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Analysis of adaptation strategies of uropathogenic Escherichia coli during growth in the bladder

Die häufigste Form von Harnwegsinfektionen, hauptsächlich verursacht durch Bakterien der Spezies Escherichia coli, ist die asymptomatische Bakteriurie (ABU). Diese ist trotz hoher Bakterienzahl im Urin durch die Abwesenheit klinischer Symptome charakterisiert. Die molekularen Mechanismen, die es E. coli Stämmen ermöglichen den menschlichen Harnweg ohne eine signifikante Wirtsantwort zu kolonisieren, sind bisher weitgehend unbekannt. Eine Anpassung der Virulenz durch Genomreduzierung scheint ein evolutionäres Prinzip zur Etablierung eines kommensalen Lebensstils darzustellen. Die vorliegende Arbeit untersucht das Zweikomponentensystem (TCS) BarA/UvrY ABU-assoziierter E. coli Stämme. Es zeigt sich, dass uvrY einer positiven Selektion während des Wachstums in den Harnwegen unterliegt und dass Mutationen in uvrY zu einem Wachstumsvorteil in Urin führen. Die Studien zeigen, dass ein Funktionsverlust des TCS BarA/UvrY zu einem selektiven Vorteil für Zellen bei Langzeitwachstum in Urin führt.

Titel: Analysis of adaptation strategies of uropathogenic Escherichia coli during growth in the bladder
Übersetzter Titel Untersuchung von Anpassungsstrategien uropathogener Escherichia coli bei Wachstum in der Harnblase
Verfasser: Ewert, Birgit GND
Gutachter: Dobrindt, Ulrich GND
Organisation: FB 13: Biologie
FB 05: Medizinische Fakultät
Dokumenttyp: Dissertation/Habilitation
Medientyp: Text
Erscheinungsdatum: 2015
Publikation in MIAMI: 02.07.2015
Datum der letzten Änderung: 27.07.2015
Schlagwörter: Harnwegsinfektion; Escherichia coli; ABU; Zweikomponentensystem; TCS BarA/UvrY
Fachgebiete: Biowissenschaften; Biologie; Medizin und Gesundheit
Sprache: Englisch
Format: PDF-Dokument
URN: urn:nbn:de:hbz:6-59219634218
Permalink: https://nbn-resolving.org/urn:nbn:de:hbz:6-59219634218
Onlinezugriff:
Inhalt:
List of Figures ................................................................................................................................ VI
List of Tables ..................................................................................................................................IX
List of Abbreviations ........................................................................................................................X
Summary ......................................................................................................................................... 1
Zusammenfassung ........................................................................................................................... 2
1 Introduction ............................................................................................................................. 4
1.1 Escherichia coli – a bacterial species of clinical relevance ...................................................... 4
1.2 Urinary tract infections ........................................................................................................... 5
1.3 Uropathogenic E. coli – the main causative of UTI ................................................................. 7
1.3.1 Virulence factors ............................................................................................................. 8
1.4 Asymptomatic bacteriuria (ABU) .......................................................................................... 10
1.4.1 E. coli 83972 .................................................................................................................. 11
1.5 Genome plasticity and bacterial adaptation ......................................................................... 11
1.6 Two‐component systems ...................................................................................................... 13
1.6.1 TCS BarA/UvrY ............................................................................................................... 16
1.6.2 Role of the carbon storage regulator system ................................................................ 18
1.7 Metabolism ........................................................................................................................... 20
1.7.1 Glycolysis ....................................................................................................................... 20
1.7.2 Gluconeogenesis ........................................................................................................... 21
1.7.3 Pentose Phosphate shunt ............................................................................................. 22
1.7.4 Tricarboxylic Acid Cycle (TCA) ....................................................................................... 22
1.7.5 Mixed Acid Fermentation .............................................................................................. 22
1.8 Aims of the study .................................................................................................................. 23
2 Material ................................................................................................................................. 25
2.1 Bacterial strains ..................................................................................................................... 25
2.2 Plasmids ................................................................................................................................. 27
2.3 Oligonucleotides .................................................................................................................... 27
2.4 Chemicals, enzymes and kits ................................................................................................. 29
2.5 Buffers and solutions ............................................................................................................. 30
2.6 Media and agar plates ........................................................................................................... 30
2.6.1 Media ............................................................................................................................ 30
2.6.2 Agar plates ..................................................................................................................... 31
2.7 Antibiotics ............................................................................................................................. 32
2.8 Antibodies ............................................................................................................................. 32
2.9 DNA / protein size markers ................................................................................................... 32
2.10 Technical equipment ............................................................................................................. 33
3 Methods ................................................................................................................................ 35
3.1 Bacterial cultures ................................................................................................................... 35
3.1.1 Cultivation ..................................................................................................................... 35
3.1.2 Bacterial preservation ................................................................................................... 35
3.1.3 Bacterial growth ............................................................................................................ 35
3.2 Molecular biological methods ‐ DNA .................................................................................... 35
3.2.1 Extraction of genomic DNA ........................................................................................... 35
3.2.2 Photometric determination of nucleic acid concentration ........................................... 36
3.2.3 Polymerase chain reaction ............................................................................................ 36
3.2.4 Analysis of PCR fragments ............................................................................................. 38
3.2.5 Gel extraction ................................................................................................................ 39
3.2.6 Ethanol precipitation of DNA ........................................................................................ 39
3.2.7 PCR clean‐up .................................................................................................................. 39
3.2.8 DNA sequencing ............................................................................................................ 39
3.2.8.1 PCR purification by EXOSAP ...................................................................................... 40
3.2.8.2 Sequencing protocol.................................................................................................. 40
3.2.9 Preparation of electrocompetent cells ......................................................................... 40
3.2.10 Transformation of electrocompetent cells ................................................................... 41
3.2.11 Isolation of plasmids ..................................................................................................... 41
3.2.12 Enzymatic hydrolysis of DNA ......................................................................................... 41
3.2.13 Ligation of DNA fragments ............................................................................................ 42
3.2.14 Colony PCR .................................................................................................................... 42
3.2.15 Dot blot .......................................................................................................................... 42
3.2.15.1 Vacuum Blotting .................................................................................................... 42
3.2.15.2 Probe labeling ........................................................................................................ 43
3.2.15.3 Hybridization and detection .................................................................................. 43
3.2.16 Southern blot................................................................................................................. 44
3.2.16.1 Capillary Blotting ................................................................................................... 44
3.2.16.2 Probe labeling, hybridization and detection ......................................................... 44
3.3 Construction of uvrY allelic variants ...................................................................................... 45
3.3.1 Site‐specific recombination by recombineering according to Datsenko & Wanner ..... 45
3.3.2 Genetic modification by a counter‐selection system .................................................... 46
3.3.3 P1 mediated transduction ............................................................................................. 48
3.4 Molecular biological methods ‐ RNA ..................................................................................... 49
3.4.1 RNA Isolation ................................................................................................................. 49
3.4.1.1 RNA isolation from urine ........................................................................................... 49
3.4.1.2 RNA isolation from LB ............................................................................................... 49
3.4.2 DNA digestion ................................................................................................................ 49
3.4.3 RNA quality check .......................................................................................................... 50
3.4.4 Reverse transcription (RT) for cDNA synthesis ............................................................. 50
3.4.5 Quantitative real‐time PCR ........................................................................................... 51
3.5 Phenotypic characterization ................................................................................................. 52
3.5.1 Growth kinetics ............................................................................................................. 52
3.5.2 Biofilm assay .................................................................................................................. 52
3.5.3 Motility assay ................................................................................................................ 53
3.5.4 Congo Red staining assay .............................................................................................. 53
3.5.5 Calcoflour assay ............................................................................................................. 53
3.5.6 In vitro competition assay ............................................................................................. 53
3.5.7 Sub‐cultivation assay ..................................................................................................... 54
3.6 Protein biochemical methods ............................................................................................... 54
3.6.1 Isolation of proteins – whole cell lysate ........................................................................ 54
3.6.1.1 BCA quantification of protein concentration ............................................................ 54
3.6.2 Tricine‐SDS‐Page ........................................................................................................... 55
3.6.3 Coomassie staining ........................................................................................................ 56
3.6.4 Western blot .................................................................................................................. 56
3.6.4.1 Transfer of proteins ‐ `semi‐dry blot` ........................................................................ 56
3.6.4.2 Immune detection ..................................................................................................... 57
3.6.4.3 ECL detection ............................................................................................................. 57
3.7 Metabolomics ........................................................................................................................ 58
3.7.1 Metabolome sample preparation ................................................................................. 58
3.8 In silico analysis ..................................................................................................................... 59
3.9 Statistics ................................................................................................................................ 59
4 Results ................................................................................................................................... 60
4.1 Extracellular metabolome ..................................................................................................... 60
4.2 Limitations for growth in urine ............................................................................................. 65
4.3 Stress response to growth in urine ....................................................................................... 66
4.4 Sequence variation in different groups of E. coli isolates ..................................................... 69
4.4.1 Amplification and sequencing of barA/uvrY ................................................................. 69
4.4.2 BarA/ UvrY sequence analysis ....................................................................................... 74
4.5 Phylogenetic classification .................................................................................................... 85
4.6 Test of selection – McDonald‐Kreitman Test ........................................................................ 87
4.7 SNP combination analysis ..................................................................................................... 90
4.8 Phenotypic characterization ................................................................................................. 91
4.8.1 Biofilm ........................................................................................................................... 91
4.8.2 Synthesis of curli fimbriae and cellulose expression ..................................................... 93
4.8.3 Motility assay ................................................................................................................ 95
4.8.4 Growth characteristics .................................................................................................. 97
4.9 Expression of csrB.................................................................................................................. 97
4.10 Impact analysis of non‐synonymous SNPs in uvrY – allelic variants ................................... 100
4.10.1 Selection of uvrY alleles ............................................................................................... 100
4.10.2 Construction of uvrY allelic variants ............................................................................ 101
4.10.3 Phenotypic assays ....................................................................................................... 103
4.10.3.1 Biofilm ................................................................................................................. 103
4.10.3.2 Expression of curli, cellulose & swarming motility .............................................. 103
4.10.3.3 Growth characteristics ........................................................................................ 106
4.10.3.4 Competition assays ............................................................................................. 107
4.10.3.5 Sub‐cultivation assay ........................................................................................... 112
4.10.4 Real‐Time PCR ............................................................................................................. 113
5 Discussion ............................................................................................................................ 117
5.1 Basic metabolomics vs. adapted metabolome ................................................................... 117
5.2 Carbon – a limiting factor for growth in urine .................................................................... 122
5.3 Stress response is growth medium‐dependent .................................................................. 124
5.4 Sequence context variation of barA and uvrY ..................................................................... 127
5.5 Sequence variability of barA and uvrY ................................................................................ 128
5.6 Correlation of SNPs and phylogeny ..................................................................................... 131
5.7 Detection of adaptive evolution for barA and uvrY ............................................................ 131
5.8 Impact of individual barA/uvrY alleles on bacterial phenotypes ........................................ 133
5.8.1 Biofilm formation ........................................................................................................ 133
5.8.2 Expression of components of the extracellular matrix ............................................... 134
5.8.3 Motility ........................................................................................................................ 134
5.8.4 Growth ........................................................................................................................ 135
5.9 Gene expression of csrB ...................................................................................................... 136
5.10 Selection of uvrY allelic variants .......................................................................................... 136
5.11 Phenotypic variations suggest “loss‐of‐function” as an adaptive trait ............................... 138
5.11.1 Biofilm formation ........................................................................................................ 138
5.11.2 Expression of components of the extracellular matrix ............................................... 139
5.11.3 Motility ........................................................................................................................ 140
5.11.4 Growth kinetics ........................................................................................................... 141
5.12 Competition assay ............................................................................................................... 142
5.13 Sub‐cultivation .................................................................................................................... 143
5.14 Mutated uvrY exhibits impact on the Csr system ............................................................... 144
5.14.1 Comparison of the expression of components of the Csr system under standard
laboratory conditions .................................................................................................................. 144
5.14.2 Comparison of the expression of components of the Csr system in urine ................. 146
6 Conclusions and outlook ...................................................................................................... 148
7 Appendix .................................................................................................................................. i
7.1 Supplements .............................................................................................................................. i
7.1.1 Amplification and sequencing of barA/uvrY ..................................................................... i
7.1.2 Evolutionary rates ............................................................................................................ ii
7.1.2.1 UvrY .............................................................................................................................. ii
7.1.2.2 BarA ............................................................................................................................. iii
7.1.3 Biofilm ............................................................................................................................. xi
7.1.4 Summary of the phenotypic assays ............................................................................... xiii
7.2 References ............................................................................................................................. xvi
7.3 Publications ........................................................................................................................ xxxiii
7.4 Presentations ..................................................................................................................... xxxiii
7.5 Curriculum vitae ................................................................................................................. xxxiv