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• W2119009760 abstract "The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC50 = 4.6 ± 0.4 ng/ml) and crustaceans (Artemia nauplii LD50 = 10 ± 2 μg/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity against ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1- and 1.25-Å resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment. The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC50 = 4.6 ± 0.4 ng/ml) and crustaceans (Artemia nauplii LD50 = 10 ± 2 μg/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity against ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1- and 1.25-Å resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment. Many pathogenic bacteria utilize secreted protein toxins (exotoxins) as components of their virulence repertoire. These toxins induce cell death or alter cellular physiology by mechanisms such as proteolysis, pore formation, and covalent modification of host proteins. Although some toxins are responsible for the complete pathology of a disease, others manipulate the host immune response, promote escape from the intracellular environment, release nutrients, or facilitate bacterial penetration of host barriers (1Alouf J.E. Freer J.H. The Comprehensive Sourcebook of Bacterial Protein Toxins. 2nd. Academic Press, San Diego, San Diego1999Google Scholar, 2Burns D.L. Barbieri J.T. Iglewski B.H. Rappuoli R. Bacterial Protein Toxins. ASM Press, Washington, DC2003Crossref Google Scholar). Since secreted toxins have been best characterized in terms of their virulence toward mammals, survival of these pathogens in the environment may provide additional selective pressures. For example, the secreted phospholipase of Pseudomonas aeruginosa encoded by plcS (now known as plcH) contributes to virulence against Candida albicans (3Hogan D.A. Kolter R. Science. 2002; 296: 2229-2232Crossref PubMed Scopus (458) Google Scholar), the greater wax moth, Galleria mellonella (4Jander G. Rahme L.G. Ausubel F.M. J. Bacteriol. 2000; 182: 3843-3845Crossref PubMed Scopus (407) Google Scholar), Arabidopsis, and mice (5Rahme L.G. Stevens E.J. Wolfort S.F. Shao J. Tompkins R.G. Ausubel F.M. Science. 1995; 268: 1899-1902Crossref PubMed Scopus (985) Google Scholar). Furthermore, secreted toxins may play roles in survival or colonization by non-pathogenic bacteria involved in symbioses (6Ruby E.G. Urbanowski M. Campbell J. Dunn A. Faini M. Gunsalus R. Lostroh P. Lupp C. McCann J. Millikan D. Schaefer A. Stabb E. Stevens A. Visick K. Whistler C. Greenberg E.P. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3004-3009Crossref PubMed Scopus (275) Google Scholar). Cholera toxin, an A-B5 toxin that is expressed by some strains of Vibrio cholerae, causes cholera disease by specifically transferring an ADP-ribose group to an Arg residue of the GTP-binding protein Gs, thereby activating adenylate cyclase. Increased concentration of cAMP leads to secretion of Cl-, (HCO3−), and water from epithelial cells at the site of colonization, resulting in dehydration and electrolyte loss from the infected patient. Production of a “rice stool” by these patients, which carries V. cholerae at concentrations as high as 108/ml (7Kaper J.B. Morris Jr., J.G. Levine M.M. Clin. Microbiol. Rev. 1995; 8: 48-86Crossref PubMed Google Scholar), promotes dissemination of the disease among people without access to clean drinking water. Cholera cases have been linked to physical and biological conditions present in aquatic environments (8Alam M. Hasan N.A. Sadique A. Bhuiyan N.A. Ahmed K.U. Nusrin S. Nair G.B. Siddique A.K. Sack R.B. Sack D.A. Huq A. Colwell R.R. Appl. Environ. Microbiol. 2006; 72: 4096-4104Crossref PubMed Scopus (90) Google Scholar, 9Huq A. Sack R.B. Nizam A. Longini I.M. Nair G.B. Ali A. Morris Jr., J.G. Khan M.N. Siddique A.K. Yunus M. Albert M.J. Sack D.A. Colwell R.R. Appl. Environ. Microbiol. 2005; 71: 4645-4654Crossref PubMed Scopus (242) Google Scholar, 10Lobitz B. Beck L. Huq A. Wood B. Fuchs G. Faruque A.S. Colwell R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1438-1443Crossref PubMed Scopus (333) Google Scholar), and the bacterium is now known to be a constituent of the aquatic microbial community (11Colwell R.R. Science. 1996; 274: 2025-2031Crossref PubMed Scopus (969) Google Scholar). Thus far, only two serogroups (O1 and O139, of more than 200 known) are thought to have been responsible for the seven pandemics occurring since 1817 (11Colwell R.R. Science. 1996; 274: 2025-2031Crossref PubMed Scopus (969) Google Scholar). In the environment, most strains of other serogroups (non-O1, non-O139), do not carry the genes encoding cholera toxin, and exhibit considerable genetic diversity. These strains are known to be capable of carrying many additional virulence factors, including hemolysin (12Faruque S.M. Chowdhury N. Kamruzzaman M. Dziejman M. Rahman M.H. Sack D.A. Nair G.B. Mekalanos J.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2123-2128Crossref PubMed Scopus (163) Google Scholar), repeats in the structural toxin (13Chow K.H. Ng T.K. Yuen K.Y. Yam W.C. J. Clin. Microbiol. 2001; 39: 2594-2597Crossref PubMed Scopus (119) Google Scholar), heat-stable enterotoxin (14Sarkar B. Bhattacharya T. Ramamurthy T. Shimada T. Takeda Y. Balakrish N.G. Epidemiol. Infect. 2002; 129: 245-251Crossref PubMed Scopus (13) Google Scholar), hemagglutinin/protease (15Halpern M. Gancz H. Broza M. Kashi Y. Appl. Environ. Microbiol. 2003; 69: 4200-4204Crossref PubMed Scopus (71) Google Scholar), a type III secretion system (16Dziejman M. Serruto D. Tam V.C. Sturtevant D. Diraphat P. Faruque S.M. Rahman M.H. Heidelberg J.F. Decker J. Li L. Montgomery K.T. Grills G. Kucherlapati R. Mekalanos J.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3465-3470Crossref PubMed Scopus (162) Google Scholar), and a novel type VI secretion system associated with virulence (17Pukatzki S. Ma A.T. Sturtevant D. Krastins B. Sarracino D. Nelson W.C. Heidelberg J.F. Mekalanos J.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1528-1533Crossref PubMed Scopus (811) Google Scholar), and have caused sporadic outbreaks of gastrointestinal disease distinct from cholera (18Dalsgaard A. Albert M.J. Taylor D.N. Shimada T. Meza R. Serichantalergs O. Echeverria P. J. Clin. Microbiol. 1995; 33: 2715-2722Crossref PubMed Google Scholar, 19Sharma C. Thungapathra M. Ghosh A. Mukhopadhyay A.K. Basu A. Mitra R. Basu I. Bhattacharya S.K. Shimada T. Ramamurthy T. Takeda T. Yamasaki S. Takeda Y. Nair G.B. J. Clin. Microbiol. 1998; 36: 756-763Crossref PubMed Google Scholar) as well as extra-intestinal infections (20Dalsgaard A. Forslund A. Hesselbjerg A. Bruun B. Clin. Microbiol. Infect. 2000; 6: 625-627Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). However, unsuccessful attempts to correlate genotypes of non-O1, non-O139 V. cholerae isolates with their virulence phenotypes in rabbit and mouse models suggest the presence of additional virulence factors (12Faruque S.M. Chowdhury N. Kamruzzaman M. Dziejman M. Rahman M.H. Sack D.A. Nair G.B. Mekalanos J.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2123-2128Crossref PubMed Scopus (163) Google Scholar). Detailed analyses of the genomes of specific non-O1, non-O139 strains of environmental origin (21Purdy A. Rohwer F. Edwards R. Azam F. Bartlett D.H. J. Bacteriol. 2005; 187: 2992-3001Crossref PubMed Scopus (51) Google Scholar) and clinical origin (22Chen Y. Johnson J.A. Pusch G.D. Morris Jr., J.G. Stine O.C. Infect. Immun. 2007; 75: 2645-2647Crossref PubMed Scopus (41) Google Scholar) have revealed the presence of a gene (chxA) encoding a novel putative secreted exotoxin (23Yates S.P. Jorgensen R. Andersen G.R. Merrill A.R. Trends Biochem. Sci. 2006; 31: 123-133Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), with similarity to exotoxin A (ExoA) 6The abbreviations used are: ExoA, P. aeruginosa exotoxin A; ExoAc, P. aeruginosa exotoxin A catalytic fragment; cholixc, V. cholerae exotoxin catalytic fragment; DTA, diphtheria toxin catalytic fragment; DT, diphtheria toxin; eEF2, eukaryotic ribosomal elongation factor 2; ADPRT, ADP-ribosyltransferase; LRP, low density lipoprotein receptor-related protein; r.m.s.d., root mean square deviation. of P. aeruginosa. ExoA is a potent ADP-ribosylating toxin that specifically modifies the post-translationally modified histidine residue, diphthamide, in the essential eukaryotic ribosomal elongation factor 2 (eEF2) (24Jorgensen R. Merrill A.R. Andersen G.R. Biochem. Soc. Trans. 2006; 34: 1-6Crossref PubMed Scopus (91) Google Scholar). The ADP-ribose moiety of NAD+ is transferred onto the diphthamide imidazole leading to inhibition of protein synthesis in susceptible eukaryotic cells (25Wilson B.A. Collier R.J. Curr. Top. Microbiol. Immunol. 1992; 175: 27-41PubMed Google Scholar, 26Jorgensen R. Yates S.P. Teal D.J. Nilsson J. Prentice G.A. Merrill A.R. Andersen G.R. J. Biol. Chem. 2004; 279: 45919-45925Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Herein, we clearly demonstrate that the chxA gene encodes a second major ADP-ribosylating toxin in V. cholerae. This toxin is catalytically active, specific for the ribosomal eEF2 substrate, and toxic against a diverse array of eukaryotes. Cholix toxin is only the third member of the eEF2-specific ADP-ribosyltransferase toxins, in addition to ExoA and diphtheria toxin (DT). Finally, we have determined the crystal structures of the full-length cholix toxin and its catalytic C-terminal domain (cholixc) to 2.1 Å and 1.25 Å, respectively. Remarkably, the latter structure is the highest resolution to date for any member of the ADPRT family and is co-crystallized in complex with a competitive inhibitor, PJ34, which binds to the NAD+ binding pocket of the toxin. Furthermore, the full-length structure demonstrates striking similarities to ExoA, which consists of a tripartite domain structure, including domains I–III that function in receptor binding, membrane translocation, and enzyme catalysis, respectively. The new crystal structures reveal that inherent flexibility of Loop 1 (L1) (and perhaps also Loop 4, L4) in the toxin is a prerequisite for enzymatic activity and that disruption of specific H-bonds to domain II, either from reduction of disulfide bonds, from furin cleavage or both, is what activates the toxin upon entry into the eukaryotic host cell. In agreement with recent structural studies of the eEF2·ExoA·NAD+ complex, 7R. Jorgensen, Y. Wang, D. Visschedyk, and A. R. Merrill, submitted for publication. L1 of cholix toxin also has the potential to interact with both NAD+ and the diphthamide target residue in eEF2 to form a solvent cover for the active site during the transferase reaction. Cloning of chxA Gene, Expression, and Purification of Cholix and Cholixc Toxins—The 208-residue catalytic fragment of cholix toxin (cholixc) gene (GB AY876053) was cloned into a pET-28a(+) vector with a N-terminal His6 tag and a Tobacco Etch Virus protease site. Escherichia coli ER2566 cells were transformed with plasmid and were harvested by centrifugation, resuspended in 50 mm Tris-HCl, pH 7.6, 200 mm NaCl, 0.1% Tween, 1.25 mm phenylmethylsulfonyl fluoride and lysed in a French press. The cell lysate was centrifuged at 4 °C at 20,000 × g for 20 min, twice. The filtered supernatant was loaded onto a nickel-charged HiTrap™ Chelating HP column (GE Healthcare) equilibrated in 20 mm Tris-HCl, pH 7.9, and 500 mm NaCl, washed with buffer and eluted with a 0–250 mm imidazole gradient. The cholixc toxin was dialyzed in 20 mm Tris-HCl, pH 7.6, 200 mm NaCl, and 0.1 mm phenylmethylsulfonyl fluoride and was digested with tobacco etch virus (1:10 ratio) at 4 °C. The protein was then separated on a HiTrap™ Chelating HP column, and the flow-through was loaded onto a Mono Q column (Amersham Biosciences) in 20 mm Tris-HCl, pH 7.6, 10% glycerol, and 25 mm NaCl and eluted with a 25–500 mm NaCl gradient. The gene encoding the 634-residue cholix toxin for structural studies (GB AY876053) was cloned into a pET-28a(+) vector with a N-terminal His6 tag. The cells were expressed and purified as for the catalytic fragment. The cholix toxin for in vivo studies was produced from a different construct possessing a tobacco etch virus protease digestion site between the His6 tag and the protein sequence. The His6 tag was cleaved off the full-length cholix toxin by tobacco etch virus digestion as described for the catalytic fragment. The cholixc and cholix toxins were both concentrated to ∼7 mg/ml in 100 mm NaCl, 20 mm Tris-HCl, pH 7.2, buffer. Cytotoxicity Assays—Mouse L-M fibroblasts (ATCC CCL-1.2) were maintained at 37 °C (5% CO2) in modified McCoy 5A media supplemented with 10% fetal calf serum, 125 units/ml penicillin, 125 μg/ml streptomycin, 25 mm HEPES, and 2 mm l-glutamine. Cells were added to 24-well cell culture dishes (1 × 105 cells/ml) and were incubated for 5 h, washed with fresh media, and incubated with 0.75 ml of media containing 1 μCi/ml of l-[4,5-3H]leucine (GE Healthcare) for 18 h. Cells were washed twice with 0.5 ml phosphate-buffered saline, and 0.25 ml of 0.1 n NaOH was added. After 5 min at 37 °C, 4 wells of each treatment were pooled together and transferred to microcentrifuge tubes. Protein was precipitated with sodium deoxycholate and 7% trichloroacetic acid, and the precipitate was washed twice with 6% trichloroacetic acid, prior to dissolving in 0.2–0.4 ml of 0.1 n NaOH for 30 min at 56 °C. Protein concentrations were determined (Bio-Rad DC Protein Assay kit), and incorporation of [3H]leucine was measured in a Beckman LS6000TA scintillation counter (Ultima Gold mixture, PerkinElmer Life Sciences). For Fig. 1, toxin was added at the indicated dilutions in fresh media and incubated with the cells for ∼48 h. Cytotoxicity Assays Comparing MEF-1 and PEA13 Cells—MEF-1 and PEA 13 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and incubated at 37 °C under 10% CO2. Then, 5 × 105 cells/ml were added to 24-well dishes in a 1-ml volume, and experiments were performed as above, except that cells were incubated with toxin for 16 h, then with [3H]leucine for 3 h. Samples were processed as above, except that ScintiSafe-30% (Fisher) scintillation mixture was utilized. Artemia Toxicity Assays—Artemia cysts were added to filtered seawater and aerated at room temperature for ∼24 h. Within several hours of hatching, A. nauplii were placed in 24-well dishes containing 400 μl of sterile sea water, with a total of 30–70 nauplii per well, and incubated at 28 °C for 42–45 h. Artemia mortality was assessed using a dissecting microscope. Detection of Biotinylated ADP-ribose-eEF2—100 μm Bio-NAD (Trevigen) was incubated with 5 μm toxin in the presence of 7 μl of CHO cell lysate in 60 mm Tris-HCl, pH 7.6, buffer for 60 min at 25 °C. The proteins were separated on by SDS-PAGE (27Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and were transferred to nitrocellulose at 125 mA for 80 min. The membrane was blocked (2% bovine serum albumin in phosphate-buffered saline) for 1 h and then was incubated in 0.5% bovine serum albumin in phosphate-buffered saline with 1:5000 dilution of streptavidin-alkaline phosphate conjugate (Promega) and mixed overnight on a Nutator at 4 °C. The blot was then washed with 0.5 mm MgCl2, 40 mm NaHCO3, pH 9.6, buffer and developed in 10 ml of 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium alkaline phosphate substrate for 3 min. Crystallography—The cholixc toxin was co-crystallized with 5 mm PJ34 (Sigma-Aldrich) by vapor diffusion against reservoirs containing 15% polyethylene glycol-8000 and 20 mm KH2PO4 at 19 °C. Before flash freezing in liquid N2, the crystals were transferred to paratone-N (Hampton research) for cryoprotection. A native 1.25-Å dataset was collected at Advanced Photon Source beamline 19-ID, and a 1.65-Å dataset on a crystal soaked for 1 h in 5 mm HgCl2 was collected at beamline 17-ID. The cholixc toxin structure was solved using single-wavelength anomalous dispersion phases. The anomalous scattering substructure search, density modification, and initial chain tracing were all performed in Phenix (28Adams P.D. Grosse-Kunstleve R.W. Hung L.W. Ioerger T.R. Mc-Coy A.J. Moriarty N.W. Read R.J. Sacchettini J.C. Sauter N.K. Terwilliger T.C. Acta Crystallogr. D. Biol. Crystallogr. 2002; 58: 1948-1954Crossref PubMed Scopus (3667) Google Scholar), and a single major and two minor sites were found. The resulting partial model was used for molecular replacement against the high resolution native dataset using the CCP4 program MolRep (29Vagin A. Teplyakov A. Acta Crystallogr. D. Biol. Crystallogr. 2000; 56: 1622-1624Crossref PubMed Scopus (690) Google Scholar), and the structure was then traced using warpNtrace (30Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2565) Google Scholar) and was iteratively rebuilt in Coot (31Emsley P. Cowtan K. Acta Crystallogr. D. Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar) and anisotropically refined in Refmac5 (32Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D. Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) at 1.25-Å resolution. Cholix toxin was crystallized by vapor diffusion against reservoirs containing 23% polyethylene glycol-10,000, 7.5% ethylene glycol, and 0.1 m HEPES, pH 7.5, at 19 °C. Before flash freezing in liquid N2 the crystals were transferred to paratone-N (Hampton research) for cryoprotection. A native 2.1-Å dataset was collected at our in-house Enraf-Nonius FR571 diffractometer equipped with a rotating copper anode and a Proteum Pt135 CCD detector (Bruker). The structure of cholix toxin was solved by molecular replacement with CNS 1.1 (33Brunger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) and Phaser (34McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. D. Biol. Crystallogr. 2005; 61: 458-464Crossref PubMed Scopus (1602) Google Scholar) using the refined structure of cholixc toxin together with the receptor binding and translocation domains from the structure of ExoA from P. aeruginosa (PDB entry 1IKQ) as search models. The solution from molecular replacement was used as input to warpNtrace, which could trace ∼80% of the structure. The model was rebuilt in Coot and refined in Refmac5 using TLS at 2.1-Å resolution. chxA Encodes a Putative ADPRT—Previously, we identified several DNA fragments from V. cholerae strains SIO and TP that have strong similarity to genes encoding virulence factors in known bacterial pathogens, including a putative ADPRT with resemblance to the toxA gene from P. aeruginosa (21Purdy A. Rohwer F. Edwards R. Azam F. Bartlett D.H. J. Bacteriol. 2005; 187: 2992-3001Crossref PubMed Scopus (51) Google Scholar). This gene, called chxA, encodes a 666-residue protein with a 32-residue leader sequence, called cholix toxin (70.7-kDa, 634-residue mature protein), and is similar to known diphthamide-specific ADPRTs (23Yates S.P. Jorgensen R. Andersen G.R. Merrill A.R. Trends Biochem. Sci. 2006; 31: 123-133Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The cholix toxin primary structure shows 32% sequence identity with Pseudomonas ExoA, has a furin protease site for cellular activation (35Inocencio N.M. Moehring J.M. Moehring T.J. J. Biol. Chem. 1994; 269: 31831-31835Abstract Full Text PDF PubMed Google Scholar), contains a C-terminal KDEL sequence that likely routes the toxin to the endoplasmic reticulum of the host cell (36Hessler J.L. Kreitman R.J. Biochemistry. 1997; 36: 14577-14582Crossref PubMed Scopus (74) Google Scholar), and possesses three classical signature regions (23Yates S.P. Jorgensen R. Andersen G.R. Merrill A.R. Trends Biochem. Sci. 2006; 31: 123-133Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 37Masignani V. Balducci E. Serruto D. Veggi D. Arico B. Comanducci M. Pizza M. Rappuoli R. Int. J. Med. Microbiol. 2004; 293: 471-478Crossref PubMed Scopus (28) Google Scholar) peculiar to the catalytic domain of the diphthamide-specific toxins (23Yates S.P. Jorgensen R. Andersen G.R. Merrill A.R. Trends Biochem. Sci. 2006; 31: 123-133Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Thus, all of these features within the primary sequence of the cholix toxin provided a powerful indication that this protein is a new member of the eEF2-specific ADPRT group (DT group) (23Yates S.P. Jorgensen R. Andersen G.R. Merrill A.R. Trends Biochem. Sci. 2006; 31: 123-133Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 37Masignani V. Balducci E. Serruto D. Veggi D. Arico B. Comanducci M. Pizza M. Rappuoli R. Int. J. Med. Microbiol. 2004; 293: 471-478Crossref PubMed Scopus (28) Google Scholar). Cholix Toxin Is a Bacterial ADPRT Enzyme—The ADPRT reaction in the DT group involves a nucleophilic substitution where the diphthamide imidazole in eEF2 is the nucleophile that replaces the nicotinamide base (leaving group) of the NAD+ substrate (23Yates S.P. Jorgensen R. Andersen G.R. Merrill A.R. Trends Biochem. Sci. 2006; 31: 123-133Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The cellular effect of the covalent modification (ribosylation) of eEF2 is inhibition of protein synthesis leading to host cell death (38Perentesis J.P. Miller S.P. Bodley J.W. Biofactors. 1992; 3: 173-184PubMed Google Scholar). To explore whether cholix toxin possessed ADPRT activity, we cloned both the full-length and truncated chxA gene into the T7-based E. coli pET28b vector for expression and purification of both the whole toxin and its C-terminal catalytic domain. The proteins were purified by immobilized metal ion chromatography and were tested for both NAD-glycohydrolase and ADPRT activities using a fluorescence-based assay with purified yeast eEF2 as substrate previously developed in our laboratory (39Armstrong S. Merrill A.R. Anal. Biochem. 2001; 292: 26-33Crossref PubMed Scopus (41) Google Scholar). The full-length recombinant toxin (634 residues, 70.7 kDa) showed only weak catalytic activity suggesting that this protein requires activation, a prerequisite for this family of enzymes/toxins (40Leppla S.H. Martin O.C. Muehl L.A. Biochem. Biophys. Res. Commun. 1978; 81: 532-538Crossref PubMed Scopus (31) Google Scholar, 41Chung D.W. Collier R.J. Infect. Immun. 1977; 16: 832-841Crossref PubMed Google Scholar, 42Beattie B.K. Merrill A.R. Biochemistry. 1996; 35: 9042-9051Crossref PubMed Scopus (22) Google Scholar). In contrast, the catalytic fragment (208 residues, 23 kDa) showed strong glycohydrolase (Km, 67 ± 4 μm; kcat, 1.92 ± 0.12 h-1) and ADPRT activities (Table 1). Cholix toxin is a slightly better enzyme than ExoA with a lower Km (3-fold), similar kcat, and specificity constant (2-fold higher) for the NAD+ substrate (23Yates S.P. Jorgensen R. Andersen G.R. Merrill A.R. Trends Biochem. Sci. 2006; 31: 123-133Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Furthermore, replacing the hallmark catalytic Glu-581 residue (corresponding to Glu-553 in ExoA and Glu-148 in DT) within the enzyme domain of cholix toxin with an Ala showed the expected result on the activity of the enzyme (∼2600-fold reduction in kcat, Table 1). Thus, cholix toxin recognizes eEF2 as the target protein substrate and possesses both glycohydrolase and ADPRT enzyme activities, which qualifies it as the third bone fide member of the DT group of bacterial ADPRT enzymes along with ExoA and DT (23Yates S.P. Jorgensen R. Andersen G.R. Merrill A.R. Trends Biochem. Sci. 2006; 31: 123-133Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar).TABLE 1Comparison of the ADPRT kinetic parameters for ExoAc and cholixc toxins The kinetic parameters were determined as described under “Experimental Procedures.” The values represent the mean ± S.D. from three independent experiments.ParameterExoAcCholixc toxinKm(NAD) (μm)121 ± 2145 ± 3Vmax (m s–1)1.3 × 10–71.03 × 10–7kcat (s–1)13 ± 210 ± 3kcat (s–1) catalytic Glu → AlaaThe catalytic Glu to Ala mutations involved Glu-553 (ExoAc) and Glu-581 (cholixc toxin)0.008 ± 0.00010.004 ± 0.003kcat/Km (m–1 s–1)1.1 × 1052.3 × 105a The catalytic Glu to Ala mutations involved Glu-553 (ExoAc) and Glu-581 (cholixc toxin) Open table in a new tab Cholix Toxin Is Active against Eukaryotic Cells—To assess whether cholix toxin possessed biological activity against eukaryotes, we dosed mouse fibroblast cells with both wild-type toxin and a catalytically inactive mutant toxin, E581A, and compared the effects on cell viability (Fig. 1). The E581A mutant toxin had little or no effect on cell viability even at high doses (50 ng/ml) (Fig. 1b), whereas the wild-type toxin showed considerable clearing of cell density (killing) at 1 ng/ml (Fig. 1c) with little or no surviving fibroblast cells at 50 ng/ml (Fig. 1f). Using the cytotoxicity assay described under “Experimental Procedures,” we quantified the sensitivity of mouse fibroblast 1.2 L-M cells to cholix toxin, and the results are shown in Fig. 2a. The IC50 is 4.6 ± 0.4 ng/ml, and this is comparable to the cytotoxicity of ExoA against this mouse fibroblast cell line (43Middlebrook J.L. Dorland R.B. Can. J. Microbiol. 1977; 23: 183-189Crossref PubMed Scopus (84) Google Scholar). Furthermore, the inactive E581A mutant toxin showed little or no activity against the mouse fibroblast cells (Fig. 2a). The protein inhibition by ExoA and cholix toxin was also tested on mouse cells lines with and without the low density lipoprotein receptor-related protein (LRP) receptor. Fig. 2b shows that cholix toxin recognizes the ubiquitous LRP receptor (44May P. Woldt E. Matz R.L. Boucher P. Ann. Med. 2007; 39: 219-228Crossref PubMed Scopus (182) Google Scholar), which is also the specific receptor that ExoA exploits to enter the target eukaryotic cell (45Kounnas M.Z. Morris R.E. Thompson M.R. FitzGerald D.J. Strickland D.K. Saelinger C.B. J. Biol. Chem. 1992; 267: 12420-12423Abstract Full Text PDF PubMed Google Scholar). This suggests that the cellular intoxication mechanism for cholix toxin is very similar to ExoA but differs from that of DT (46Collier R.J. Toxicon. 2001; 39: 1793-1803Crossref PubMed Scopus (263) Google Scholar). However, because the LRP-deficient strain also showed some sensitivity to cholix toxin, it is possible that there may be other avenues, besides the LRP receptor, by which cholix toxin can access the host cell cytoplasm. V. cholerae is an aquatic organism that is often found attached to the exoskeletons of zooplankton (47Tamplin M.L. Gauzens A.L. Huq A. Sack D.A. Colwell R.R. Appl. Environ. Microbiol. 1990; 56: 1977-1980Crossref PubMed Google Scholar), and this behavior may provide nutrients and protection against environmental challenges (48Kirn T.J. Jude B.A. Taylor R.K. Nature. 2005; 438: 863-866Crossref PubMed Scopus (239) Google Scholar). Therefore, we tested the ability of purified cholix toxin to act on A. nauplii (brine shrimp), and the results are shown in Fig. 2c. Remarkably, cholix toxin was toxic to A. nauplii, because doses near 50 μg/ml killed all of the crustaceans, yet the E581A cholix toxin mutant had no effect on their viability. We next examined whether cholix toxin was able to specifically and covalently modify the ribosomal eEF2 protein in a mam" @default.
• W2119009760 created "2016-06-24" @default.
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• W2119009760 date "2008-04-01" @default.
• W2119009760 modified "2023-10-17" @default.
• W2119009760 title "Cholix Toxin, a Novel ADP-ribosylating Factor from Vibrio cholerae" @default.
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