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

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4.6.2 The role of PAPI-2 during acute respiratory infection A reduction in virulence in PA14 was only seen when PAPI-2 was deleted, mutants ∆PAPI-2 and ∆PAPI-1∆PAPI-2. The only genes highlighted in PAPI-2 within the literature as contributing to virulence are ExoU which is a potent cytotoxin plus SpcU its chaperone. The literature appears to suggest that the majority of the reduction in virulence could be due to the loss of ExoU. ExoU has been previously been shown to be an important factor in terms of acute respiratory illness within murine acute respiratory models (Allewelt et al. 2000, Schulert et al. 2003, Shaver, Hauser 2004). Allewelt et al, 2000 reported a clear difference in LD50 for a panel of P. aeruginosa strains between those expressing ExoU and those strains that did not and that introducing the exoU gene with its chaperone spcU into a non-carrying strain could significantly reduce the LD50. The loss of ExoU from the P. aeruginosa strain, PA99, was also shown to be significantly attenuated in a murine acute respiratory model (Shaver, Hauser 2004). This model is very similar to the one described in this thesis, they used a similar dose and culling timepoint (18 hours). They evaluated differences in virulence by performing CFU counts on lung, liver and spleen homogenate. They did not evaluate the nasopharynx or directly assess dissemination from the lungs to the blood by cardiac puncture. The data generated during this project support these observations as they show a loss of PAPI-2 results in an attenuated strain in comparison with PA14. The data suggest that PAPI-2 plays a number of important roles in acute respiratory infection. Infection with ∆PAPI-2 in comparison with wild-type PA14 led to a loss of one log in nasopharynx as well as the lungs and a two log in blood at 18 hours post- infection. Consideration of the data generated for ∆PAPI-1, suggests that the presence of PAPI-2 solely is enough to ensure persistence in nasopharynx and lungs as well as successful dissemination to the blood. This data is the first to recognise that PAPI-2 is important for upper respiratory tract (nasopharynx) infection. 124
The intravenous sepsis model data seemed to demonstrate that all the isogenic mutants can survive equally well within the blood; it is merely the ability to get into the bloodstream where the mutants differ. This data is supported by Shaver et al. (2004) who observed that the presence of ExoU was important for dissemination from the lungs. They constructed mutants in PA99, so the strain solely excreted ExoS, ExoT or ExoU. They observed dissemination by evaluating CFU recovered from the spleen and liver. They observed that in 60% of the mice showed dissemination when secreting ExoU solely in comparison to ExoS (40%) and ExoT (29%). In addition to having a similar ability to survive within the blood, both the mutants lacking PAPI-2 showed a decrease in CFU within the blood at 18 hours post- infection in comparison to PA14 in the acute respiratory model. ∆PAPI-1∆PAPI-2 has lower CFU numbers than ∆PAPI-2, but this could be explained by increased susceptibly to the immune system decreasing the number of bacteria within the lungs as previously discussed. An alternative is that the CFU within the lungs correlates with the CFU recovered within the blood. An additional explanation is PAPI-1 encodes additional factors that allows successful dissemination from the lungs to occur, still allowing ∆PAPI-2 to dissemination, but not ∆PAPI-1∆PAPI-2. This factor would be less potent than ExoU, which is would explain the lack of difference blood dissemination seen with ∆PAPI-1. In summary, PAPI-2 contributes significantly to the virulence of PA14. ∆PAPI-2 was the only single mutant to be attenuated in comparison to wild-type, PA14. The important of this pathogenicity island is solidified as the role of PAPI-1 in acute respiratory infection is masked till PAPI-2 is deleted. The data generated from this project suggest that PAPI-2 is important for upper respiratory infection and for the ability of PA14 to disseminate from the lungs. The data generated during this project is the first to highlight that PAPI-2 (ExoU) contributes to the ability of PA14 to get into the bloodstream, but not essential for survival in the bloodstream. 4.6.3 PAO1 as virulent as PA14 ∆PAPI-2 in an acute respiratory model 125
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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
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 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: morphology. ∆PAPI-1∆PAPI-2 leav
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 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
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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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