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

ii
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.









iii
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
iv
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

v
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

vi
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
vii
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 IC
50

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.






viii
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

ix
List of Figures


Figure Page

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 S
N
1 and S
N
2-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

x
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
























xi
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

ddH
2
O 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 guanosine triphosphate
h hour
xii

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
P phosphorus-32 radionuclide

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

xiii
TEM transmission electron microscopy

Tris tris [hydroxymethyl] amino-methane

3
H/
3
H tritium/tritiated

UV ultraviolet

UTP uridine triphosphate