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

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A challenge dose of 2x10 5 CFU/50µl was attempted with PA14 and the results are shown in Figure 4-5. CFU numbers in the nasopharynx and lungs were detected up to 48 hours post-infection. log CFU/mg 4 3 2 1 0 1 2 Days post-infection 100 7 Nasopharynx Lungs Figure 4-5 shows the CFU numbers recovered from the nasopharynx and lungs at 1, 2 and 7 days post-infection for Pseudomonas aeruginosa PA14 at 2x10 5 CFU/50µl ; n=5 mice for each timepoint. Each symbol the mean log CFU/mg and the error bar represents the standard error of the mean. In summary, a pilot experiment with PAO1 at a challenge dose of 2x10 4 CFU/50µl allowed recovery of bacteria 7 days post-infection. Subsequent repeat experiments did not achieve this initial success. Similar experiments were attempted with PA14 and ∆PAPI-1∆PAPI-2. The chronic infection model was shown not to be robust and the subsequent experiments focussed solely on the acute respiratory model of infection. 4.3 Preliminary studies of acute respiratory infection model The acute respiratory model used during this project is modified from Shaver et al. (2004). To develop a murine acute respiratory model, preliminary experiments were conducted using P. aeruginosa PAO1. The pilot studies involved determining the dose and the organs to recover for further analysis. The mice were symptom scored and the organs were recovered at 18 hours post-infection. The challenge dose was 2x10 6 CFU/50µl. The results of the pilot experiment are shown in Figure 4-6. The
mice exhibited visible symptoms at 18 hours post-infection with an average symptom score of 4. The maximum symptom score a mouse can achieve is 6. A small survival experiment resulted in the mice surviving till approximately 24 hours post-infection. The recovery of the bacteria from the nasopharynx, lungs and blood were selected for assessment of virulence. log CFU/mg 4 3 2 1 0 Nasopharynx Lungs Blood 101 Liver Spleen Figure 4-6 shows the CFU numbers recovered from the nasopharynx, lungs, blood, liver and spleen at 18 hours post-infection with Pseudomonas aeruginosa PAO1 at 2x10 6 CFU; n=8 mice. The bars represent the mean log CFU and the error bar represents the standard error of the mean. Scale for blood is log CFU/ml. All data was generated over 2 independent experiments for each strain. The acute respiratory model was then tested with P. aeruginosa PA14. As previously stated, this strain has been described as being more virulent than PAO1 in a number of infection models. Figure 4-7 shows the difference in CFU number recovered from the chosen tissues and clearly demonstrates that the chosen tissues can differentiate virulence at this dose as there was significant differences (P
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Characterisation of pathogenicity i
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Statement of Originality This accom
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% Percent Abbreviations ºC Degrees
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Table of contents 1 INTRODUCTION 1
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4 THE CONTRIBUTION OF PAPI-1 AND PA
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1 Introduction 1 INTRODUCTION 1 1.1
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Cell-surface Proteases Haemolysins
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1.2.1 Pathogenicity islands: a subg
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appear to be related and there have
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assessed in number of assays and mo
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TAR cloning is a method designed by
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Figure 1-3 Top plate shows colony g
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Figure 1-5 depicts the principles o
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Balb/c mice. The models described b
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urns-sepsis model, but not in the n
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ORF ID Gene name Gene function PA14
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functional activities it could be s
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Figure 1-6 Schematic showing the OR
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RhlI Autoinducer RhlR Rhl Regulon G
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1.5.2 Pseudomonas aeruginosa LES Th
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2 Material and Methods 2 MATERIAL A
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2.1.2 Strains used Pseudomonas aeru
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Plasmids Plasmid Description Refere
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In the case of capture vector const
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Primer name Sequence Capture vector
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The relevant homology sequence was
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Important to the use of the capture
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2.3.4 Electroporation: Transfer of
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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
Page 99 and 100: cloning and their findings was inco
Page 101 and 102: The genomic island capture method c
Page 103 and 104: unpublished). This was achieved by
Page 105 and 106: Pseudomonas aeruginosa PA14 (UCBPP-
Page 107 and 108: stressful’ to the bacteria and th
Page 109: log CFU/mg 4 3 2 1 0 0 2 4 6 8 Days
Page 113 and 114: 4.4.1 Survival times post-infection
Page 115 and 116: (P
Page 117 and 118: 4.4.4 Intravenous infection A quest
Page 119 and 120: Symptom score Symptom score Symptom
Page 121 and 122: 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
Page 127 and 128: Figure 4-19 Leukocytes numbers moni
Page 129 and 130: interesting result is that the ∆P
Page 131 and 132: Unfortunately, 65% of the ORFs with
Page 133 and 134: morphology. ∆PAPI-1∆PAPI-2 leav
Page 135 and 136: The intravenous sepsis model data s
Page 137 and 138: in PAO1(Lee et al. 2006). They show
Page 139 and 140: shown that deletion of the exoU gen
Page 141 and 142: 5 The role of quorum-sensing in the
Page 143 and 144: Figure 5-1 shows overnight growth o
Page 145 and 146: Quorum-sensing is bacterial cell de
Page 147 and 148: Interestingly, the quorum-sensing d
Page 149 and 150: Symptom score Symptom score Symptom
Page 151 and 152: LES431 LESB58 LESB65 LES400 � �
Page 153 and 154: Additional comparisons of the CFU r
Page 155 and 156: log CFU/mg log CFU/mg log CFU/ml 5
Page 157 and 158: For LESB58-infected mice the histop
Page 159 and 160: 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
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AQ: 18 AQ: 19 AQ: 20 F1 AQ: 21 tapr
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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
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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
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specificity of chaperone-usher mach
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VAN HEECKEREN, A.M. and SCHLUCHTER,
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