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Functional genome analysis of the intracellular lifestyle of Pseudomonas aeruginosa in phagocytes [Elektronische Ressource] / von Prabhakar Salunkhe

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Functional genome analysis of the intracellularlifestyle of Pseudomonas aeruginosa in phagocytesDem Fachbereich Chemie der Universität Hannover zur Erlangung des Grades Doktor der NaturwissenschaftenDr. rer.nat.genehmigte DissertationvonMasters in Biochemistry Prabhakar SalunkheBorn on June 01, 1974 in Kalkheda, India (MS)Hannover 2003Die vorliegende Arbeit wurde in der Klinischen Forschergruppe MolekularePathologie der Mukoviszidose, Zentrum Biochemie und Zentrum Kinderheilkunde derMedizinischen Hochschule Hannover in der Zeit vom 01.10.2000 bis zum 30.09.2003unter der Leitung von Prof. Dr. Dr. Burkhard Tümmler angefertigt.Tag der Promotion: 19.11.2003Referent: Prof. Dr. Dr. Burkhard TümmlerKlinische Forschergruppe OE 6711Zentrum Biochemie und Zentrum KinderheilkundeMedizinische Hochschule HannoverKorreferent: Prof. Dr. Peter Valentin-WeigandInstitut für Mikrobiologie Zentrum für Infektionsmedizin Tierärztliche Hochschule HannoverAcknowledgementsAcknowledgementsI would like to express my deep and sincere gratitude to my supervisor, Prof. Dr.Burkhard Tümmler, of Klinische Forschergruppe, Medizinische HochschuleHannover, for giving me the opportunity to commence this thesis. He has a sharp eyefor details and scientific knowledge, these have been instrumental in the success ofthe thesis.

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Published 01 January 2003
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Functional genome analysis of the intracellular
lifestyle of Pseudomonas aeruginosa in phagocytes
Dem Fachbereich Chemie der Universität Hannover
zur Erlangung des Grades
Doktor der Naturwissenschaften
Dr. rer.nat.
genehmigte Dissertation
von
Masters in Biochemistry Prabhakar Salunkhe
Born on June 01, 1974 in Kalkheda, India (MS)
Hannover 2003Die vorliegende Arbeit wurde in der Klinischen Forschergruppe Molekulare
Pathologie der Mukoviszidose, Zentrum Biochemie und Zentrum Kinderheilkunde der
Medizinischen Hochschule Hannover in der Zeit vom 01.10.2000 bis zum 30.09.2003
unter der Leitung von Prof. Dr. Dr. Burkhard Tümmler angefertigt.
Tag der Promotion: 19.11.2003
Referent: Prof. Dr. Dr. Burkhard Tümmler
Klinische Forschergruppe OE 6711
Zentrum Biochemie und Zentrum Kinderheilkunde
Medizinische Hochschule Hannover
Korreferent: Prof. Dr. Peter Valentin-Weigand
Institut für Mikrobiologie
Zentrum für Infektionsmedizin
Tierärztliche Hochschule HannoverAcknowledgements
Acknowledgements
I would like to express my deep and sincere gratitude to my supervisor, Prof. Dr.
Burkhard Tümmler, of Klinische Forschergruppe, Medizinische Hochschule
Hannover, for giving me the opportunity to commence this thesis. He has a sharp eye
for details and scientific knowledge, these have been instrumental in the success of
the thesis.
I am thankful to the Deutsche Forschungsgemeinschaft (DFG)-sponsored European
Graduate College “Pseudomonas: Pathogenicity and Biotechnology“ for providing
funding for my research throughout this Ph. D. work from October 2000 to September
2003.
I would like to express my warmest thanks to my colleagues Dr. Lutz Wiehlmann and
Dr. Franz von Goetz who taught me the basic techniques in STM and Microarray
experiments respectively.
My project acquired different expert assistance from different scientific groups. I wish
to thank Prof. Dr. Niels Hoiby, University Hospital of Copenhagen (Rigshospitalet),
Denmark, for providing me the lab facility and Dr. Claus Moser for scientific
supervision during the mouse experiments. I am also thankful to Peter Jensen, Kenji
Kishi, Thomas Bjarnsholt and Lars Christophersen for their assistance during the
mouse experiments. I warmly thank Dr. L. Eberl (Munich) for quorum sensing
experiments, Dr. G. Brandes (MHH, Hannover) for electron microscopy and Dr. I.
Attree (Grenoble) for cytotoxicity experiments carried out during this thesis. I could
not forget Dr. Joerg Lauber for his valuable assistance in the establishment of gene
expression analysis by using Affymetrix microarrays and Tanja Toepfer for her
experimental support during microarray hybridization.
I thank all the members who donated blood for these experiments otherwise it would
have been difficult to carry out this study.
My Ph. D. study was carried out at the Klinische Forschergruppe, Medizinische
Hochschule Hannover. I wish to thank all the members and ex-members of the groupAcknowledgements
for their motivation, support, advice and many helpful discussions and for maintaining
excellent research environment.
The support provided by Cystic Fibrosis Therapeutics Inc. (USA) and the
Mukoviszidose e.V. (Germany) for subsidized Pseudomonas aeruginosa PAO1
genome arrays is gratefully acknowledged.
I have enjoyed many Ph. D. workshops, summer schools, scientific seminars and
social events arranged by the Co-ordinator of the European Graduate College. I
extend my appreciation to all the EGK members for the scientific discussions during
the summer schools and monthly seminars.
Finally I must realize that the achievements I got were closely linked with the help
from my family, my wife Kavita Salunkhe, my parents Latabai and Budha Salunkhe,
uncle Shivaji Salunkhe. They gave me not only love but also caring, understanding,
encouragement and support throughout the studies.
Once again, thank all of you!
Prabhakar Salunkhe, October, 2003
Hannover, GermanyAbstract
Abstract
The objectives of the present study were to identify genes that enable the opportunistic
pathogen P. aeruginosa TB to survive intracellularly in polymorphonuclear granulocytes
(PMNs). A signature-tagged mutant (STM) library made up of 2304 mutants was screened
for intracellular survival in PMNs. Out of the identified 44 mutants, 35 mutants which have
reduced survival significantly, while 9 mutants which survived even better as compared to the
TB wild type. The genes identified encode elements of oxidative stress response, flagella
and type IV pili biogenesis. Most of them are belong to the class of conserved hypotheticals
of unknown function. Almost all genes that are needed by TB are present in the sequenced
PAO1 core genome, only very few are strain-or clone-specific. This indicates that subtle
shifts in the genetic repertoire are sufficient to cause a smooth transition from extracellular
lifestyle to intracellular survival and growth in the major antipseudomonal host defense cell.
The genome wide transcriptional profile of P. aeruginosa against paraquat was performed
using PAO1 GeneChips which uncovered one operon (PA0939-PA0942) encoding four
proteins of previously unknown function being 20-200 fold up-regulated in all three analyzed
P. aeruginosa strains TB, 892 and PAO1. It confers most resistance to paraquat-induced
superoxide stress in addition to constitutively highly expressed genes sodB, katA and ahpC.
The transcriptional profile of the three P. aeruginosa strains against hydrogen peroxide
stress identified significantly more number of genes up-regulated in TB as compared to 892
and PAO1.
The GeneChip experiments with the P. aeruginosa TB mutants harboring transposon
insertions in strain specific genes revealed that there were virtually no interference with the
expression of the PAO1 defined core genome under standard growth conditions. Murine
infection experiments indicated that the analyzed mutants are even more virulent than the TB
wild type.
The transcriptome data together with murine infection model described in this study could be
useful for experiments to dissect the intracellular survival mechanisms in P. aeruginosa TB
and the biochemical function of the strain specific genes.
Key-words: Pseudomonas aeruginosa, Polymorphonuclear granulocytes, GeneChip Zusammenfassung
Zusammenfassung
Das Ziel dieser Arbeit war die Identifizierung von Genen, die es dem opportunistischen
Pathogen P. aeruginosa TB ermöglichen, intrazellulär in polymorphonukleären Granulozyten
(PMNs) zu überleben. 2304 Transposonmutanten einer STM- Bibliothek wurden auf ihre
intrazelluläre Überlebensfähigkeit in PMNs untersucht. Hierbei wurden 44 Mutanten mit
deutlichen Veränderungen des Phänotyps identifiziert. 35 hatten eine deutlich verringerte
intrazelluläre Überlebensfähigkeit, wogegen 9 Mutanten sogar besser überlebten als der TB-
Wildtyp. Die Funktionen der identifizierten Gene beinhalten die Abwehr von oxidativem
Stress, den Aufbau von Flagellen und Typ IV Pili. Die meisten der identifizierten Gene haben
aber bisher eine unbekannte Funktion. Es war auffällig, daß die meisten Gene, die von P.
aeruginosa TB zum intrazellulären Überleben benötigt werden, aus dem sequenzierten
PAO1 – Genom bekannt sind und nur sehr wenige Gene Stamm- oder Klon- spezifisch sind.
Dies ist ein Hinweis darauf, daß nur geringste Veränderungen im genetischen Hintergrund
ausreichend sind, um einen Übergang von einer extrazellulären Lebensweise zu einer
intrazellulären auszulösen, bei der die P. aeruginosa Bakterien sogar in der Lage sind, sich
in der wichtigsten Abwehrzelle des Menschen gegenüber Pseudomonas-Infektionen zu
vermehren.
In PAO1 GeneChips wurde das genomweite Transkriptionsprofil von P. aeruginosa nach
Exposition mit Paraquat analysiert. Ein Operon aus vier Genen (PA0939-PA0942) mit bisher
unbekannter Funktion wurde in den drei analysierten Stämmen TB, 892 und TB 20-200fach
stärker exprimiert. Es verleiht neben den konstitutiv hoch exprimierten Gene sodB, katA und
ahpC den größten Anteil an Resistenz gegen Paraquat-induzierten Superoxid Stress. Das
transkriptionelle Profil der drei P. aeruginosa Stämme während Wasserstoffperoxid-
induziertem Stress zeigte signifikant mehr hochregulierte Gene bei TB als bei 892 und
PAO1.
GeneChip Experimente mit zwei P. aeruginosa TB Mutanten, die Transposon-Insertionen in
non-PAO1 Genen beherbergen, zeigten praktisch keine Interferenz mit der globalen
Expression des PAO1 Kerngenoms unter Standard-Wachstumsbedingungen. Die Produkte
dieser beiden Gene schützen P. aeruginosa TB vor dem bakteriziden Zugriff durch PMNs,,
attenuieren aber gleichzeitig die Virulenz im Mäuseinfektionsversuch.
Die Transkriptom-Daten können zusammen mit den in dieser Studie beschriebenen
Mausinfektions-Versuchen nützlich für gezielte Experimente sein, um die intrazellulären
Überlebensmechanismen von P. aeruginosa TB sowie die biochemischen Funktionen der
Stamm-spezifischen Gene aufzuklären.
Schlüsselwörter: Pseudomonas aeruginosa, polymorphnukleäre Granulozyten, GeneChipTable of contents
Table of contents
1. Introduction____________________________________________________ 1
1.1. Pseudomonas aeruginosa............................................................................ 1
1.1.1. P. aeruginosa and cystic fibrosis........................................................ 2
1.1.2. Pathogenesis of P. aeruginosa infections.......................................... 3
1.1.2.1. Role of cell-associated virulence factors............................... 3
1.1.2.1. Role of extracellular virulence factors....................................4
1.2. Type III secretion system.............................................................................. 6
1.2.1. Proteins secreted by P. aeruginosa type III pathway......................... 7
1.2.2. P. aeruginosa type III secretion system genes...................................8
1.2.3. Transcriptional regulation in P. aeruginosa........................................ 8
1.3. Quorum sensing in Gram-negative bacteria................................................. 9
1.3.1. What is quorum sensing......................................................................9
1.3.2. Quorum sensing systems in P. aeruginosa....................................... 10
1.4. Intracellular lifestyle of bacterial pathogens................................................12
1.4.1. P. aeruginosa TB:Survival in PMN.................................................... 15
1.5. Methods for functional genome analysis.....................................................18
1.5.1. In vivo expression technology............................................................18
1.5.2. Differential fluorescence induction.....................................................19
1.5.3. Signature tagged mutagenesis..........................................................19
1.5.4. Differential display............................................................................. 20
1.5.5. DNA microarray technology...............................................................21
1.6. P. aeruginosa pathogenicity in murine infection model.............................. 22
1.7. Objectives of the present investigation.......................................................23
2. Materials and methods__________________________________________ 26
2.1. Materials..................................................................................................... 26
2.1.1. Equipments........................................................................................26
2.1.2. Consumables.....................................................................................26
2.1.3. Chemicals and enzymes................................................................... 27
2.1.4. Media and solutions...........................................................................28
2.1.4.1. Media................................................................................... 28
2.1.4.2. Solutions for DNA work........................................................29
2.1.4.3. Solutions for RNA work........................................................32
2.1.4.4. Solutions for animal experiments.........................................35
ITable of contents
2.1.5. Bacterial cultures............................................................................... 35
2.1.5.1. Escherichia coli DH5α......................................................... 35
2.1.5.2. P. aeruginosa.......................................................................36
2.1.5.3. P. putida...............................................................................36
2.2. Methods...................................................................................................... 36
2.2.1. Microbiological methods.................................................................... 37
2.2.1.1. Bacterial growth conditions..................................................37
2.2.1.2. Determination of bacterial cell density................................. 37
2.2.1.3. Maintenance of bacterial cultures........................................ 37
2.2.2. Separation of DNA.............................................................................37
2.2.2.1. Agarose gel electrophoresis................................................ 37
2.2.2.2. Polyacrylamide gel electrophoresis..................................... 38
2.2.3. Transformation.................................................................................. 38
2.2.3.1. Generation of transformation competent cells..................... 38
2.2.3.2. Transformation by heat shock method................................ 38
2.2.4. DNA isolation.................................................................................... 39
2.2.4.1. Isolation of genomic DNA from Gram-negative bacteria..... 39
2.2.4.2. Isolation of plasmid DNA..................................................... 39
2.2.4.3. Quantitation of DNA and RNA.......................................... 40
2.2.5. Polymerase chain reaction................................................................ 41
2.2.5.1. Construction of primers....................................................... 41
2.2.5.2. PCR reaction....................................................................... 41
2.2.5.3. PCR parameters.................................................................. 42
2.2.6. Restriction digestion of DNA............................................................. 43
2.2.7. Plasmid rescue.................................................................................. 44
2.2.8. Generation of DIG-labeled probe...................................................... 44
2.2.8.1. Isolation of DNA fragments from agarose............................44
2.2.8.2. Separation of small DNA fragments.................................... 45
2.2.8.3. Random primer labeling by DIG-DNA labeling kit................46
2.2.8.4. DIG-3‘ end labeling of oligonucleotides............................... 47
2.2.9. DNA fixation and hybridization.......................................................... 47
2.2.9.1. Southern transfer................................................................. 47
2.2.9.2. Dot-blot preparation............................................................. 49
2.2.9.3. Southern and dot-blot hybridization..................................... 49
IITable of contents
2.2.9.4. Immunological detection of the hybridized blot....................50
2.2.9.5. Washing of Southern and dot blots......................................51
2.3. Screening of the STM library for intracellular survival in PMNs.................. 51
2.3.1. Growth of transposon mutants.......................................................... 51
2.3.2. Isolation of granulocytes from human blood......................................51
2.3.3. Determination of granulocyte concentration......................................52
2.3.4. Phagocytosis assay in granulocytes..................................................52
2.3.5. Cloning, sequencing and analysis of mutated P. aeruginosa DNA... 55
2.4. Screening of STM library for mutants defective in protease secretion........55
2.5. Characterization of STM knockout mutants................................................56
2.5.1. In silico analysis.................................................................................56
2.5.2. Sensitivity towards hydrogen peroxide.............................................. 56
2.5.3. Motility assay..................................................................................... 57
2.5.4. Cytotoxicity towards macrophage J774 cells.....................................57
2.6. Transcriptional analysis of P. aeruginosa...................................................58
2.6.1. RNA handling and storage................................................................ 58
2.6.2. P. aeruginosa PAO1 microarray........................................................58
2.6.3. Bacterial growth conditions for RNA isolation....................................59
2.6.3.1. Bacterial growth in LB medium............................................ 59
2.6.3.2. Bacterial growth in presence of superoxide and oxidative
stress generating agents...................................................................60
2.6.3.3. Bacterial growth in iron depleted LB medium...................... 60
2.6.3.4. Bacterial growth in presence of PMNs.................................61
2.6.3.5. Bacterial growth in minimal medium.................................... 61
2.6.4. RNA isolation.....................................................................................61
2.6.5. Separation of RNA by formaldehyde agarose gel electrophoresis....62
2.6.5.1. Northern transfer..................................................................64
2.6.5.2. RNA-DNA hybridization....................................................... 64
2.6.5.3. Detection of the Northern blot..............................................64
2.6.6. cDNA generation............................................................................... 65
2.6.7. Fragmentation of cDNA..................................................................... 65
2.6.8. Labeling of fragmented cDNA with biotin.......................................... 66
2.6.9. P. aeruginosa PAO1 GeneChip hybridization and washing.............. 66
2.6.10. GeneChip analyses......................................................................... 68
IIITable of contents
2.7. Examination of P. aeruginosa pathogenicity in murine infection model......69
2.7.1. Mice................................................................................................... 69
2.7.2. Bacterial inoculation and challenge procedure..................................69
2.7.2.1. Free bacteria........................................................................69
2.7.2.2. Immobilization of bacteria in alginate beads........................70
2.7.3. Infection of mice with P. aeruginosa TB............................................70
2.7.4. Evaluation of different parameters.....................................................71
2.7.4.1. Collection of peripheral blood.............................................. 71
2.7.4.2. of sera................................................................. 72
2.7.5. Detection of lung bacteriology........................................................... 72
2.7.6. Collection of bronchoalveolar lavage............................................... 72
2.7.7. Measurement of leukocytes from blood and BAL-fluid......................73
2.7.8. Estimation of leukocyte phenotypes..................................................73
2.7.9. Measurement of respiratory burst......................................................74
2.7.10. Measurement of cytokine production...............................................74
2.7.11. Flow cytometric measurements.......................................................76
2.7.12. Statistical analysis........................................................................... 76
3. Results and discussion_________________________________________ 77
3.1. Screening of STM library for intracellular survival in PMN..........................78
3.1.1. Evaluation of selection.......................................................................79
3.2. Characterization of STM knockout mutants................................................82
3.2.1. In silico characterization of P. aeruginosa TB mutants......................82
3.2.2. Oxidative stress response in P. aeruginosa TB.................................86
3.2.3. Flagellar export and synthesis machinery......................................... 88
3.2.4. Cytotoxicity towards macrophage J774 cells.....................................92
3.3. Screening of STM library for mutants defective in protease secretion........93
3.4. Transcriptional analysis of P. aeruginosa...................................................97
3.4.1. Global transcriptional analysis of P. aeruginosa strains against
superoxide and oxidative stress........................................................97
3.4.1.1. GeneChip analysis...............................................................99
3.4.1.2. Paraquat mediated superoxide stress response................101
3.4.1.3. Hydrogen peroxide mediated oxidative stress response... 109
3.4.2. Transcriptional analysis of non-PAO1 ORFs...................................122
3.4.2.1. Gene organization of non-PAO1 sequences......................122
IV