Uncovering novel pathogenicity associated loci among Yersinia enterocolitica species by subtractive hybridization [Elektronische Ressource] / von Azuka Iwobi

Iwobi, Azuka

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2003
• Nombre de lectures: 9
• Langue: English

Extrait

Dissertation der Fakultät für Biologie der
Ludwig-Maximilians-Universität München















Uncovering novel pathogenicity-associated loci among
Yersinia enterocolitica species by subtractive hybridization









von
Azuka Iwobi
aus Lagos, Nigeria








Submitted on 12 March 2003





























Dissertation eingereicht am 12. März 2003


Erstgutachterin: Prof. Elizabeth Weiss
Zweitgutachter: Prof. Anton Hartmann
Sondervotum: Prof. Jürgen Heesemann

Tag der mündlichen Prüfung: 12. Mai 2003 TABLE OF CONTENTS I
Uncovering novel pathogenicity-associated loci among
Yersinia enterocolitica species by subtractive hybridization
A. INTRODUCTION 1
1. Yersinia 1
1.1 Y. enterocolitica 2
1.2 Representational difference analysis as a tool in the elucidation
of the genetic variability among Y. enterocolitica strains. 3
1.3 State of the Art 5
1.3.1 Overview of the pathogenesis of Yersinia species. 5
1.3.1. Invasin (Inv) 5
1.3.2 Attachment invasion locus (Ail) 6
1.3.3 Mucoid Yersinia factor (Myf) 6
1.3.4 Yersinia adhesin (YadA) 6
1.3.5 Yst Enterotoxin 7
1.3.6 Yersinia outer proteins (Yops) 7
1.3.7 The Yersiniabactin iron acquisition system 8
2 Protein Secretion 9
2.1 Type III secretion systems in Y. enterocolitica 9
2.2 Type II secretion systems 10
3 Aims of this research study 13
B. MATERIALS AND METHODS 14
1. Material 14
1.1 Equipment 14
1.2 Other materials 15
1.3 Chemicals and Enzymes 15
2. Bacteria, Plasmids and Primers 16
2.1 Bacterial strains and plasmids 16
2.2 Primers 18
3. Culture media, Antibiotics, Strain Cultivation and Storage 19
3.1 Culture media 19
3.2 Antibiotics 20
3.3 Cultivation and long term storage of bacteria 20TABLE OF CONTENTS II
4. Molecular genetic methods 21
4.1 Isolation of Chromosomal DNA 21
4.2 Isolation of plasmid DNA 22
4.2.2 Plasmid isolation with QIAprep Spin Miniprep kit (Qiagen) 22
4.2.3 Plasmid isolation with Nucleobond AX100 Kit (Machery-Nagel) 22
4.3 Purification of DNA and determination of DNA
concentration and purity 22
4.3.1 Phenol extraction and ethanol precipitation of DNA 22
4.3.2 Determination of DNA concentration and purity 23
4.4 Polymerase Chain Reaction 23
4.5 Agarose gel electrophoresis 24
4.6 Enzymatic modification of DNA 25
4.6.1 Restriction digestion of DNA 25
4.6.2 Dephosphorylation of DNA 26
4.6.3 Ligation of DNA molecules 26
4.7 DNA sequencing 26
4.8 RNA analysis 26
4.8.1 RNA Isolation 26
4.8.2 DNase reaction 27
4.8.3 Reverse Transcription 27
4.8.4. Reverse Transcription analysis from bacterial RNA
isolated from an infected HeLa cell line 28
4.9 Bacterial transformation 29
4.9.1 Production of electrocompetent cells 29
4.9.2 Production of calcium-competent cells 30
4.10 Southern Blot hybridization (Southern, 1975) 30
4.10.1 Preparation of DNA probe 31
4.10.1.1 Digoxigenin-labeling of DNA through PCR 31
4.10.1.2 Random-primed method of DNA labeling 31
4.10.2 Southern (Vacuum) Blot 32
4.10.3 Hybridization and detection 32
4.11 Cosmid gene bank of Y. enterocolitica WA-314 33
4.11.1 Preparation of cosmid vector DNA 34TABLE OF CONTENTS III
4.11.2 Preparation of genomic DNA 34
4.11.3 Ligation and packaging of DNA 35
4.12 Representational difference analysis
(suppressive subtractive hybridization) 36
4.12.1 Hybridization 37
4.12.2 PCR amplification 37
4.12.3 Preparation of X-gal / IPTG plates for blue-white screening
of recombinants 38
5. Enzyme activity assays 41
5.1 Chitinase assay 41
5.2 Detection of chitinolytic activity with Methylumbelliferyl (MU)-
diacetyl-chitobioside and MU-triacetyl-chitotrioside 41
5.3 Cellulase assay 42
6. Protein biochemical studies 42
6.1 Sodium-dodecyl-sulphate Polyacrylamide Gel Electrophoresis 42
6.2 Western Blot 44
6.3 The Glutathione-S-transferase Gene Fusion System
(Pharmacia Biotech) 45
6.3.1 Cultivation and induction of bacteria 45
6.3.2 Purification of the GST-fusion protein 45
6.4 Preparative SDS-PAGE, electroelution and protein recovery 46
6.4.1 Preparative SDS-PAGE 46
6.4.2 Electroelution 46
6.4.3 Protein precipitation 46
6.5 Rabbit immunization 47
7. Mouse experiments 47
7.1 Mobilization of the pYV plasmid into recipient
Y. enterocolitica strains 47
7.2 Confirmation of the presence of the pYV plasmid 48
7.2.1 Agglutination test 48
7.2.2 Proof of Yops - secreted proteins encoded by the pYV plasmid 48
8. QuantitativeTissue Culture Invasion (TCI) Assay 49
9. Bioinformatics 49TABLE OF CONTENTS IV
10. Nucleotide sequence accession numbers 50
C. RESULTS 51
1. Construction of a library of subtracted fragments unique to
Y. enterocolitica WA-314 and absent from the genome of NF-O 51
2. Analysis of the subtracted library for tester-specific sequences 52
3. Sequence analysis of the subtracted fragments 53
4. A novel IS10-like element - IS1330 uncovered by
subtractive hybridization 55
5. IS1330 is associated with a novel ars operon in
Y. enterocolitica 8081 59
6. Subtractive Hybridization uncovers a novel type II secretion
apparatus unique to highly pathogenic Y. enterocolitica strains 61
7. yts2 - a species-specific type II secretion cluster 64
7.1 The yts2 gene cluster 64
7.2 Comparison of yts1 and yts2 65
8. Reverse transcription analysis of the yts1 and yts2 genes 68
9. A putative chitin-binding protein flanks downstream the
Yts1 secreton 69
10. Construction of Y. enterocolitica mutants 70
r10.1 WA-CS yts1E::Kan 71
r10.2 WA-CS chiY::Kan 74
r 10.3 WA-CS hns::Kan 77
11. The yts1 secretion cluster may not be involved in
house-keeping functions of the harboring species 80
12. Mouse virulence assays 81
13. Impact of ChiY on the virulence of Y. enterocolitica WA-314 83
14. Reporter fusion studies with chiY-gfp 84
15. ChiY-Antibody production and assessment of
chitin-binding activity 85
15.1 Recombinant fusion between GST and ChiY 86
15.2 Chitin binding activity of the ChiY protein 87TABLE OF CONTENTS V
D. DISCUSSION 90
1.1 Representational difference analysis to identify genomic
differences among Yersinia species 90
1.2 Representational difference analysis applied to
Y. enterocolitica strains 91
2. IS1330 is a novel insertion sequence unique to the assemblage
of pathogenic Y. enterocolitica strains 92
2.1 IS elements and genetic rearrangements 92
2.2 IS1330 flanks novel sequences in Y. enterocolitica 8081
that could have consequences on the pathogenicity of the species 94
3. The yts1 gene cluster 95
3.1 Expression of the yts1 genes is temperature-enhanced 96
3.2 Role of the Yts1 secreton in virulence 98
3.3 Is yts1 a pathogenicity island? 99
3.4 The yts2 gene cluster 99
4.1 ChiY – a putative chitin-binding protein 100
4.2 The ChiY protein might play a virulence-enhancing role in
Y. enterocolitica species 101
E. SUMMARY 103
F. REFERENCES 107
G. ABBREVIATIONS 120
Published aspects of this work 122
Curriculum vitae 123A. INTRODUCTION 1
1. Yersinia
Yersinia species are gram-negative bacteria belonging to the Enterobacteriaceae family.
They are facultative non-sporulating anaerobes with optimal growth at 27 °C. Based on
differentiating biochemical traits, 11 species have been identified to date which are
further divided into different biotypes. On the basis of antibody reactions to different
lipopolysaccharide structures, Yersinia species can also be divided into different
serological groups. Typically, the serotypes are linked to geographical distribution,
severity of human disease, and animal reservoir (Boyd and Cornelis, 2001, Salyers and
Whitt, 1994).
Three species of Yersinia cause disease in humans: Y. enterocolitica,
Y. pseudotuberculosis and Y. pestis. Y. pestis is the agent of the bubonic plague, the
infamous black death that swept through the Middle Ages, killing millions of people.
Today, isolated cases of Y. pestis infection are reported sporadically in the US, India and
Madagascar (Perry, 1997). Human infection with Y. pestis can be acquired in two ways:
by transmission from rats or other wild animals to humans by flea bite or by direct
human-to-human transmission via aerosols. Y. pestis is an obligate parasite, in contrast to
Y. enterocolitica and Y. pseudotuberculosis, which ca