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

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mutant with both PAPI-1 and PAPI-2 deleted. The results show that both pathogenicity islands contribute to virulence in a murine acute respiratory model of infection. The results demonstrated that the presence of PAPI-2, possibly ExoU, is enough to maintain wild-type virulence. The data presented suggested that recovery of the nasopharynx can provide an important variable for assessing virulence of P. aeruginosa. The results from this project suggest that PAPI-2 have a role in infection within the upper respiratory tract. The data generated also suggests that PAPI-2 is important for dissemination of PA14 from the lungs to the blood, but not for survival within the blood as demonstrated by testing in an intravenous sepsis model of infection. The results supported the literature by showing the difference in virulence between PAO1 and PA14 is more complex than the presence or absence of pathogenicity islands alone. The virulence of PAO1 was more closely mimicked by PA14 ∆PAPI-2 than PA14 ∆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 within PAPI-1. The third project focused on in vivo virulence of P. aeruginosa. The aim of the project was to determine if quorum-sensing over-expression was a reliable marker of virulence within P. aeruginosa Liverpool Epidemic Strain (LES) isolates in a murine acute respiratory model. The results suggest that this theory is generally true in an acute respiratory model of infection. The over-expressing isolates (LES431 and LESB65) were more virulent than the deficient isolate (LES400), the exception being the over-expressing isolate LESB58. The results also showed that LES isolates (as well as PAO1 and PA14) from acute infection were more virulent than those from chronic infection (CF patients). The project has highlighted a need to determine the genomic differences between the isolates to explain the continuum of virulence. Further work that could expand the data generated during this thesis is to evaluate LES isolate, LES416 in the acute respiratory model. This LES isolate was taken from the CF patient who parents also developed a respiratory infection, to compare its virulence with LES 431, which was taken from one of the CF patient’s parents (McCallum et al. 2002). 166
Hopefully the methodologies described during this thesis will enable future project aimed at evaluating pathogenicity islands to link in vivo and in vitro data analysis. The in vitro work could enable rapid sequencing of genomic islands as well as allowing detailed analysis of the functional role of the genes present. When combined with the in vivo work, known and newly discovered genomic islands could be described as pathogenicity islands. This is especially important for P. aeruginosa where for example in PAO1, 44% of the ORFs within the genome have no known function (Class 4). P. aeruginosa is currently the largest of all sequenced bacterial genomes. It contains a large portion of genes with no known homologues within other bacterial species. Both the in vivo projects described in this thesis could contribute to elucidating the role of virulence genes, at least in an acute respiratory model. A more detailed comparison between the LES isolates used during this thesis could highlight key genomic differences between the most virulent isolate (LES431) and least virulent (LESB58), as well as highlight new markers of virulence within P. aeruginosa. 167
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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
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Figure 3-15 depicts the codon usage
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ORF Gene range Blastx Description y
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ecognition sequences are found with
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(S: 150, E: 1e-37). This predicts t
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having multiple copies of itself. A
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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
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Symptom score Symptom score Symptom
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Fold increase Fold increase 15 10 5
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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
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: 6 Thesis conclusion The underlying
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
Page 189 and 190: AQ: 48 tapraid5/z9d-jid/z9d-jid/z9d
Page 191 and 192: tapraid5/z9d-jid/z9d-jid/z9d01009/z
Page 193 and 194: 8 Bibliography AL-ALOUL, M., CRAWLE
Page 195 and 196: D'ARGENIO, D.A., WU, M., HOFFMAN, L
Page 197 and 198: genomic islands in Pseudomonas aeru
Page 199 and 200: 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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