GTPases by mono-O-glucosylation at specific threonine residues (13). Glycosylated Rho GTPases are locked in an inactive state, inhibiting downstream signaling pathways, resulting in the disruption of the cell cytoskeleton, the induction of apoptotic cell death, and fluid accumulation in the large intestine (14).The transfer of carbohydrate components to proteins usually occurs at the oxygen nucleophile of the hydroxyl group of a serine or threonine side chain, resulting in the formation of an O-linked glycosidic linkage, or at the nitrogen nucleophile of the amide group of an asparagine side chain, forming an N-linked glycosidic linkage (15)(16)(17)(18)(19). O-GlcNAc modification, unlike classic O-or Nlinked protein glycosylation, involves the transfer of a single GlcNAc moiety to the hydroxyl group of a serine or threonine residue, and this modification is not elongated (17,20). A nonclassical N-linked modification involves the addition of single glycosyl moieties from activated nucleotide sugars to asparagine residues of the target protein (21-23). NleB1 catalyzes an unusual glycosylation reaction that results in the addition of GlcNAc in an N-glycosidic linkage to arginine 117 (R117) of FADD (8,9). Th...

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Section: Discussionmentioning

confidence: 99%

Mutagenesis and Functional Analysis of the Bacterial Arginine Glycosyltransferase Effector NleB1 from Enteropathogenic Escherichia coli

et al. 2016

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“…Like many other A-B toxins that mediate their own delivery into cells, high-resolution structures of the enzymic A domains (11)(12)(13) and the receptor-binding portion of the B domains of glucosylating toxin family members are known (14,15), whereas the structure and mechanism of the pore-forming translocation domain remains poorly characterized. These interconnected processes have been proposed to be mediated by the central ∼1,000-aa D domain (i.e., amino acids 801-1,850); however, with the absence of any structural information for this domain in either the prepore or pore state, no framework exists for resolving the functional determinants for this large domain that govern pore formation and translocation.…”

mentioning

confidence: 99%

Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore

Zhang

Park

Tam

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Disease associated with Clostridium difficile infection is caused by the actions of the homologous toxins TcdA and TcdB on colonic epithelial cells. Binding to target cells triggers toxin internalization into acidified vesicles, whereupon cryptic segments from within the 1,050-aa translocation domain unfurl and insert into the bounding membrane, creating a transmembrane passageway to the cytosol. Our current understanding of the mechanisms underlying pore formation and the subsequent translocation of the upstream cytotoxic domain to the cytosol is limited by the lack of information available regarding the identity and architecture of the transmembrane pore. Here, through systematic perturbation of conserved sites within predicted membrane-insertion elements of the translocation domain, we uncovered highly sensitive residues-clustered between amino acids 1,035 and 1,107-that when individually mutated, reduced cellular toxicity by as much as >1,000-fold. We demonstrate that defective variants are defined by impaired pore formation in planar lipid bilayers and biological membranes, resulting in an inability to intoxicate cells through either apoptotic or necrotic pathways. These findings along with the unexpected similarities uncovered between the pore-forming "hotspots" of TcdB and the wellcharacterized α-helical diphtheria toxin translocation domain provide insights into the structure and mechanism of formation of the translocation pore for this important class of pathogenic toxins.T he primary virulence determinants of pathogenic Clostridium difficile are two protein toxins, TcdA and TcdB, which are responsible for the symptoms associated with infection, including diarrhea and pseudomembranous colitis (1). TcdA and TcdB are large (i.e., 308 and 270 kDa, respectively) homologous toxins (sharing 48% sequence identity) that appear to intoxicate target cells using a strategy that is similar in principle to that described for a number of smaller A-B toxins, such as anthrax toxin (2) and diphtheria toxin (DT) (3). In addition to a cytotoxic enzymic A domain and receptor-binding B domain responsible for binding and translocating the A domain into cells, TcdA and TcdB are additionally equipped with an internal autoprocessing domain that proteolytically cleaves and releases the N-terminal glucosyltransferase domain in response to intracellular inositol hexakisphosphate (4).The series of events leading to the delivery of the A domain into cells begins with toxin binding to an as yet unidentified receptor on target cells via the C-terminal receptor-binding domain (i.e., the B domain), which triggers toxin internalization into acidified vesicles via clathrin-mediated endocytosis (5). In the endosome, cryptic regions from within the large ∼1,000-aa translocation domain emerge and insert into the endosomal membrane, creating a pore that is believed to enable translocation of the N-terminal glucosyltransferase (i.e., the A domain) into the cytosol. Processed and released A chains enzymatically glucosylate and thereby inactivat...

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“…Until recently, the structural reasons for this difference in substrate targeting were not known. A study by Pruitt and colleagues found that the overall structure of the glucosyltransferase domain (GTD) in TcdA is similar to that in TcdB; however, the GTD of TcdA has a larger net negative charge of surface-exposed residues than that in TcdB (137). Moreover, the UDP-glucosebinding pocket of TcdA is positively charged, but the corresponding region in TcdB exhibits a large net negative charge.…”

Section: Mechanism Of Action Of Tcda and Tcdbmentioning

confidence: 99%

Variations in Virulence and Molecular Biology among Emerging Strains of Clostridium difficile

Hunt

Ballard

2013

Microbiol Mol Biol Rev

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SUMMARY Clostridium difficile is a Gram-positive, spore-forming organism which infects and colonizes the large intestine, produces potent toxins, triggers inflammation, and causes significant systemic complications. Treating C. difficile infection (CDI) has always been difficult, because the disease is both caused and resolved by antibiotic treatment. For three and a half decades, C. difficile has presented a treatment challenge to clinicians, and the situation took a turn for the worse about 10 years ago. An increase in epidemic outbreaks related to CDI was first noticed around 2003, and these outbreaks correlated with a sudden increase in the mortality rate of this illness. Further studies discovered that these changes in CDI epidemiology were associated with the rapid emergence of hypervirulent strains of C. difficile , now collectively referred to as NAP1/BI/027 strains. The discovery of new epidemic strains of C. difficile has provided a unique opportunity for retrospective and prospective studies that have sought to understand how these strains have essentially replaced more historical strains as a major cause of CDI. Moreover, detailed studies on the pathogenesis of NAP1/BI/027 strains are leading to new hypotheses on how this emerging strain causes severe disease and is more commonly associated with epidemics. In this review, we provide an overview of CDI, discuss critical mechanisms of C. difficile virulence, and explain how differences in virulence-associated factors between historical and newly emerging strains might explain the hypervirulence exhibited by this pathogen during the past decade.

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Structural Determinants of Clostridium difficile Toxin A Glucosyltransferase Activity

Cited by 74 publications

References 51 publications

Mutagenesis and Functional Analysis of the Bacterial Arginine Glycosyltransferase Effector NleB1 from Enteropathogenic Escherichia coli

Mutagenesis and Functional Analysis of the Bacterial Arginine Glycosyltransferase Effector NleB1 from Enteropathogenic Escherichia coli

Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore

Variations in Virulence and Molecular Biology among Emerging Strains of Clostridium difficile

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