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

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This subsection aims to address the hypothesis raised by He et al. (2004) that suggested the major differences in virulence between PAO1 and PA14 is the presence of PAPI-1 and PAPI-2. PAO1 lacks both PAPI-1 and PAPI-2; based on this hypothesis PAO1 should have similar virulence to ∆PAPI-1∆PAPI-2. The data generated during this project shows that PAO1 is more virulent than ∆PAPI-1∆PAPI-2. PAO1 has a lower survival time than ∆PAPI-1∆PAPI-2. PAO1 has a significantly higher CFU (P
in PAO1(Lee et al. 2006). They showed that PA14 had 58 additional gene clusters than PAO1. They tried to analyse these 58 gene clusters in two ways. Firstly, they screened 20 P. aeruginosa strains (including PA14 and PAO1) and compared them for virulence in a C. elegans model of infection and ranked their virulence. They then analysed the strains for the presence of the identified PA14-specific gene regions. The results showed no correlation between virulence and the presence of the PA14-specific gene regions. A second approach taken was to create a transposon insertion mutant library for PA14. They screened the library using the C. elegans model of infection for those in the PA14-specific gene regions for attenuation. They discovered 9 genes, 2 of which are present in PAPI-1 that caused attenuation. The present of these regions within the other P. aeruginosa strains, showed no set trend for correlation for virulence within C. elegans model of infection. This led the group to conclude that the presence of these gene regions within another P. aeruginosa strain was not an indicator of its virulence within a C. elegans model of infection. The publication by Lee et al. (2006) justify the need to create P. aeruginosa pathogenicity islands mutants. To evaluate the true impact of a pathogenicity island, it is essential to remove the whole island. Pathogenicity islands have evolved over time to become single elements that encode all the genes that are required to increase virulence. The island has evolved and continued to function as a single element because it functions better as a whole. Lee et al. (2006) discovered 9 genes that were important for virulence in a C. elegans model of infection, 2 of which were in PAPI-1. The data could represent ‘kinks in the armour’ of the pathogenicity island that causes the whole island to be rendered non-functional. The availability of the technique to create unmarked mutants within P. aeruginosa is available and our laboratory has proven that 100kb of genome can be successfully removed. This could be used to explore the roles of the 58 clusters of PA14-specific regions within the strain by removing them directly, especially as so few genes within the PA14- specific regions have an assigned function; 63% have unknown function (Lee et al. 2006). The use of PA14-specific region mutants could be used as a cost-effective method to decide on individual genes to target for further virulence characterisation. 127
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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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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: The intravenous sepsis model data s
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
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
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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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