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

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In conclusion the data shows that PAPI-1 and PAPI-2 are not the only differences between PAO1 and PA14 and that these differences may be more complex than a straight forward absent of the pathogenicity islands, PAPI-1 and PAPI-2 (Choi et al. 2002, Lee et al. 2006). For example, previous studies have also shown that in acute respiratory models, strains harbouring ExoU are more virulent than strains harbouring ExoS (Hauser et al. 2002, Schulert et al. 2003); PA14 has ExoU and PAO1 has ExoS. A study has demonstrated that there is a 4 fold lower LD50 of ExoU positive strains in comparison with ExoS positive strains (Schulert et al. 2003) when tested in a murine acute respiratory model, on a panel of clinical isolates from hospital-acquired pneumonia. 4.6.4 The immune response during acute respiratory infection This subsection is to discuss the data generated based on lung histopathology and the cellular infiltration into the lungs. The total amount of cellular infiltration into the lungs did not differ significantly (P
shown that deletion of the exoU gene can lead to a decrease in histopathology (Finck-Barbancon et al. 1997). The group measured levels of the oedema within the lungs caused by exoproducts. The PA103 mutant in the exoU gene had significantly lower amount of damage in comparison to the wild-type as measured 4 hour post- infection. The presence of PAPI-2 and therefore ExoU is likely the cause of the additional histopathology damage seen with the isogenic mutants, PA14 and ∆PAPI-1. 4.7 Conclusions and further work The aim of this project was to explore the role of pathogencity islands on the virulence of Pseudomonas aeruginosa within a murine respiratory model. This is first study to use P. aeruginosa pathogenicity island deletant mutants in vivo. The project involved developing an acute respiratory model and its use demonstrated that PAPI-1 and PAPI-2 contribute to the virulence of PA14 in a murine acute respiratory model. This project reports a role for PAPI-1 in an acute respiratory model of infection. This is the first acute respiratory model developed for P. aeruginosa to evaluate the upper respiratory tract. The data generated highlighted that PAPI-2 plays an important role in infection of the nasopharynx. The reduction in dissemination from lungs to blood in mutants lacking PAPI-2 led to the development of an intravenous sepsis model. The sepsis model demonstrated that PAPI-2 (ExoU) was essential for dissemination from the lungs but was not essential for survival within the blood. The results also support the literature suggesting that the difference in virulence between PAO1 and PA14 is more complex than the presence or absence of pathogenicity islands. The virulence of PAO1 was more closely mimicked by ∆PAPI-2 than ∆PAPI-1∆PAPI-2. This is despite PAO1 itself not containing either PAPI-1 or PAPI-2. The project has shown that the use of pathogenicity island mutants in virulence models is feasible. The use of this model in the future could aid the labelling of known genomic islands as pathogenicity islands. The data generated could also be used as a starting point to pin point virulence genes. Further experiments based on this premise, could be to evaluate the 58 PA14-specific regions highlighted by Lee et al. (2006). This could produce interesting results especially as our laboratory has 129
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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
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mould was then added to the metal c
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3 Analysis of genomic islands captu
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3.1 Capture experiments involving g
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DNA No of colonies Control No DNA 0
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were designed based on the assumpti
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3.2.1 KR115-lys10 and KR159-lys10 A
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estriction digestion with I-SceI to
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generated. The most significant nuc
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they both produced three fragments
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elated genomic islands and this res
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All the Yersinia species available
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Leicester). E105-leuX was the first
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The strains highlighted with alignm
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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 and 110: log CFU/mg 4 3 2 1 0 0 2 4 6 8 Days
Page 111 and 112: mice exhibited visible symptoms at
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: in PAO1(Lee et al. 2006). They show
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
Page 161 and 162: Score: 4 HB Score: 4 Score: 4 B HCI
Page 163 and 164: post-infection to that of healthy m
Page 165 and 166: Tang et al. (1996) performed additi
Page 167 and 168: Figure 5-14 PFGE comparison of the
Page 169 and 170: isolates have a similar virulence i
Page 171 and 172: The data presented in this thesis s
Page 173 and 174: than PAO1. Despite the bacterial lu
Page 175 and 176: 6 Thesis conclusion The underlying
Page 177 and 178: Hopefully the methodologies describ
Page 179 and 180: 1 2 3 4 5 6 7 8 9 10 11 12 13 λHin
Page 181 and 182: 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
Page 187 and 188: 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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