5 The role of quorum-sensing in the virulence of Pseudomonas ...

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Escherichia coli Laboratory strain Description Source MG1655 K12 (E101) Reference strain of E. coli Lab 212 E105 Clinical blood isolate from Leicester Royal Infirmary E106 Clinical blood isolate from Leicester Royal Infirmary DH10B Laboratory strain of E. coli used for transformation of plasmids 34 Dept. of Infection, Immunity and Inflammation, University of Leicester Lab 212 Dept. of Infection, Immunity and Inflammation, University of Leicester Lab 212 Dept. of Infection, Immunity and Inflammation, University of Leicester Lab 212 Dept. of Infection, Immunity and Inflammation, University of Leicester K12:leuX (KR507) This study E105:leuX (KR510) This study K12:serW (KR506) This study E106:serW This study PA01:lys10 (KR508) This study PA14:lys10 (KR509) This study KR115:lys10 (KR559) This study KR159:lys10 (KR560) This study Table 2-4 shows the Escherichia coli strains used and created during this study Saccharomyces cerevisiae Laboratory strain Description Source CRY1-2 Mating type (MATα), cycloheximide resistant (cyh2 R ) and uracil prototrophic (ura3∆) Stephen Lory Table 2-5 shows the Saccharomyces cerevisiae strain used during this study Dept. of Microbiology and Molecular Genetics Harvard Medical School (Wolfgang et al 2003)
Plasmids Plasmid Description Reference pLLX13 pLLX8 pWSK29 lys10 capture vector lys47 capture vector asnV capture vector leuX capture vector serT capture vector serW capture vector (9.9kb) provides the backbone for the capture vector. It contains the tetAR genes that confer tetracycline resistance (10µg/ml) in E. coli. It has the URA3 gene that will allow S. cerevisiae CRY1- 2 to grow on uracil –deficient media. The plasmid also contains genes to allow replication in both types of organisms. CEN6 and ARSH4 allow replication and segregation of the plasmid within yeast. There are two origin of replication; oriV for vegetative replication in E. coli and oriT which acts as an origin of transfer. (4.6kb) is used to produce an amplicon of 2.9kb that contain the cyh2, which confers sensitivity to cycloheximide (2.5 µg/ml) in S. cerevisiae CRY 1-2 and bla (b-lactamase) confer resistance to carbenicllin (50µg/ml) in E. coli. Low copy vector used for the sub-cloning of E106-serW genomic island Capture vector that targets the 10 th tRNA for lysine in Pseudomonas aeruginosa PAO1; adjacent to PA0976 Capture vector that targets the 47 th tRNA for lysine in Pseudomonas aeruginosa PAO1; adjacent to PA4541 Capture vector that targets asnV in Escherichia coli K12 MG1655 Capture vector that targets leuX in Escherichia coli K12 MG1655 35 Stephen Lory Dept. of Microbiology and Molecular Genetics Harvard Medical School (Wolfgang et al 2003) Stephen Lory Dept. of Microbiology and Molecular Genetics Harvard Medical School (Wolfgang et al 2003) Lab 212 Dept. of Infection, Immunity and Inflammation, University of Leicester (Wang, Kushner 1991) This study This study This study This study Capture vector that targets serT in Escherichia coli K12 MG1655 This study Capture vector that targets serW in Escherichia coli K12 MG1655 Table 2-6 shows plasmids used and created during this study 2.1.3 Preparation of electrocompetent cells This study A 5ml culture of E. coli DH10B was grown overnight 200rpm, 37ºC in LB. The overnight culture was added to 495ml of LB and incubated at 200rpm, 37ºC for approximately 2-3 hours until culture reached an OD600 of 0.5. The culture was
Page 1 and 2: Characterisation of pathogenicity i
Page 3 and 4: Statement of Originality This accom
Page 5 and 6: % Percent Abbreviations ºC Degrees
Page 7 and 8: Table of contents 1 INTRODUCTION 1
Page 9 and 10: 4 THE CONTRIBUTION OF PAPI-1 AND PA
Page 11 and 12: 1 Introduction 1 INTRODUCTION 1 1.1
Page 13 and 14: Cell-surface Proteases Haemolysins
Page 15 and 16: 1.2.1 Pathogenicity islands: a subg
Page 17 and 18: appear to be related and there have
Page 19 and 20: assessed in number of assays and mo
Page 21 and 22: TAR cloning is a method designed by
Page 23 and 24: Figure 1-3 Top plate shows colony g
Page 25 and 26: Figure 1-5 depicts the principles o
Page 27 and 28: Balb/c mice. The models described b
Page 29 and 30: urns-sepsis model, but not in the n
Page 31 and 32: ORF ID Gene name Gene function PA14
Page 33 and 34: functional activities it could be s
Page 35 and 36: Figure 1-6 Schematic showing the OR
Page 37 and 38: RhlI Autoinducer RhlR Rhl Regulon G
Page 39 and 40: 1.5.2 Pseudomonas aeruginosa LES Th
Page 41 and 42: 2 Material and Methods 2 MATERIAL A
Page 43: 2.1.2 Strains used Pseudomonas aeru
Page 47 and 48: In the case of capture vector const
Page 49 and 50: Primer name Sequence Capture vector
Page 51 and 52: The relevant homology sequence was
Page 53 and 54: Important to the use of the capture
Page 55 and 56: 2.3.4 Electroporation: Transfer of
Page 57 and 58: 2.4.2 Infection dose preparation Th
Page 59 and 60: mould was then added to the metal c
Page 61 and 62: 3 Analysis of genomic islands captu
Page 63 and 64: 3.1 Capture experiments involving g
Page 65 and 66: DNA No of colonies Control No DNA 0
Page 67 and 68: were designed based on the assumpti
Page 69 and 70: 3.2.1 KR115-lys10 and KR159-lys10 A
Page 71 and 72: estriction digestion with I-SceI to
Page 73 and 74: generated. The most significant nuc
Page 75 and 76: they both produced three fragments
Page 77 and 78: elated genomic islands and this res
Page 79 and 80: All the Yersinia species available
Page 81 and 82: Leicester). E105-leuX was the first
Page 83 and 84: The strains highlighted with alignm
Page 85 and 86: predict this protein to be a dnaJ-c
Page 87 and 88: Figure 3-15 depicts the codon usage
Page 89 and 90: ORF Gene range Blastx Description y
Page 91 and 92: ecognition sequences are found with
Page 93 and 94: (S: 150, E: 1e-37). This predicts t
Page 95 and 96:
having multiple copies of itself. A
Page 97 and 98:
The capture vectors used during thi
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cloning and their findings was inco
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The genomic island capture method c
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unpublished). This was achieved by
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Pseudomonas aeruginosa PA14 (UCBPP-
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stressful’ to the bacteria and th
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log CFU/mg 4 3 2 1 0 0 2 4 6 8 Days
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mice exhibited visible symptoms at
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4.4.1 Survival times post-infection
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(P
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4.4.4 Intravenous infection A quest
Page 119 and 120:
Symptom score Symptom score Symptom
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Fold increase Fold increase 15 10 5
Page 123 and 124:
Figure 4-15 Histopathology lung sec
Page 125 and 126:
4.4.7 Progression of disease post-i
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Figure 4-19 Leukocytes numbers moni
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interesting result is that the ∆P
Page 131 and 132:
Unfortunately, 65% of the ORFs with
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morphology. ∆PAPI-1∆PAPI-2 leav
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The intravenous sepsis model data s
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in PAO1(Lee et al. 2006). They show
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shown that deletion of the exoU gen
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5 The role of quorum-sensing in the
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Figure 5-1 shows overnight growth o
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Quorum-sensing is bacterial cell de
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Interestingly, the quorum-sensing d
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Symptom score Symptom score Symptom
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LES431 LESB58 LESB65 LES400 � �
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Additional comparisons of the CFU r
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log CFU/mg log CFU/mg log CFU/ml 5
Page 157 and 158:
For LESB58-infected mice the histop
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Strain Mouse Timepoint HB CI GC Sco
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Score: 4 HB Score: 4 Score: 4 B HCI
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post-infection to that of healthy m
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Tang et al. (1996) performed additi
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Figure 5-14 PFGE comparison of the
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isolates have a similar virulence i
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The data presented in this thesis s
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than PAO1. Despite the bacterial lu
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6 Thesis conclusion The underlying
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Hopefully the methodologies describ
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1 2 3 4 5 6 7 8 9 10 11 12 13 λHin
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AQ: 1 AQ: 2 AQ: 3 AQ: 4 AQ: 5 AQ: 6
Page 183 and 184:
AQ: 18 AQ: 19 AQ: 20 F1 AQ: 21 tapr
Page 185 and 186:
AQ: 32 AQ: 33 AQ: 34 tapraid5/z9d-j
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AQ: 40 AQ: 41 DISCUSSION tapraid5/z
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AQ: 48 tapraid5/z9d-jid/z9d-jid/z9d
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tapraid5/z9d-jid/z9d-jid/z9d01009/z
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8 Bibliography AL-ALOUL, M., CRAWLE
Page 195 and 196:
D'ARGENIO, D.A., WU, M., HOFFMAN, L
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genomic islands in Pseudomonas aeru
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MURRAY, A.W. and SZOSTAK, J.W., 198
Page 201 and 202:
specificity of chaperone-usher mach
Page 203 and 204:
VAN HEECKEREN, A.M. and SCHLUCHTER,
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