Cytophysiologic effects and molecular inhibition of a functional actin specific ADP ribosyltransferase CDT from clostridium difficile 1

CYTOPHYSIOLOGIC EFFECTS AND MOLECULAR INHIBITION OF A FUNCTIONAL ACTIN-SPECIFIC ADP-RIBOSYLTRANSFERASE CDT FROM CLOSTRIDIUM DIFFICILE DARIO CRUZ ANGELES [M.Sc.,(UP), M.Phil., (ANU), RM (AAM), RMT (AMT)] A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2005 Acknowledgements Foremost is a most sincere thanks to my supervisor, Dr. Song Keang Peng for giving me the opportunity to work in this engaging project. His support, guidance, patience, kind words and encouragements are truly appreciated. Indeed, I can profess that several years of toil had not waned to futility as I have not only gained a mentor but a lifetime friend. I am grateful to the technical assistance extended by our endearing technicians, Ms. Boon, Ms. Nalini, Ms. Soo and Mr. Go Ting Kiam. I truly hope that the department would have more staff like them who would go out of their way to help. Allow me to also extend a heartfelt gratitude to my labmates who have made my stay bearable, particularly to Gan Bong Hwa whom I have learned to consider as my little sister primarily due to her forthright and unpretentious ways and to the department’s aunties and uncles who have taught me by example, much about humility, contentment and simplicity. Ultimately, I am eternally indebted to God almighty for His unwavering love and compassion, my beloved parents and Boom Boom for their relentless sacrifices and from whom I have derived most of my strength and inspiration through all these trying years. ii Table of Contents Title page . i Acknowledgements . ii Table of Contents iii Summary vi List of Tables viii List of Figures ix List of Abbreviations . xi Chapter Review of Literature 1.1. Clostridium difficile . 1.2. Diagnosis . 1.3. Epidemiology 1.4. Pathogenesis 1.5. Disease management 1.6. Virulence factors 1.6.1. Large clostridial cytotoxins . 1.6.1.1. Toxin A 11 1.6.1.2. Toxin B 15 1.6.1.3. The Pathogenicity Locus (PaLoc)………………………………………… 16 1.6.1.3.1. Genetic profile of PaLoc 16 1.6.1.3.2. Regulation of PaLoc genes 18 1.6.2. CDT toxin 19 1.6.2.1. ADP-ribosyltransferase (ADPRT) 20 1.6.2.2. Actin as substrate 21 1.6.2.3. Biology of ADPRT 23 1.6.2.4. Genetics and regulation 24 1.6.2.5. Conserved structures and function 25 1.6.3. Other virulence factors . 30 1.7. Objectives of the study . 33 Chapter Materials and Methods 34 2.1. General protocols and molecular techniques 34 2.1.1. Laboratory procedures 34 2.1.2. Bacterial culture and growth conditions . 34 2.1.3. General cell culture techniques . 36 2.1.4. Isolation of DNA . 36 2.1.5. Standard techniques for DNA manipulation . 37 2.1.5.1. Quantitation 37 2.1.5.2. Restriction endonuclease digestion 37 2.1.5.3. Agarose gel electrophoresis 38 2.1.5.4. Gel extraction 38 2.1.5.5. Dephosphorylation of linearized DNA 38 2.1.5.6. Ligation of fragment into plasmid 39 2.1.6. Production of competent cells . 39 2.1.6.1. Preparation of competent cells 39 iii 2.1.6.2. Transformation of competent cells 40 2.1.7. Blotting and hybridization . 40 2.1.7.1. Colony and dot blotting 40 2.1.7.2. Fixation of blots 41 2.1.7.3. Probe labelling 41 2.1.7.4. Hybridization 41 2.1.7.5. Autoradiography 42 2.1.8. Polymerase chain reaction (PCR) . 42 2.1.8.1. Primer preparation 42 2.1.8.2. Reaction conditions 42 2.1.8.3. Purification of PCR product 43 2.1.9. DNA sequencing . 43 2.1.9.1. Preparation of template 43 2.1.9.2.Dye Terminator sequencing 44 2.1.9.3. Purification of sequencing product 44 2.2. Specific protocols used in results chapter . 45 2.2.1. Genomic subtraction: target, subtractor, adapter preparation . 45 2.2.2. Genomic subtraction: hybridization . 47 2.2.3. Library construction 48 2.2.4. Screening of clones 48 2.2.5. Total RNA purification . 50 2.2.6. Reverse transcription-PCR (RT-PCR) 51 2.2.7. Real-time PCR 51 2.2.8. Derivation of CDT 52 2.2.9. Site-directed mutagenesis . 53 2.2.10. ADP-ribosyltransferase assay (ARTase) 54 2.3. Specific protocols used in results chapter . 55 2.3.1. Cells and reagents . 55 2.3.2. Cell culture 56 2.3.3. Cytotoxicity assay and confocal microscopy . 56 2.3.4. Thymidine assimilation 57 2.3.5. ARTase . 57 2.3.6. Flow cytometry 58 2.3.7. Enzyme-linked immunosorbent assay (ELISA) . 59 2.3.8. Caspase-3 assay . 59 2.3.9. Multiplex immunoassay 60 2.4. Specific protocols used in results chapter . 60 2.4.1. ARTase 60 2.4.2. NAD glycohydrolase assay (NADse) . 60 2.4.3. Photoaffinity labelling . 61 Chapter Molecular characterization of cdt genes from CCUG 20309 . 62 3.1. Introduction . 62 3.2. Results . 63 3.2.1. Isolation of 19126-specific virulenceDNA 63 3.2.2. Identification of inserts with putative virulence function 64 3.2.3. Variant forms of cdt . 67 3.2.4. Analysis of cdt regulatory region 69 3.2.5. Growth dependent transcription of cdt 69 3.2.6. Transcription of cdt relative to PaLoc 72 iv 3.2.7. Expression of CDTa . 76 3.2.8. Catalytically essential amino acid residues of CDTa . 76 3.3. Discussion . 82 Chapter Effects of CDT on actin dynamics and signal transduction 92 4.1. Introduction . 92 4.2. Results . 93 4.2.1.Differential cell sensitivity related toCDTb-receptor variability . 93 4.2.2. Changes in morphology and actin isoform ratio . 95 4.2.3. Effects of cdt with actin modifying agents . 98 4.2.4. Changes in expression of signal proteins 99 4.2.5. Stimulation of stress-related proteins 100 4.3. Discussion . 104 Chapter Peptide antibiotics and actin binding proteins are mixed-type CDT inhibitors 114 5.1. Introduction . 114 5.2. Results . 115 5.2.1. Nucleotide binding in CDT 115 5.2.2. Role of divalent metal ions in CDT-NAD binding . 117 5.2.3. Inhibitor compounds for CDT activities 117 5.2.4. Actin binding proteins showed diverse modes of CDT inhibition 122 5.3. Discussion . 125 Chapter General discussion 132 Chapter Bibliography . 139 Appendix A: Buffers, solutions and media . 191 Appendix B: Curriculum vitaé 195 v Summary The emerging clinical importance of Clostridium difficile has prompted the application of targeted strategy to rapidly identify virulence determinants from an otherwise poorly characterized genome. Using subtractive hybridization, putative virulence-encoding gene fragments were identified which led to the isolation of a binary ADP-ribosyltransferase cdtA and cdtB (cdt locus) from CCUG 20309. Transcriptional studies of cdtA and cdtB showed lower expression compared to a Pathogenicity Locus (PaLoc) gene tcdE suggesting accessory role of CDT toxin in pathogenesis. Unison mRNA expression of cdtA and cdtB was suggestive of bicistronic conformation of the cdt operon, supported by regulatory elements found upstream of cdtA and not in cdtB, inverted repeats flanking cdt genes but not the intergenic region of the cdt locus and detection of readthrough cDNA. Higher and early tcde expression which was detectable as early as the late lag growth phase, implied multiplicity of PaLoc promoters with more efficient activity and predominant role of PaLoc toxins in pathogenesis. Information on comparative toxin expression patterns would be useful for both research and clinical applications. Using ADP-ribosyltransferase assay (ARTase), analyses of wild-type and mutant CDTa activities revealed conserved amino acid residues that are crucial for its ability to hydrolyze cofactor nicotinamide adenine dinucleotide (NAD) and attach the ADP-ribose moiety onto Gactin to cause disruption of the microfilament structure and cell rounding. Cytometric quantitation of G:F-actin ratio by binary CDT (CDTa and CDTb) with or without established actin modifiers showed CDT-induced shift in the level of actin isoform in favor of monomeric Gactin. Since actin reorganization is important for proper cell physiologic functions, ADP- ribosylation may well be an innate but well-regulated process among eukaryotic cells. CDTb was important in intracellular translocation of the enzymatic protein CDTa. When binary CDT were applied, colonic cells showed signs of cytotoxicity, remodelled actin, and stress effector vi activation via the integrin-cAMP-stress-related MAPK-ATF2 but not the MEK2-ERK1/2-AKTStat3 route. In addition, activation of caspase-3 and inability of CDT to mediate phosphoinactivation of BAD (Bcl-activated death promoter) is suggestive of CDT’s commitment to apoptosis. Activation of opposing mitogenic signal pathway by the CCUG20309 total lysate suggests the presence of endogenous pro-mitogenic factors. Noting the deleterious effects of CDT toxin on various mammalian cell lines, we searched for potential antagonists of CDTa ADP-ribosyltransferase and NAD glycohydrolase activities. Compounds including heterocyclic peptide antibiotics with modified amino acid (polymyxin B and β–lactam cephalosporins) which neutralized CDTa transferase and glycohydrolase activities at consistently and significantly high mean inhibition and low IC50 values were found. The strongest inhibitors were actin-binding proteins having extensive interfaces with G-actin adjoining the CDT-ADP-ribose+ acceptor site (R177) and nucleotide cleft. It was also realized that efficient NAD-CDTa interaction required specific divalent cations which can be substituted by ATP but not ADP. Data generated on the extent of actin interaction sites and different modes of inhibition of CDTa actions provided fresh evidences in support of the designation of actin interface domains with actin binding proteins. Overall, we have demonstrated the applicability of genomic subtraction in isolating differential DNA between between closely related but phenotypically diverse organisms. This had resulted in the isolation and characterization of CDT, a potent molecule that may enhance C. difficile pathogenicity by complementing the deleterious effects of large clostridial toxins, partly explaining the increased isolation of an otherwise attenuated non-toxin A-producing pathogenic C. difficile strain. vii List of Tables Table Page 1.1. Bacterial toxins with A-B structures . 22 2.1. Bacterial strains, recombinant clones and plasmids used in this study . 35 2.2. Oligonucleotides used in PCR amplification of cdt, tcd and 16S rDNA genes . 46 3.1. Protein homologies of CCUG 19126 library inserts . 66 3.2. Promoter sequences among different bacteria 71 5.1. Effects of various compounds on CDT enzymatic functions . 120 viii List of Figures Figure Page 1.1. Steps in the development and outcomes of PMC . 1.2. Morphological views of PMC . 1.3. Genetic map of C. difficile PaLoc . 1.4. Mode of action of toxin A and toxin B . 12 1.5. Genetic map of C. difficile cdt locus . 20 1.6. Structural model of Escherichia coli heat-labile toxin 27 1.7. Stereo view of Ia complex with NADH . 28 1.8. Superimposed stereo view of NAD binding site of ADPRT C-domains . 29 1.9. Enzymatic processes involving SN1 and SN2-type reaction . 31 2.1. Schematic representation of the genomic subtraction process 49 3.1. Colony PCR of C. difficile reference strains for tcdB gene portion 64 3.2. Colony blot hybridization for the detection of genomic library clones encoding putative virulence DNA insert by subtraction products 65 3.3. Genetic map of cdt in CCUG 19126 and CCUG 20309 . 68 3.4. Features of cdt regulatory region 70 3.5. Comparative transcription of cdt variants using RT-PCR 73 3.6. Quantitative comparison of cdt and tcdE transcription using real-time RT-PCR . 74 3.7. Alignment of conserved ADPRT regions . 78 3.8. Comparative analysis of wild-type and mutated cdt genes and mutant proteins 79 3.9. Electropherogram of wild-type and mutated cdtA nucleotide 80 3.10. Protein profiles and autoradiograms showing ADP-ribosylated actin by various CDTa isoforms and NAD photolabeled CDTa 81 3.11. Proposed mechanism of ADP-ribosylation of actin by ADPRT . 90 4.1. Effects of CDT on cytoskeletal structure 94 ix 4.2. Confocal images of variably treated and double-stained cells showing spatial distribution of actin isoforms 96 4.3. Cytometric analysis of HCT 116 on dual signal detection . 97 4.4. Comparison of G and F-actin level in CDT-treated HCT 116 cells . 101 4.5. Analysis of relative expression of talin, cAMP, Rho and phospho-p38 MAPK in CDTtreated HCT 116 . 102 4.6. Analysis of relative expression of caspase-3, signal phosphoproteins and transcription factors in CDT and lys20309-treated HCT 116 103 4.7. Western blot analysis of lysate proteins of CDT-treated HCT 116 105 4.8. Hypothetical model of CDT-induced stress transduction cascades in HCT 116 113 5.1. Kinetics of recombinant CDT . 116 5.2. Phosphorimages showing effects of various compounds on CDT actions . 118 5.3. Lineweaver-Burk plots of initial velocity patterns for inhibited CDT with respect to labeled NAD . 124 5.4. Schematic illustration of G-actin showing representative interface sites with myosin 128 5.5. Schematic drawing of SN1-type mechanism for Ia . 130 x List of Abbreviations AAD antibiotic-associated diarrhea ADP adenosine diphosphate ATP adenosine triphosphate ADPRT ADP-ribosyltransferase ARTase ADP-ribosylation assay amp ampicillin bp base pairs BHI brain heart infusion CIAP calf intestinal alkaline phosphatase cDNA complementary deoxyribonucleic acid cpm counts per minute Ci curie CCFA cycloserine cefoxitin fructose agar CDAD Clostridium difficile-associated diarrhea dNTP 2’-deoxyribonucleoside-5’-triphosphate DEPC diethylpyrocarbonate DMSO dimethyl sulphoxide ddH2O double deionized distilled water DMEM Dulbecco’s Modified Eagle’s Medium ELISA enzyme-linked immunosorbent assay EDTA ethylene diamine tetraacetic acid FCS fetal calf serum FITC fluorescein isothiocyanate GTP h guanosine triphosphate hour xi pl isoelectric point IPTG isopropyl-β-D-thiogalactoside Kb kilobase kDa kilodalton L liter mRNA messenger ribonucleic acid µ micro m milli mol mole MW molecular weight n nano NAD nicotinamide adenine dinucleotide orf open reading frame PaLoc pathogenicity locus PBS phosphate buffered saline 32 phosphorus-32 radionuclide P p pico PMC pseudomembranous colitis RT-PCR reverse transcription-polymerase chain reaction rpm revolutions per minute RBS ribosomal binding site sec second SD Shine Dalgarno SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis TSS transcriptional start site xii TEM transmission electron microscopy Tris tris [hydroxymethyl] amino-methane tritium/tritiated H/3H UV ultraviolet UTP uridine triphosphate xiii [...]... of Abbreviations AAD antibiotic-associated diarrhea ADP adenosine diphosphate ATP adenosine triphosphate ADPRT ADP- ribosyltransferase ARTase ADP- ribosylation assay amp ampicillin bp base pairs BHI brain heart infusion CIAP calf intestinal alkaline phosphatase cDNA complementary deoxyribonucleic acid cpm counts per minute Ci curie CCFA cycloserine cefoxitin fructose agar CDAD Clostridium difficile- associated... IPTG isopropyl-β-D-thiogalactoside Kb kilobase kDa kilodalton L liter mRNA messenger ribonucleic acid micro m milli mol mole MW molecular weight n nano NAD nicotinamide adenine dinucleotide orf open reading frame PaLoc pathogenicity locus PBS phosphate buffered saline 32 phosphorus-32 radionuclide P p pico PMC pseudomembranous colitis RT-PCR reverse transcription-polymerase chain reaction rpm revolutions... Clostridium difficile- associated diarrhea dNTP 2’-deoxyribonucleoside-5’-triphosphate DEPC diethylpyrocarbonate DMSO dimethyl sulphoxide ddH2O double deionized distilled water DMEM Dulbecco’s Modified Eagle’s Medium ELISA enzyme-linked immunosorbent assay EDTA ethylene diamine tetraacetic acid FCS fetal calf serum FITC fluorescein isothiocyanate GTP h guanosine triphosphate hour xi pl isoelectric point... transcription-polymerase chain reaction rpm revolutions per minute RBS ribosomal binding site sec second SD Shine Dalgarno SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis TSS transcriptional start site xii TEM transmission electron microscopy Tris tris [hydroxymethyl] amino-methane 3 tritium/tritiated H/3H UV ultraviolet UTP uridine triphosphate xiii . 3 .10 . Protein profiles and autoradiograms showing ADP- ribosylated actin by various CDTa isoforms and NAD photolabeled CDTa 81 3 .11 . Proposed mechanism of ADP- ribosylation of actin by ADPRT. of Abbreviations AAD antibiotic-associated diarrhea ADP adenosine diphosphate ATP adenosine triphosphate ADPRT ADP- ribosyltransferase ARTase ADP- ribosylation assay amp ampicillin. Cytometric analysis of HCT 11 6 on dual signal detection 97 4.4. Comparison of G and F -actin level in CDT- treated HCT 11 6 cells 10 1 4.5. Analysis of relative expression of talin, cAMP, Rho and phospho-p38