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

104 Chapter 5 Peptide antibiotics and actin binding proteins are mixed-type CDT inhibitors 114 5.1.. Actin binding proteins showed diverse modes of CDT inhibition .... Using subtractive

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

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 1 Review of Literature 1

1.1 Clostridium difficile 1

1.2 Diagnosis 2

1.3 Epidemiology 3

1.4 Pathogenesis 4

1.5 Disease management 6

1.6 Virulence factors 8

1.6.1 Large clostridial cytotoxins 8

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 2 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

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 3 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 4 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 5 60

2.4.1 ARTase 60

2.4.2 NAD glycohydrolase assay (NADse) 60

2.4.3 Photoaffinity labelling 61

Chapter 3 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

3.2.7 Expression of CDTa 76

3.2.8 Catalytically essential amino acid residues of CDTa 76

3.3 Discussion 82

Chapter 4 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 5 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 6 General discussion 132

Chapter 7 Bibliography 139

Appendix A: Buffers, solutions and media 191

Appendix B: Curriculum vitaé 195

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 G-actin 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 G-actin 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

activation via the integrin-cAMP-stress-related MAPK-ATF2 but not the MEK2-ERK1/2-AKT-Stat3 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

List of Tables

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

List of Figures

1.1 Steps in the development and outcomes of PMC 5

1.2 Morphological views of PMC 7

1.3 Genetic map of C difficile PaLoc 9

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

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 CDT-treated 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

List of Abbreviations

CDAD Clostridium difficile-associated diarrhea

ddH2O double deionized distilled water

pl isoelectric point

32

RT-PCR reverse transcription-polymerase chain reaction

SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis

TEM transmission electron microscopy

3