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«(Über die Bedeutung der bakteriellen Genomplastizität für die Adaptation und Evolution asymptomatischer Bakteriurie (ABU) Escherichia coli ...»

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Bacterial Genome Plasticity and its Role for Adaptation

and Evolution of Asymptomatic Bacteriuria (ABU)

Escherichia coli Strains

(Über die Bedeutung der bakteriellen Genomplastizität

für die Adaptation und Evolution asymptomatischer

Bakteriurie (ABU) Escherichia coli Isolate)

Doctoral thesis for submission to a doctoral degree

at the Graduate School of Life Sciences,

Julius Maximilian University Würzburg,

Section: Infection and Immunity

submitted by

Jarosław Maciej Zdziarski

from Góra, Poland Würzburg, 2008   Submitted on: …………………………………………………………….

office stamp

Members of the Promotionskomitee:

Chairperson: ……………………………………………………………… to be completed by the office Primary Supervisor: Prof. Dr. Dr. h. c. mult. Jörg Hacker Supervisor (second): Prof. Catharina Svanborg Supervisor (third): PD Dr.rer.nat. Ulrich Dobrindt Day of Rigorosum: ……………………………………………………….

Certificates were handed out on: ……………………………………..

Erklärung Gemäß § 4 Abs. 3 Ziff. 3, 5 und 8 der Promotionsordnung der Fakultät für Biologie der Bayerischen Julius-Maximilians-Universität Würzburg.

Ich versichere hiermit, dass ich die vorliegende Arbeit selbständig und nur unter Verwendung der angegebenen Quellen und Hilfsmittel verfasst habe.

Weiterhin versichere ich, dass die Dissertation bisher nicht in gleicher oder ähnlicher Form in einem anderen Prüfungsverfahren vorgelegen hat und ich bisher keine akademischen Grade erworben oder zu erwerben versucht habe.

Würzburg, im Juli 2008 (Jaroslaw Zdziarski) Acknowledgement This study was carried out at the Institute of Molecular Biology of Infectious Diseases at the University of Würzburg, Germany from September 15th, 2004 until May 31st, 2008. In this time period, many people helped me to accomplish my PhD dissertation.

First of all I am grateful to Prof. Jörg Hacker, who gave me the opportunity to join his group and for the whole support that allowed me to complete my PhD.

Secondly, I am very happy that Prof. Catharina Svanborg (Institute of Laboratory Medicine, Department of Microbiology, Immunology and Glycobiology, Lund University, Sweden) gave me the opportunity to stay in her laboratory and learn more about asymptomatic bacteriuria. By her extraordinary scientific input she made my work very interesting and ‘colourful’.

I kindly thank Prof. Björn Wullt (Department of Urology, Lund University Hospital, Sweden) for providing with consecutive re-isolates of strain 83972 and the corresponding patient data.

Every scientific and non-scientific discussion, we had together, was just fantastic.

I would love to express my deepest gratitude to Dr. Ulrich Dobrindt who is a great supervisor.

In the time of my PhD, he has helped me to develop scientific ideas and together we drilled into the depths of science. Despite his very tight schedule, we could go through all crazy things....and that he polished his polish...

Many thanks to all ‘Colis’ and ‘Staphis’, especially to Barbara Plaschke, whose experience and engagement significantly contributed to my work.

Torben – thanks for the “Zusammenfassung” and all that happy times.

In addition, to all ‘Lunds’ friends, especially Mattias Gustafsson, Bryndis Ragnarsdottir and Eva Ljunggren - thank you very much.

Special thanks to Hilde Merkert for many helpful advices and solving ‘unsolvable’ problems.

The German bureaucracy would be far more difficult without Elke Stahl, Claudia Borde and Wilma Samfass. Jozef handyman and his tools were also often needed in laboratory – thanks.

Lissi and Gabi from ‘die Kuche’ – thank you very much and sorry for that ‘difficult material’ to be sometimes autoclaved.

Importantly, special thanks to my parents who gave me the opportunity to study and for the whole help – DZIEKUJE RODZICE!

And finally, thank you Kerstin for being there for me, and all support and love I have got from you!

–  –  –

TABLE OF CONTENT 

1.  SUMMARY 

1.  ZUSAMMENFASSUNG 

2.  INTRODUCTION

2.1. EPIDEMIOLOGY OF URINARY TRACT INFECTION 

2.2. ESCHERICHIA COLI AS A PATHOGEN 

2.2.1. Virulence factors of uropathogenic E. coli 

Adhesins 

Flagella 

Toxins 

Iron acquisition systems 

O‐, K‐antigens and serum resistance 

.

2.3. ASYMPTOMATIC BACTERIURIA (ABU) 

2.3.1. UTI versus ABU 

2.3.2. Escherichia coli strain 83972: a model ABU E. coli isolate 

2.4. MECHANISMS OF PATHOGEN RECOGNITION 

2.5. NITRIC OXIDE ‐ A HOST DEFENCE MECHANISM 

2.6. GENOME PLASTICITY AND BACTERIAL EVOLUTION 

2.7. BACTERIAL POPULATION DYNAMICS 

.

2.8. AIMS OF THIS WORK 

.

3.  MATERIAL 

3.1.  STRAINS 

3.2.  PLASMIDS 

3.3.  OLIGONUCLEOTIDES 

.

3.4.  CHEMICALS AND ENZYMES 





3.5.  MEDIA, AGAR PLATES AND ANTIBIOTICS 

3.5.1. Media 

3.5.2. Agar plates 

3.5.3. Antibiotics

3.5.4. DNA Markers 

3.6.  TECHNICAL EQUIPMENT 

4.  METHODS 

4.1.  WORKING WITH DNA 

4.1.1. Isolation of chromosomal DNA 

4.1.2. Precipitation of DNA with alcohol 

4.1.3. Determination of nucleic acid concentration and quality control 

4.1.4. Polymerase chain reaction (PCR) 

.

Standard PCR 

.

PCR with proof‐reading polymerases 

Box PCR 

Triplex PCR 

Multiplex PCR 

Inverse PCR (IPCR) 

4.1.5. Sequence analysis 

.

4.1.6. Multi locus sequence typing (MLST) 

4.1.7. Isolation of plasmids 

4.1.8. Enzymatic digest of DNA with restriction nucleases 

4.1.9. Horizontal gel electrophoresis 

4.1.10. Plus Field Gel Electrophoresis (PFGE) 

4.1.11. Restriction of high molecular weight DNA 

4.1.12. Separation of restriction fragments by gel electrophoresis 

4.1.13. Isolation of DNA fragments from agarose gels 

4.1.14. Ligation of DNA fragments 

4.1.15. Preparation of electrocompetent cells and electroporation 

Content   4.1.16 Gene inactivation by λ Red recombinase‐mediated mutagenesis using linear DNA fragments ........ 54  4.1.17. Southern Blot analysis 

Vacuum blotting 

Probe labelling (ECLTM Kit, Amersham Biosciences) 

Hybridization and detection of the membrane 

4.1.18. Comparative Genome Hybridization 

Membrane Layout 

Probes synthesis

Hybridization and detection 

Quantification of hybridization signals 

.

Data analysis 

4.2. WORKING WITH RNA 

4.2.1. Isolation of total RNA with RNAeasy Kit 

4.2.2. Removal of contaminating DNA by DNase treatment and RNA cleanup 

4.2.3. Reverse transcription (RT) for cDNA synthesis 

4.2.4. Quantitative Real‐Time PCR 

4.2.5. Expression profiling using DNA arrays 

Array Layout 

RNA isolation and cDNA labelling 

.

Array pre‐hybridisation 

Array hybridisation 

Post‐Hybridization washing 

.

Scanning 

Data analysis 

4.3. 2D PROTEIN GEL ELECTROPHORESIS

4.3.1. Isolation of intracellular proteins and rehydration 

4.3.2. Isolation of outer membrane proteins and rehydration 

4.3.3. Determination of protein concentrations 

4.3.4. Protein rehydration 

4.3.5. Isoelectric focusing (IEF) 

4.3.6. Equilibration 

4.3.7. Second dimension ‐ separation based on size 

4.3.8. Proteins staining 

.

4.3.9. Analysis of 2‐D Gels with the Delta‐2D® Software (Decodon) 

4.3.10. Protein identification by MALDI‐TOF‐MS 

4.4. ANALYSIS OF LIPOPOLYSACCHARIDES (LPS) 

4.4.1. Isolation of LPS 

4.4.2. Electrophoresis and staining with silver nitrate 

4.5. PHENOTYPIC ASSAYS 

4.5.1. Detection of type 1 fimbrial expression 

4.5.2. Detection of F1C and P fimbrial expression 

4.5.3. Detection of secreted α‐hemolysin 

4.5.4. Detection of biofilm forming abilities 

In M63 defined media 

In pooled, sterile human urine 

4.6. CONTINOUS CULTURE OF E. COLI IN MICROFERMENTERS 

.

4.7. IN SILICO ANALYSIS 

5. RESULTS 

5.1. DIVERSITY OF CLINICAL ABU E. COLI ISOLATES 

5.1.1. Analysis of relatedness of different ABU isolates 

5.1.2. Comparative Genomic Hybridization (CGH) 

5.1.3. Genomic fingerprints of different ABU isolates 

5.1.4. Genome size of different ABU isolates 

5.2. PHENOTYPIC VS. GENOTYPIC CHARACTERISTICS OF ABU E. COLI ISOLATES 

5.2.1. Type 1 fimbriae 

.

5.2.2. P fimbriae 

5.2.3. F1C fimbriae 

Content   5.2.4. Expression of α‐hemolysin 

5.2.5. LPS O side chain expression 

5.2.6. Biofim formation 

5.2.7. Growth characteristics of ABU E. coli isolates 

5.3. ADAPTIVE FLEXIBILITY AND GENOME PLASTICITY OF MODEL STRAIN 83972 

5.3.1. Patient colonization

5.3.2. Patients’ immune response upon colonization with strain 83972

5.3.3. Verification of the re‐isolates 

5.3.4. Genome structure of in vivo 83972 re‐isolates 

5.3.5. Phenotypes of different in vivo re‐isolates 

Motility 

Growth characteristics 

Competitiveness 

.

Biofilm formation 

5.3.6. Host independent growth of E. coli strain 83972 

5.3.7. Genomic and phenotypic properties of ABU strain 83972 grown in vitro 

Genetic structure of the in vitro 83972 re‐isolates 

Motility 

Growth characteristics 

Biofilm formation 

5.4. TRANSCRIPTOME ANALYSIS OF 83872 RE‐ISOLATES

5.4.1. Significant changes in the expression pattern 

5.4.2. Individual adaptation of re‐isolates 

Transcriptome changes of in vitro re‐isolate 4.9 relative to parent strain 83972 

Transcriptome changes of in vivo re‐isolate SR12 relative to parent strain 83972 

Transcriptome changes of in vivo re‐isolates KA25 and CK12 relative to parent strain 83972 

5.4.3. Common adaptive patterns in re‐isolates 

5.4.4. Verification of microarray results by quantitative RT‐PCR 

5.5. CHANGES IN THE CYTOPLASMIC PROTEIN EXPRESSION OF THE 83972 RE‐ISOLATES 

5.5.1. Cytoplasmic proteome changes of in vivo re‐isolate KA25 relative to parent strain 83972 .............. 126  5.5.2. Cytoplasmic proteome changes of in vivo re‐isolate SR12 relative to parent strain 83972 .............. 128  5.5.3. Cytoplasmic proteome changes of in vivo re‐isolate CK12 relative to parent strain 83972 .............. 130 

5.6. OUTER MEMBRANE PROTEOME CHANGES OF THE IN VIVO RE‐ISOLATES OF STRAIN 83972. 

6. DISCUSSION 

6.1. ASYMPTOMATIC BACTERIURIA IS CAUSED BY A HETEROGENEOUS GROUP OF E. COLI ISOLATES 

.

6.2. IMPAIRED ABILITY OF ABU ISOLATES TO EXPRESS TYPICAL UPEC VIRULENCE FACTORS 

6.3. GENOME REDUCTION AND EVOLUTION OF ST 73 ABU STRAINS 

.

6.4. HOST IMMUNE RESPONSE DURING BACTERIAL COLONISATION 

.

6.5. HOST‐BACTERIUM INTERACTIONS 

6.5.1. Bacterial variability and host response 

6.5.2. Flagella expression / motility 

6.5.3. Biofilm formation 

6.5.4. Growth characteristics 

6.6. METABOLIC ACTIVITY OF ABU ISOLATES 

.

6.7. OUTER MEMBRANE PROTEIN PROFILE AND IRON UPTAKE 

6.8. HOST DEFENCE‐DRIVEN BACTERIAL GENE EXPRESSION 

6.9. IMPLICATIONS AND OUTLOOK 

7. REFERENCES 

8. APPENDIX 

8.1. LEGENDS TO FIGURES AND TABLES 

.

8.2. EXPRESSION PROFILING DATA 

8.3. CURRICULUM VITAE 

8.4. PUBLICATIONS 

8.5. ABBREVIATIONS 

Summary  

1. Summary 

Asymptomatic bacteriuria (ABU) represents the long term bacterial colonization of the urinary tract, frequently caused by Escherichia coli (E. coli), without typical symptoms of a urinary tract infection (UTI). To investigate characteristics of ABU E. coli isolates in more detail, the geno- and phenotypes of eleven ABU isolates have been compared. Moreover, consecutive in vivo re-isolates of the model ABU strain 83972 were characterized with regard to transcriptomic, proteomic and genomic alterations upon long term in vivo persistence in the human bladder. Finally, the effect of the human host on bacterial adaptation/evolution was assessed by comparison of in vitro and in vivo-propagated strain 83972.

ABU isolates represent a heterologous group of organisms. The comparative analysis of different ABU isolates elucidated the remarkable genetic and phenotypic flexibility of E. coli isolates. These isolates could be allocated to all four major E. coli phylogenetic lineages as well as to different clonal groups. Accordingly, they differed markedly in genome content, i.e., the genome size as well as the presence of typical UPEC virulence-associated genes.



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