Cytophysiologic effects and molecular inhibition of a functional actin specific ADP ribosyltransferase CDT from clostridium difficile 5

Chapter Bibliography Ailenberg, M., and Silverman, M. (2003). Cytochalasin D disruption of actin filaments in 3T3 cells produces an anti-apoptotic response by activating gelatinase A extracellularly and initiating intracellular survival signals. Biochim Biophys Acta 1593, 249-258. Aktories, K., Ankenbauer, T., Schering, B., and Jakobs, K. H. (1986a). ADP-ribosylation of platelet actin by botulinum C2 toxin. Eur J Biochem 161, 155-162. Aktories, K., Barmann, M., Ohishi, I., Tsuyama, S., Jakobs, K. H., and Habermann, E. (1986b). Botulinum C2 toxin ADP-ribosylates actin. Nature 322, 390-392. Aktories, K., and Just, I. (1995). Monoglucosylation of low-molecular-mass GTP-binding Rho proteins by clostridial cytotoxins. Trends Cell Biol 5, 441-443. Aktories, K., Rosener, S., Blaschke, U., and Chhatwal, G. S. (1988). Botulinum ADPribosyltransferase C3. Eur J Biochem 172, 445-450. Aktories, K., Weller, U., and Chhatwal, G. S. (1987). Clostridium difficile type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin. FEBS Lett 212, 109-113. Al Saif, N., and Brazier, J. S. (1996). The distribution of Clostridium difficile in the environment of South Wales. J Med Microbiol 45, 133-137. Alfa, M. J., Kabani, A., Lyerly, D., Moncrief, S., Neville, L. M., Al-Barrak, A., Harding, G. K. H., Dyck, B., Olekson, K., and Embil, J. M. (2000). Characterization of a Toxin A-Negative, 139 Toxin B-Positive Strain of Clostridium difficile Responsible for a Nosocomial Outbreak of Clostridium difficile-Associated Diarrhea. J Clin Microbiol 38, 2706-2714. Alfa, M. J., Swan, B., van Dekerkhove, B., Pang, P., and Harding, G. K. M. (2002). The diagnosis of Clostridium difficile-associated diarrhea: comparison of Triage Clostridium difficile panel, enzyme immunoassay for toxin A/B and cytotoxin assays. Diagnostic Microbiol Infect Dis 43, 257-263. Al-Hasani, K., Rajakumar, K., Bulach, D., Robins-Browne, R., Adler, B., and Sakellaris, H. (2001). Genetic organisation of the she pathogenicity island in Shigella flexneri 2a. Microb Pathog 30, 1-8. Allen, W. E., Jones, G. E., Pollard, J. W., and Ridley, A. J. (1997). Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages. J Cell Sci 110 ( Pt 6), 707-720. Altschuler, D. L., Peterson, S. N., Ostrowski, M. C., and Lapetina, E. G. (1995). Cyclic AMPdependent activation of Rap1b. J Biol Chem 270, 10373-10376. Angeles, D. C., Leong, W. Y., Koay, E., and Song, K. P. (2004). Comparative and quantitative transcription analyses of the binary ADP-ribosyltransferase cdt from a variant Clostridium difficile strain with truncated pathogenicity locus. J Infect Dis Submitted. Angeles, D. C., and Song, K. P. (2005a). Clostridium difficile ADP-ribosylating CDT toxin induced shift in actin dynamics and stress signal transduction in colorectal carcinoma cell. Res Microbiol Submitted. 140 Angeles, D. C., and Song, K. P. (2005b). Peptide antibiotic and actin-binding protein as mixedtype inhibitors of Clostridium difficile CDT toxin activities. Biochem Biophys Res Commun 327, 361-370. Apisarnthanarak, A., Razavi, B., and Mundy, L. M. (2002). Adjunctive intracolonic vancomycin for severe Clostridium difficile colitis: case series and review of literature. Clin Infect Dis 35, 690-696. Bachellier, S., Clement, J. M., Hofnung, M., and Gilson, E. (1997). Bacterial interspersed mosaic elements (BIMEs) are a major source of sequence polymorphism in Escherichia coli intergenic regions including specific associations with a new insertion sequence. Genetics 145, 551-562. Bacon, A. E., Fekety, R., Schaberg, D. R., and Faix, R. G. (1988). Epidemiology of Clostridium difficile colonization in newborns: results using a bacteriophage and bacteriocin typing system. J Infect Dis 158, 349-354. Bagrodia, S., Derijard, B., Davis, R. J., and Cerione, R. A. (1995). Cdc42 and PAK-mediated signaling leads to Jun kinase and p38 mitogen-activated protein kinase activation. J Biol Chem 270, 27995-27998. Banasik, M., Komura, H., Shimoyama, M., and Ueda, K. (1992). Specific inhibitors of poly(ADP-ribose) synthetase and mono(ADP-ribosyl)transferase. J Biol Chem 267, 1569-1575. Banasik, M., Komura, H., and Ueda, K. (1990). Inhibition of poly(ADP-ribose) synthetase by unsaturated fatty acids, vitamins and vitamin-like substances. FEBS Lett 263, 222-224. 141 Barroso, L. A., Moncrief, J. S., Lyerly, D. M., and Wilkins, T. D. (1994). Mutagenesis of the Clostridium difficile toxin B and effect on cytotoxic activity. Microb Pathog 16, 297-303. Barroso, L. A., Wang, S.-Z., Phelps, C. J., Johnson, J. L., and Wilkins, T. D. (1990). Nucleotide sequence of Clostridium difficile toxin B gene. Nucleic Acids Res 18, 4004. Barth, H., Preiss, J. C., Hofmann, F., and Aktories, K. (1998). Characterization of the catalytic site of the ADP-ribosyltransferase Clostridium botulinum C2 toxin by site-directed mutagenesis. J Biol Chem 273, 29506-29511. Bartlett, J. G. (1998). Pseudomembranous enterocolitis and antibiotic-associated colitis. In Sleisenger and Fordtran's Gastrointestinal and Liver Disease: Pathophysiology/ Diagnosis/ Management., M. Feldman, B. F. Schar-Schmidt, and M. H. Sleisenger, eds. (Philadelphia, USA, W.B. Saunders Co.), pp. 1633-1647. Bartlett, J. G., Chang, T. W., Gurwith, M., Gorbach, S. L., and Onderdonk, A. B. (1978). Antibiotic-associated pseudomembranous colitis due to a toxin producing clostridia. New England J Med 298, 531-534. Beckerle, M. C., and Yeh, R. K. (1990). Talin: role at sites of cell-substratum adhesion. Cell Motil Cytoskeleton 16, 7-13. Bell, C. E., and Eisenberg, D. (1996). Crystal structure of diphtheria toxin bound to nicotinamide adenine dinucleotide. Biochemistry 35, 1137-1149. 142 Berg, J. M., and Shi, Y. (1996). The galvanization of biology: a growing appreciation for the roles of zinc. Science 271, 1081-1085. Bethards, L. A., Skadsen, R. W., and Scandalios, J. G. (1987). Isolation and characterization of a cDNA clone for the Cat2 gene in maize and its homology with other catalases. Proc Natl Acad Sci 84, 6830-6834. Bingley, P. J., and Harding, G. M. (1987). Clostridium difficile colitis following treatment with metronidazole and vancomycin. Postgrad Med J 63, 993-994. Binz, T., Kurazono, H., Wille, M., Frevert, J., Wernars, K., and Niemann, H. (1990). The complete sequence of botulinum neurotoxin type A and comparison with other clostridial neurotoxins. J Biol Chem 265, 9153-9158. Bishai, W. R., Rappuoli, R., and Murphy, J. R. (1987). High-level expression of a proteolytically sensitive diphtheria toxin fragment in Escherichia coli. J Bacteriol 169, 5140-5151. Blattner, F. R., Plunkett, G., III, Bloch, C. A., Perna, N. T., Burland, V., Riley, M., ColladoVides, J., Glasner, J. D., Rode, C. K., Mayhew, G. F., et al. (1997). The Complete Genome Sequence of Escherichia coli K-12. Science 277, 1453-1462. Blocker, D., Behlke, J., Aktories, K., and Barth, H. (2001). Cellular uptake of the Clostridium perfringens binary iota-toxin. Infect Immun 69, 2980-2987. Bochner, B. R., Gadzinski, P., and Panomitros, E. (2001). Phenotype microarrays for highthroughput phenotypic testing and assay of gene function. Genome Res 11, 1246-1255. 143 Bond, F., Payne, G., Borriello, S. P., and Humphreys, H. (1995). Usefulness of culture in the diagnosis of Clostridium difficile infection. Eur J Clin Microbiol Infect Dis 14, 223-226. Bongaerts, G. P. A., and Lyerly, D. M. (1997). Role of bacterial metabolism and physiology in the pathogenesis of Clostridium difficile disease. Microb Pathog 22, 253-256. Borriello, S. P., and Carman, R. J. (1983). Association of iota-like toxin and Clostridium spiroforme with both spontaneous and antibiotic-associated diarrhea and colitis in rabbits. J Clin Microbiol 17, 414-418. Borriello, S. P., Davies, H. A., and Barclay, F. E. (1988). Detection of fimbriae amongst strains of Clostridium difficile. FEMS Microbiol Lett 49, 65-67. Borriello, S. P., Davies, H. A., Kamiya, S., Reed, P. J., and Seddon, S. (1990). Virulence factors of Clostridium difficile. Rev Infect Dis 12 Suppl 2, S185-S191. Borriello, S. P., Wren, B. W., Hyde, S., Seddon, S. V., Sibbons, P., Krishna, M. M., Tabaqchali, S., Manek, S., and Price, A. B. (1992). Molecular, immunological, and biological characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile. Infect Immun 60, 41924199. Boshans, R. L., Szanto, S., van Aelst, L., and D'Souza-Schorey, C. (2000). ADP-ribosylation factor regulates actin cytoskeleton remodeling in coordination with Rac1 and RhoA. Mol Cell Biol 20, 3685-3694. 144 Botteri, F. M., Ballmer-Hofer, K., Rajput, B., and Nagamine, Y. (1990). Disruption of cytoskeletal structures results in the induction of the urokinase-type plasminogen activator gene expression. J Biol Chem 265, 13327-13334. Bowman, T., Garcia, R., Turkson, J., and Jove, R. (2000). STATs in oncogenesis. Oncogene 19, 2474-2488. Braun, M., Herholz, C., Straub, R., Choisat, B., Frey, J., Nicolet, J., and Kuhnert, P. (2000). Detection of the ADP-ribosyltransferase toxin gene (cdtA) and its activity in Clostridium difficile isolates from Equidae. FEMS Microbiol Lett 184, 29-33. Braun, V., Hundsberger, T., Leukel, P., Sauerborn, M., and von Eichel-Streiber, C. (1996). Definition of the single integration site of the pathogenicity locus in Clostridium difficile. Gene 181, 29-38. Brazier, J. S. (1998). The diagnosis of Clostridium difficile associated disease. J Antimicrob Chemother 4, 29-40. Briceland, L. L., Quintiliani, R., and Nightingale, C. H. (1988). Multidisciplinary costcontainment program promoting oral metronidazole for treatment of antibiotic-associated colitis. Amer J Hosp Pharma 45, 122-125. Bubb, M. R., Senderowicz, A. M., Sausville, E. A., Duncan, K. L., and Korn, E. D. (1994). Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin. J Biol Chem 269, 14869-14871. 145 Buggy, B. P., Fekety, R., and Silva, J. J. (1987). Theraphy of relapsing Clostridium difficileassociated diarrhea and colitis with the combination of vancomycin and rifampin. J Clin Gastroenterol 9, 155-159. Busch, C., Hofmann, F., Selzer, J., Munro, S., Jeckel, D., and Aktories, K. (1998). A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins. J Biol Chem 273, 19566-19572. Caiazza, N. C., and O'Toole, G. A. (2003). Alpha-toxin is required for biofilm formation by Staphylococcus aureus. J Bacteriol 185, 3214-3217. Calabi, E., Calabi, F., Phillips, A. D., and Fairweather, N. F. (2002). Binding of Clostridium difficile surface layer proteins to gastrointestinal tissues. Infect Immun 70, 5770-5778. Carlier, M. F. (1991). Actin: protein structure and filament dynamics. J Biol Chem 266, 1-4. Carraway, K. L. (1990). Membranes and microfilaments: interactions and role in cellular dynamics. Bioessays 12, 90-92. Carroll, S. F., and Collier, R. J. (1984). NAD binding site of diphtheria toxin: identification of a residue within the nicotinamide subsite by photochemical modification with NAD. Proc Natl Acad Sci U S A 81, 3307-3311. Caspar, M., Florin, I., and Thelestam, M. (1987). Calcium and calmodulin in cellular intoxication with Clostridium difficile toxin B. J Cell Physiol 132, 168-172. 146 Castagliuolo, I., Karalis, K., Valenick, L., Pasha, A., Nikulasson, S., Wlk, M., and Pothoulakis, C. (2001). Endogenous cortocosteroids modulate Clostridium difficile toxin A-induced enteritis in rats. Am J Physiol Gastrointest Liver Physiol 280, G539-G545. Castagliuolo, I., Keates, A. C., Wang, C. C., Pasha, A., Valenick, L., Kelly, C. P., Nikulasson, S. T., LaMont, J. T., and Pothoulakis, C. (1998). Clostridium difficile toxin A stimulates macrophage-inflammatory protein-2 production in rat intestinal epithelial cells. J Immunol 160, 6039-6045. Cerquetti, M., Molinari, A., Sebastianelli, A., Diociaiuti, M., Petruzzelli, R., Capo, C., and Mastrantonio, P. (2000). Characterization of surface layer proteins from different Clostridium difficile clinical isolates. Microb Pathog 28, 363-372. Chaves-Olarte, E., Weidmann, M., Eichel-Streiber, C., and Thelestam, M. (1997). Toxins A and B from Clostridium difficile differ with respect to enzymatic potencies, cellular substrate specificities, and surface binding to cultured cells. J Clin Invest 100, 1734-1741. Choi, J. Y., Sifri, C. D., Goumnerov, B. C., Rahme, L. G., Ausubel, F. M., and Calderwood, S. B. (2002). Identification of virulence genes in a pathogenic strain of Pseudomonas aeruginosa by representational difference analysis. J Bacteriol 184, 952-961. Ciesielski-Treska, J., Ulrich, G., Rihn, B., and Aunis, D. (1989). Mechanism of action of Clostridium difficile toxin B: role of external medium and cytoskeletal organization in intoxicated cells. Eur J Cell Biol 48, 191-202. 147 Ciesla, W. P., Jr., and Bobak, D. A. (1998). Clostridium difficile toxins A and B are cationdependent UDP-glucose hydrolases with differing catalytic activities. J Biol Chem 273, 1602116026. Clark, G. F., Krivan, H. C., Wilkins, T. D., and Smith, D. F. (1987). Toxin A from Clostridium difficile binds to rabbit erythrocyte glycolipids with terminal Gal alpha 1-3Gal beta 1-4GlcNAc sequences. Arch Biochem Biophys 257, 217-229. Clement, J.-M., Wilde, C., Bachellier, S., Lambert, P., and Hofnung, M. (1999). IS1397 Is Active for Transposition into the Chromosome of Escherichia coli K-12 and Inserts Specifically into Palindromic Units of Bacterial Interspersed Mosaic Elements. J Bacteriol 181, 6929-6936. Collier, R. J. (1967). Effect of diphtheria toxin on protein synthesis: inactivation of one of the transfer factors. J Mol Biol 25, 83-98. Cooper, J. A. (1987). Effects of cytochalasin and phalloidin on actin. J Cell Biol 105, 1473-1478. Crawford Jr, E. W., and Shimkets, L. J. (2000). The Myxococcus xanthus socE and csgA genes are regulated by the stringent response. Mol Microbiol 37, 788-799. Dai, Z., Sirard, J. C., Mock, M., and Koehler, T. M. (1995). The atxA gene product activates transcription of the anthrax toxin genes and is essential for virulence. Mol Microbiol 16, 11711181. Dailey, D. C., Kaiser, A., and Schloemer, R. H. (1987). Factors influencing the phagocytosis of Clostridium difficile by human polymorphonuclear leukocytes. Infect Immun 55, 1541-1546. 148 Thomas, D. R., Bennett, R. G., Laughon , B. E., Greenough, W. B. I., and Bartlett, J. G. (1990). Postantibiotic colonization with Clostridium difficile in nursing home patients. J Amer Geriatric Soc 38, 415-420. Triadafilopoulos, G., Pothoulakis, C., O'Brien, M. J., and LaMont, J. T. (1987). Differential effects of Clostridium difficile toxins A and B on rabbit ileum. Gastroenterology 93, 273-279. Tsuge, H., Nagahama, M., Nishimura, H., Hisatsune, J., Sakaguchi, Y., Itogawa, Y., Katunuma, N., and Sakurai, J. (2003). Crystal structure and site-directed mutagenesis of enzymatic components from Clostridium perfringens iota-toxin. J Mol Biol 325, 471-483. Tucker, K. D., and Wilkins, T. D. (1991). Toxin A of Clostridium difficile binds to the human carbohydrate antigens I, X, and Y. Infect Immun 59, 73-78. Turner, S. A., Luck, S. N., Sakellaris, H., Rajakumar, K., and Adler, B. (2001). Nested deletions of the SRL pathogenicity island of Shigella flexneri 2a. J Bacteriol 183, 5535-5543. Tvede, M., and Rask-Madsen, J. (1989). Bacteriotherapy for chronic relapsing Clostridium difficile diarrhoea in six patients. Lancet 1, 1156-1160. van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980). ADP-ribosylation of elongation factor by diphtheria toxin: NMR spectra and proposed structures of ribosyl-diphthamide and its hydrolysis products. J Biol Chem 255, 10710-10716. Vandekerckhove, J., Schering, B., Barmann, M., and Aktories, K. (1988). Botulinum C2 toxin ADP-ribosylates cytoplasmic beta/gamma-actin in arginine 177. J Biol Chem 263, 696-700. 186 Vandekerckhove, J., and Weber, K. (1979). The complete amino acid sequence of actins from bovine aorta, bovine heart, bovine fast skeletal muscle, and rabbit slow skeletal muscle. A protein-chemical analysis of muscle actin differentiation. Differentiation 14, 123-133. Viscidi, R., Laughon, B. E., Yolken, R., Bo-Linn, P., Moench, T., Ryder, R. W., and Bartlett, J. G. (1983). Serum antibody response to toxins A and B of Clostridium difficile. J Infect Dis 148, 93-100. Vitale, G., Pellizzari, R., Recchi, C., Napolitani, G., Mock, M., and Montecucco, C. (1998). Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages. Biochem Biophys Res Commun 248, 706711. von Eichel-Streiber, C., Harperath, U., Bosse, D., and Hadding, U. (1987). Purification of two high molecular weight toxins of Clostridium difficile which are antigenically related. Microb Pathog 2, 307-318. von Eichel-Streiber, C., Laufenberg-Feldmann, R., Sartingen, S., Schulze, J., and Sauerborn, M. (1992). Comparative sequence analysis of the Clostridium difficile toxins A and B. Mol Gen Genet 233, 260-268. von Eichel-Streiber, C., Meyer zu Heringdorf, D., Habermann, E., and Sartingen, S. (1995). Closing in on the toxic domain through analysis of a variant Clostridium difficile cytotoxin B. Mol Microbiol 17, 313-321. 187 von Eichel-Streiber, C., and Sauerborn, M. (1990). Clostridium difficile toxin A carries a Cterminal repetitive structure homologous to the carbohydrate binding region of streptococcal glycosyltransferases. Gene 96, 107-113. von Eichel-Streiber, C., Suckau, D., Wachter, M., and Hadding, U. (1989). Cloning and characterization of overlapping DNA fragments of the toxin A gene of Clostridium difficile. J Gen Microbiol 135, 55-64. Waligora, A. J., Hennequin, C., Mullany, P., Bourlioux, P., Collignon, A., and Karjalainen, T. (2001). Characterization of a cell surface protein of Clostridium difficile with adhesive properties. Infect Immun 69, 2144-2153. Walker, J. C., and Verma, N. K. (2002). Identification of a putative pathogenicity island in Shigella flexneri using subtractive hybridisation of the S. flexneri and Escherichia coli genomes. FEMS Microbiol Lett 213, 257-264. Wedel, N., Toselli, P., Pothoulakis, C., Faris, B., Oliver, P., Franzblau, C., and LaMont, T. (1983). Ultrastructural effects of Clostridium difficile toxin B on smooth muscle cells and fibroblasts. Exp Cell Res 148, 413-422. Wei, J., Goldberg, M. B., Burland, V., Venkatesan, M. M., Deng, W., Fournier, G., Mayhew, G. F., Plunkett III, G., Rose, D. J., Darling, A., et al. (2003). Complete Genome Sequence and Comparative Genomics of Shigella flexneri Serotype 2a Strain 2457T. Infect Immun 71, 27752786. 188 White, O., Eisen, J. A., Heidelberg, J. F., Hickey, E. K., Peterson, J. D., Dodson, R. J., Haft, D. H., Gwinn, M. L., Nelson, W. C., Richardson, D. L., et al. (1999). Genome Sequence of the Radioresistant Bacterium Deinococcus radiodurans R1. Science 286, 1571-1577. Wiggins, C. A., and Munro, S. (1998). Activity of the yeast MNN1 alpha-1,3mannosyltransferase requires a motif conserved in many other families of glycosyltransferases. Proc Natl Acad Sci U S A 95, 7945-7950. Wilcox, M. H., Cunniffe, J. G., Trundle, C., and Redpath, C. (1996). Financial burden of hospitalacquired Clostridium difficile infection. J Hosp Infect 34, 23-30. Williams, R. C., Rees, M. L., Jacobs, M. F., Pragai, Z., Thwaite, J. E., Baillie, L. W., Emmerson, P. T., and Harwood, C. R. (2003). Production of Bacillus anthracis protective antigen is dependent on the extracellular chaperone, PrsA. J Biol Chem 278, 18056-18062. Wilson, B. A., Reich, K. A., Weinstein, B. R., and Collier, R. J. (1990). Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine. Biochemistry 29, 8643-8651. Wood, D. W., Setubal, J. C., Kaul, R., Monks, D. E., Kitajima, J. P., Okura, V. K., Zhou, Y., Chen, L., Wood, G. E., Almeida, N. F., Jr., et al. (2001). The Genome of the Natural Genetic Engineer Agrobacterium tumefaciens C58. Science 294, 2317-2323. Wretlind, B., Bjorklind, A., and Pavlovskis, O. R. (1987). Role of exotoxin A and elastase in the pathogenicity of Pseudomonas aeruginosa strain PAO in experimental mouse burn infection. Microb Pathog 2, 397-404. 189 Wu, J., Dent, P., Jelinek, T., Wolfman, A., Weber, M. J., and Sturgill, T. W. (1993). Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate. Science 262, 1065-1069. Xia, Y., Makris, C., Su, B., Li, E., Yang, J., Nemerow, G. R., and Karin, M. (2000). MEK kinase is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration. Proc Natl Acad Sci U S A 97, 5243-5248. Xu, Y., Barbancon-Finck, V., and Barbieri, J. T. (1994). Role of histidine 35 of the S1 subunit of pertussis toxin in the ADP-ribosylation of transducin. J Biol Chem 269, 9993-9999. Yamakawa, K., Karasawa, T., Ikoma, S., and Nakamura, S. (1996). Enhancement of Clostridium difficile toxin production in biotin-limited conditions. J Med Microbiol 44, 111-114. Yanisch-Perron, C., Vieira, J., and Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-119. Yassin, S. F., Young-Fadok, T. M., Zein, N. N., and Pardi, D. S. (2001). Clostridium difficileassociated diarrhea and colitis. Mayo Clinic Proc 76, 725-730. Young, M., Minton, N. P., and Staudenbauer, W. L. (1989). Recent advances in the genetics of the clostridia. FEMS Microbiol Rev 5, 301-325. Zimmerman, R. K. (1991). Risk factors for Clostridium difficile cytotoxin-positive diarrhea after control for horizontal transmission. Infect Control Hosp Epidemiol 12, 96-100. 190 Appendix A Common buffers, solutions and media All solutions were dissolved in ddH2O and were autoclaved or filter sterilized unless otherwise specified. 1% Agarose gel 1% (w/v) Agarose 0.5xTAE Buffer 0.25µg/ml Ethidium bromide 2% Agarose gel 2% (w/v) Agarose 0.5xTAE Buffer 0.25µg/ml Ethidium bromide BCIG/IPTG plates 20 µl BCIG (20 mg/ml) 20 µl IPTG (20 mg/ml) spread plate immediately Carbonate coating buffer per liter, 0.05 M 5.3 g anhydrous Na2CO3 adjust to pH9.6 10XCIP dephosphorylation buffer (Boehringer Mannheim) 0.5M Tris.Cl 1mM Ethylenediaminetetraacetic acid (EDTA) pH8.5 (at 20oC) Denaturing solution 1.5M Sodium chloride 0.5M Sodium hydroxide 100XDenhardt’s solution 2% (w/v) Bovine serum albumin (BSA) 2% (w/v) Ficoll 2% (w/v) Polyvinylpyrrolidone 10XEE 100mM EPPS 10mM EDTA, pH8.0 EEN 2.5xEE 0.5M NaCl LB (Luria-Bertani) media 1% (w/v) Tryptone 0.5% (w/v) Yeast extract 191 0.5% (w/v) Sodium chloride 1.5% (w/v) Bacto-agar (plates only) LB/Ampicillin LB 100µg/ml Ampicillin 1.5% (w/v) Bacto-agar (plates only) LB/Glycerol (for -70oC storage of bacterial stocks) 50% (v/v) LB 50% (v/v) Glycerol LB/Kanamycin LB 50µg/ml Kanamycin 1.5% (w/v) Bacto-agar (plates only) Loading dye per ml in 1X TE buffer 0.25 % (w/v) Bromophenol Blue, 7.5 mg 20% Glycerol 0.1mg/ml RNase A (DNase-free) Lysis solution 5% Sodium dodecyl sulphate (SDS) 50mM Tris, pH8.0 62.5mM EDTA, pH8.0 Minimal media (MM) 1.5% (w/v) Bacto-agar, autoclaved 1xMinimal Salts 0.5% (w/v) Glucose 0.1% (w/v) Magnesium chloride 10XMinimal Salts 7.0% (w/v) K2HPO4 3.0% (w/v) KH2PO4 1.0% (w/v) (NH4)2SO4 0.5% (w/v) Sodium citrate.3H2O pH7.5 (adjusted with sodium hydroxide) Neutralising solution 1.5M Sodium chloride 0.5M Tris.Cl, pH7.2 1mM EDTA 10XPBS 8% (w/v) Sodium chloride, g/L 0.2% (w/v) Potassium chloride, 0.2 g/L 1.44% (w/v) Na2HPO4, 1.44 g/L 0.24% (w/v) KH2PO4, 0.24 g/L Adjust to pH7.4 192 Pre-hybridisation solution 5xSSPE 5xDenhardt’s solution 0.5% (w/v) Sodium dodecyl sulphate (SDS) Protein purification lysis, wash and elution buffers derived from the QIAexpressionist handbook (Qiagen) SDS-PAGE destaining solution per liter 100 ml methanol 100 ml glacial acetic acid 800 ml ddH2O SDS-PAGE loading buffer 50 mM Tris-HCl, pH8.0 2% SDS 0.1% bromophenol blue 10% glycerol 4% mercaptoethanol, final concentration SDS-PAGE staining solution per liter 2.5 g Coomassie brilliant blue R250 360 ml methanol 80 ml glacial acetic acid 360 ml ddH2O Solution I 50mM Glucose 25mM Tris-HCl, pH8.0 10mM EDTA, pH8.0 Solution II 0.2M Sodium hydroxide 1% Sodium dodecyl sulphate (SDS) freshly prepared from 10N NaOH and 10% SDS stocks Solution III per 100 ml, pH4.8 60% (v/v) 5M Potassium Acetate, 60 ml 11.5% (v/v) Glacial acetic acid, 11.5 ml ddH2O, 28.5 ml 20XSSC 3M Sodium chloride 0.3M Sodium citrate.3H2O 20XSSPE 3.6M Sodium chloride 0.2M Sodium phosphate 0.02M EDTA, pH7.7 193 10XT4 DNA ligase buffer 250mM Tris.Cl 100mM Magnesium chloride 2mM Dithiothreitol (DTT) 4mM ATP pH7.8 (at 37oC) 10XTaq DNA Polymerase buffer 100mM Tris.Cl 15mM Magnesium chloride 500mM Potassium chloride pH9.0 50X TAE buffer per liter 242 g Tris, pH8.0 57.1 ml glacial acetic acid 0.05M EDTA, pH8.0 TBS per liter, pH7.4 g NaCl 0.2 g KCl 3.0 g TES buffer 50mM Tris, pH8.0 5mM EDTA, pH9.0 50mM Sodium chloride 194 Appendix B Curriculum Vitaé Profession : Molecular Microbiologist/Medical Lab. Microbiologist PERSONAL DATA Name Date of Birth Present Address : : : E-mail Address : EDUCATION Post-Graduate DARIO CRUZ ANGELES December 16, 1965 Microbiology Department, Faculty of Medicine The National University of Singapore Science Drive 2, 117597, Singapore Tel. (65) 9049-9938 Fax. (65) 6776-6872 medp0194@nus.edu.sg : Master of Philosophy in Biochemistry and Molecular Biology The Australian National University School of Biochemistry and Molecular Biology Faculty of Science, Canberra, 0200 Australian Capital Territory January 1999 – March 2001 (AUSAID Scholar) : Master of Science in Public Health (Medical Microbiology) University of the Philippines, College of Public Health Department of Medical Microbiology, Phil. General Hospital Pedro Gil Street, Ermita, Manila June 1987- April 1992 (UP-GIA Scholar) : Advance Genetics and Cell Biology Course, University of the Philippines, Institute of Biology March-May 1998 (UP-GIA Scholar) Tertiary : Bachelor of Science in Biology University of the Philippines, Institute of Biology June 1983- March 1987 (College Scholar, Dean’s List) Secondary : MIT Pre-Engineering High School, 1979-1983 (Valedictorian, Editor-in-Chief) Primary : Northern Yorklin Chinese School, 1971-1979 (Valedictorian, Gold Medalist) ELIGIBILITY Technologist in Microbiology : Microbiology (Passed, #7220981) American Society for Clinical Pathology Board of Registry P.O. Box 12277 Chicago IL 60612-0277 195 : Registered Microbiologist (Passed, #5285176) Clinical and Public Health Category American Academy for Microbiology National Registry of Microbiologists (ASM) 1325 Massachusetts Avenue, N.W. Washington D.C., 20005-4171 USA RegisteredMedical Technologist: Medical Technologist (Passed, #83078) American Medical Technologists 710 Higgins Road, Park Ridge Illinois, 60068-5765 USA : Membership American Society for Microbiology Philippine Society of Microbiology Australian Society for Microbiology WORK EXPERIENCE Research Scientist : SINGHEALTH Research Facilities Neuroscience laboratory Blk A,#02-02, Hospital Drive 169611, Singapore (June 2005- present) Research Assistant : National University of Singapore Microbiology Dept., Faculty of Medicine Science Drive 2, 117597, Singapore March 2005- May 2005 Teaching Assistant : National University of Singapore Microbiology Dept., Faculty of Medicine Science Drive, 117597, Singapore (June 2001-December 2004) Course Tutor : National University of Singapore Biochemistry Dept., Faculty of Medicine Science Drive 2, 117597, Singapore Demystifying the New Genetics (June 2001-June 2002) Teaching Assistant : The Australian National University School of Biochemistry and Molecular Biology Faculty of Science, Canberra 0200 ACT (March 2000- February 2001) Assistant Professorial Lecturer : De La Salle University, College of Science Biology Department, Microbiology 2401 Taft Avenue, 1004, Manila Philippines (May 1992 - October 1993; January 1998-January 1999) 196 Laboratory Specialist : Saudi Arabia National Guard Medical Services King Fahad National Guard Hospital Laboratory Dept. P.O. Box 22490, Riyadh 11426, K.S.A. (October 1993 - September 1997) Senior Microbiologist : King Saud University, College of Medicine Dept. of Clinical Microbiology and Parasitology Asir Central Hospital, P.O. Box 641, Abha, K.S.A. (March 1991 - February 1992) Medical Technologist : U.S. Naval Medical Research Unit-2 San Lazaro Hospital, Microbiology & Clinical Laboratory American Embassy, 1201Roxas Blvd., Manila (February 1989 - January 1991) Lecturer/ Instructor (Part-Time) : Perpetual Help Medical Center & College of Medicine Department of Human Biochemistry and Nutrition Perpetual Help Medical Center, Biñan, Laguna (June 1988 - March 1990) Laboratory Technician : Dr. Annilie Arcangel Clinic and Laboratory 1381 Quiricada Street, Sta. Cruz, Manila (November 1987 - January 1989) RECENT POSTERS Angeles Dario, Chang Siao Yun and Song Keang Peng. “Characterization of Clostridium difficile actin-specific binary ADP-ribosylating toxin detected through genomic DNA products derived from subtractive hybridization between a pathogenic and non-pathogenic strain”. 103rd ASM General Meeting, May 18-22, 2003, Washington Convention Center, Washington D.C. (ASM Student Travel Awardee). Angeles Dario and Song Keang Peng. "Activation of colonic stress signal pathway through Clostridium difficile CDT toxin-Induced ADP-ribosylation and universal shift in actin dynamics". 8th National University of Singapore-National University Hospital Annual Scientific Meeting, Oct. 7-9, 2004. Clinical Research Center Auditorium, Faculty of Medicine. (Best Basic Science Poster, shortlisted). Angeles Dario, Song Keang Peng and Naresh Verma. “The role of serotype-converting temperate bacteriophage SFV genome-encoded factors in Shigella flexneri cell invasion”. 4th Combined Scientific Meeting BRETSS/SSBMB/SSMB, Jan. 15-18, 2003, Raffles Convention Center, Singapore. (Poster Merit Prize, shortlisted). RESEARCH & PUBLICATIONS Angeles, D.C. and Song, K.P. 2005. Peptide antibiotic and actin-binding protein as mixed-type inhibitors of Clostridium difficile CDT toxin activities. Biochemical and Biophysical Research Communications. 327: 361-370. 197 Angeles, D.C. 2005. Cytophysiologic effects and molecular inhibition of a functional actinspecific ADP-ribosylstransferase cdt from Clostridium difficile. Department of Microbiology, Faculty of Medicine, National University of Singapore. Doctoral thesis for the degree: Doctor of Philosophy in Microbiology. In progress. Angeles, D.C. and Song, K.P. 2005. Clostridium difficile CDT toxin-induced ADP-ribosylation of actin distinctly activates colonic stress signal pathway. Research in Microbiology. Submitted. Angeles, D. C., Leong, W.Y., Koay, E., and Song, K.P. 2004. Comparative and quantitative transcription analyses of the binary ADP-ribosyltransferase cdt from a variant Clostridium difficile strain with truncated pathogenicity locus. Journal of Infectious Diseases. Submitted. Angeles, D.C., Ng, K.Y., and Song K.P. 2005. Comparative proteome analysis of Clostridium difficile subcellular fraction and characterization of membrane-associated TcdE of the pathogenicity locus. Manuscript in preparation. Allison, G.E., Angeles, D.C., Huan, P. and Verma, N.K. 2003. Morphology of temperate bacteriophage SFV and characterization of the DNA packaging and capsid genes: The structural genes evolved from two different phage families. The complete genomic sequence of SfV, a serotype-converting temperate bacteriophage of Shigella flexneri. Virology. 308(1): 114-127. Allison, G.E., Angeles, D.C., and Verma, N.K. 2002. The complete genomic sequence of SfV, a serotype-converting temperate bacteriophage of Shigella flexneri. Journal of Bacteriology. 184(7): 1974-1987. Angeles, D.C. 2001. Molecular characterization of the serotype-converting Shigella flexneri bacteriophage SfV. School of Biochemistry and Molecular Biology, The Australian National University. Masteral thesis for the degree: Master of Philosophy in Biochemistry and Molecular Biology. Angeles, D.C., & Montejo, M.M. 2001. Genomic DNA sequencing and physical mapping of Shigella flexneri serotype-specific antigen V temperate bacteriophage. Manila Journal of Science, Agham. 115: 24-36. Angeles, D.C. 1995. Comparison of plasmid profile analysis and antimicrobial resistance pattern in characterizing enterotoxigenic Escherichia coli. Manila Journal of Science, Agham. 19: 37-47. Angeles, D.C., 1994. Molecular characterization of enterotoxigenic Escherichia coli (Philippine isolates) by plasmid DNA and restriction endonuclease fingerprinting. Manila Journal of Science, Agham. 17: 1-7. Angeles, D.C., & Bautista, S. 1993. Use of alkaline phosphatase conjugated oligonucleotide probe for the detection of enterotoxigenic Escherichia coli in fecal samples. Manila Journal of Science, Agham, 16: 7-11. Angeles, D.C., 1992. Detection of heat-stable toxin producing ETEC from watery and formed stools using DNA hybridization and the infant mouse assay. Department of Medical 198 Microbiology, College of Public Health, University of the Philippines. Masteral thesis for the degree: Master of Science in Public Health major in Medical Microbiology. Angeles, D.C. 1987. Effects of high caffeine doses on chromosomal aberrations in mice. Dept., of Biological Sciences, Institute of Biology, The University of the Philippines. Undergraduate thesis for the degree: Bachelor of Science in Biology. REFEREES Tan Eng King, M.D. Associate Professor Singapore General Hospital Neurology Department SINGHEALTH Research Facilities Neurology Laboratory Blk A, #02-02, Hospital Drive 169611, Singapore E-mail: gnrtek@sgh.com.sg Song Keang Peng, Ph.D. Assistant Professor National University of Singapore Microbial Pathogenesis Laboratory Microbiology Dept., Faculty of Medicine Science Drive 2, 117597, Singapore Tel: (65) 6874-3278, Fax: (65) 6776-6872 E-mail: micskp@nus.edu.sg Naresh K. Verma, Ph.D. Associate Professor The Australian National University School of Biochemistry and Molecular Biology Canberra, 0200, ACT, Australia Tel: (612) 6249-2666, Fax: (612) 6249-0313 E-mail: naresh.verma@anu.edu.au Kevin S.W. Tan, Ph.D. Assistant Professor National University of Singapore Lab. of Molecular & Cellular Parasitology Microbiology Dept., Faculty of Medicine Science Drive 2, 117597, Singapore Tel: (65) 6874-6780, Fax: (65) 6776-6872 E-mail: mictank@nus.edu.sg Adelwisa Ortega, M.D., Ph.D. Chairman, Professor University of the Philippines Institute of Public Health Dept. of Medical Microbiology 625 Pedro Gil Ermita, Manila, Philippines Tel: (632) 525-5874 E-mail: Aortega@lagundi.cph.upm.edu.ph Esperanza C. Cabrera, Ph.D. Chairman, Professor De La Salle University College of Science Biology Department, Microbiology 2401 Taft Ave.,1004, Manila, Philippines Tel:(632)524-4611 ext 460; 5360228 199 Fax: (632) 524-0451 E-mail: cosecc@mail.dlsu.edu.ph Stephen E. Walz, Ph.D. Principal Scientist U.S. Naval Medical research Unit-2 NAMRU-2, Box 3, APO AP, CA 96520 U.S.A. Tel: (301) 626-8632 Jocelyn Andrada Secretary to the Counselor for Public Affairs U.S. Information Service American Embassy 1201 Roxas Blvd. Manila, Philippines Tel: (632) 523-1326 E-mail: jmamani@usia.gov Naser El Din Bilal, M.D. Professor King Saud University College of Medicine Dept. of Clinical Microbiology & Parasitology P.O. Box 641, Abha, K.S.A. Tel: (966-07) 224-7800 Manuel J. Padilla, MSMT Medical Laboratory Supervisor Saudi Arabia National Guard Medical Services SANG Health Affairs P.O. Box 8994, Riyadh 11492 KSA Tel: (966-01) 482-9641 ext 409 laboratory Theresita A. Lariosa, M.D. Executive Dean University of Perpetual help & Medical Center Dept. of Clinical Biochemistry & Nutrition Sto.Nino, Binan, Laguna, Philippines Tel: (63) 495118636 ext Col of Medicine LABORATORY DUTIES: (Research/Clinical) • Gene cloning, GST, GFP, 6X His, myc, HA fusion tags • Genome, DNA, RNA extraction • Plasmid and cosmid library production and screening • Site-directed mutagenesis, PCR restriction site linker, Gene-tailor, transposon insertion, SiRNA knock outs • Subtractive hybridization-biotin streptavidin extraction • Colony blot, Southern, Northern, Western hybridization • Genomic and plasmid extraction and purification • Bacteriophage isolation and genome extraction-Induction, PEG purification, dialysis, CsCl ethidium bromide gradient centrifugation. • DNA processing and manipulation: transposon insertion, DNA modification, RT, RealTime PCR quantitation • • • • • • Protein overexpression, Protein purification, affinity chromatography SDS-PAGE – silver stain, coommassie blue stain, ponceau S stain 2-D gel electrophoresis Yeast two hybrid Co-immunoprecipitation 200 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Phage plaque assay and sensitivity assay Single and multiplex ELISA microplate assay Western Immunoblotting and hybridization, acetone powder antisera adsorption PCR (Perkin Elmer, BioRad, Hybaid)- colony, gene amplification, sequencing Competent cell prep.- rubidium chloride, calcium chloride, electrocompetent cells Electron microscopy-transmission EM (Hitachi JOELX-200) Confocal microscopy Immunogold labelling DNA sequencing Big dye terminator (ABI prism) and bioinformatics analysis (DNA star, WebANGIS, NCBI program) Protein Sequencing Edman degradation Polyclonal monospecific Antibody production Tissue culture-splitting, passage, feeding Vero, HeLa, McCoy, BHK, Hep-2, Chang Conjunctiva, HCT 116, N1E-115, HepG2 Lyophilization HeLa cell invasion assay Indirect, direct FA- VZ, Mycoplasma, anti-DNA, HSV, RSV, Chlamydia, Leishmania, HIV, HCV, anti-HBc, Hbe, rotavirus Flow cytometry-FACS, T4,T8 ratio FA staining Hemagglutination and indirect hemagglutination assay- Toxoplasma, Schistosoma, Amoeba, Echinococcus, Chikungunya, Rubella, Dengue hemorrhagic, JapB encephalitis Embryonated egg technique-influenza Single radial immunodiffusion-complement and immunoglobulin detection Immunohistochemistry Protein fusion and expression, Western hybridization Radioactive works (3H, 32P, 14C), photoaffinity labeling, ADP-ribosylation assay Phosphorimaging (Typhoon, Amersham) Liquid scintillation counting (MicroBeta, PerkinElmer) Routine microbiological isolation, morphological, biochemical, serological identification Fluorescent microscopy Routine urinalysis (Urotron RL9), fecalysis Hematology: complete blood count, differential count, coagulation tests:prothrombin, thromboplastin, fibrinogen, thrombin/bleeding/clotting time, fibrin degradation, ESR, reticulocyte count, sickle cell, L.E.prep, (Coulter STKS/T890, Cell Dyne 400, Diagnostica Stago Chemistry: serum level of biochemical metabolites-RBS, FBS, BUN, creatinine, cholesterol, triglyceride, AST, ALT, alk phosphatase, etc. (Gemeni, Reflotron, Hitachi 911, Kodak Ectachem, Johnson & Johnson 750 xrc). Endocrinology-LH, FSH, prolactin, cortisol, testosterone, estradiol, ammonia, Mg, Fe,C3, TIBC, etc. (Cobas Mira/DuPont Dimension). Tumor markers- CEA/colon, AFP/liver, MCA/breast, β-HCG/chorion, PSA/prostate, CA19-9/pancreas, CA-15-3/breast, CA125/ovary, etc. Drug assay. Serology: thyroid tests-T3, T4, FT1, 3, 4, TSH,etc (Cobas core, Bm Es300) Flame photometry Histology: H&E staining 201 [...]... A. , Ahmad, M F., Avruch, J., and Woodgett, J R (1994) The stress-activated protein kinase subfamily of c-Jun kinases Nature 369, 156 -160 Lachumanan, R., Armugam, A. , Durairaj, P., Gopalakrishnakone, P., Tan, C H., and Jeyaseelan, K (1999) In situ hybridization and immunohistochemical analysis of the expression of cardiotoxin and neurotoxin genes in Naja naja sputatrix J Histochem Cytochem 47, 55 1 -56 0... L-plastin -actin interaction shows a resemblance with that of alpha-actinin and allows a distinction to be made between the two actin- binding domains of the molecule Biochemistry 43, 2428-2437 163 Lebart, M C., Mejean, C., Roustan, C., and Benyamin, Y (1993) Further characterization of the alpha-actinin -actin interface and comparison with filamin-binding sites on actin J Biol Chem 268, 56 42 -56 48 Lee, R J., Albanese,... cloning of Clostridium difficile toxin A gene fragment in lambda gt11 FEBS Lett 213, 249- 253 Nagahama, M., Sakaguchi, Y., Kobayashi, K., Ochi, S., and Sakurai, J (2000) Characterization of the Enzymatic Component of Clostridium perfringens Iota-Toxin J Bacteriol 182, 2096-2103 Nakajima, H., Nagaso, H., Kakui, N., Ishikawa, M., Hiranuma, T., and Hoshiko, S (2004) Critical role of the automodification of. .. critical evaluation of mechanisms and functions Annu Rev Biochem 55 , 987-10 35 Pomerance, M., Abdullah, H B., Kamerji, S., Correze, C., and Blondeau, J P (2000) Thyroidstimulating hormone and cyclic AMP activate p38 mitogen-activated protein kinase cascade Involvement of protein kinase A, rac1, and reactive oxygen species J Biol Chem 2 75, 4 053 94 054 6 Popoff, M R., and Boquet, P (198 8a) Clostridium spiroforme... J., Karandikar, M., Xu, S., and Cobb, M H (1999) New insights into the control of MAP kinase pathways Exp Cell Res 253 , 255 -270 Enslen, H., Brancho, D M., and Davis, R J (2000) Molecular determinants that mediate selective activation of p38 MAP kinase isoforms Embo J 19, 1301-1311 Estes, J E., Selden, L A. , and Gershman, L C (1981) Mechanism of action of phalloidin on the polymerization of muscle actin. .. structure of the catalytic sites of mammalian and bacterial toxin ADP- ribosyltransferases Mol Cell Biochem 138, 177-181 Ono, S (2003) Regulation of actin filament dynamics by actin depolymerizing factor/cofilin and actin- interacting protein 1: new blades for twisted filaments Biochemistry 42, 13363-13370 Oppenheimer, N.J (1994) NAD hydrolysis: chemical and enzymatic mechanisms Mol Cell Biochem 138,2 45- 251 ... L., and Wilkins, T D (1991) Construction and expression of the complete Clostridium difficile toxin A gene in Escherichia coli Infect Immun 59 , 150 - 153 172 Poilane, I., Karjalainen, T., Barc, M.-C., Bourlioux, P., and Collignon, A (1998) Protease activity of Clostridium difficile strains Canad J Microbiol 44, 157 -161 Pollard, T D., and Cooper, J A (1986) Actin and actin- binding proteins A critical evaluation... D G., Stergachis, A. , and McFarland, L V e a (2000) Antibiotics and Clostridum difficile diarrhea in the ambulatory care setting Clin Ther 22, 91-102 164 Lima, A A., Lyerly, D M., Wilkins, T D., Innes, D J., and Guerrant, R L (1988) Effects of Clostridium difficile toxins A and B in rabbit small and large intestine in vivo and on cultured cells in vitro Infect Immun 56 , 58 2 -58 8 Limaye, A P., Turgeon,... 69-77 Karjalainen, T., Waligora-Dupriet, A J., Cerquetti, M., Spigaglia, P., Maggioni, A. , Mauri, P., and Mastrantonio, P (2001) Molecular and genomic analysis of genes encoding surfaceanchored proteins from Clostridium difficile Infect Immun 69, 3442-3446 Karlsson, S., Lindberg, A. , Norin, E., Burman, L.G., and Akerlund, T (2000) Toxins, butyric acid and other short-chained fatty acids are coordinately... Nguyen, B K., and Mercurio, A M (2000) RhoA function in lamellae formation and migration is regulated by the alpha6beta4 integrin and cAMP metabolism J Cell Biol 148, 253 - 258 Ogunniyi, A D., Giammarinaro, P., and Paton, J C (2002) The genes encoding virulenceassociated proteins and the capsule of Streptococcus pneumoniae are upregulated and differentially expressed in vivo Microbiology 148, 20 45- 2 053 Ohishi, . G487-4 95. Goncalves, C., Decre, D., Barbut, F., Burghoffer, B., and Petit, J. C. (2004). Prevalence and characterization of a binary toxin (actin- specific ADP- ribosyltransferase) from Clostridium. (1992). Specific inhibitors of poly (ADP- ribose) synthetase and mono (ADP- ribosyl)transferase. J Biol Chem 267, 156 9- 157 5. Banasik, M., Komura, H., and Ueda, K. (1990). Inhibition of poly (ADP- ribose). Fekety, R., and Silva, J. J. (1987). Theraphy of relapsing Clostridium difficile- associated diarrhea and colitis with the combination of vancomycin and rifampin. J Clin Gastroenterol 9, 155 - 159 .