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

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Score: 3 B Score: 3 B HB Score: 4 MGC B LGC HB HCI HGC MCI HB 152 HCI MCI MCI MCI V HB HCI V HB HCI HB B MCI B HB MCI Figure 5-13 Histopathology lung sections prodcued from mice infected with 2x10 6 CFU/50µl at 96 hours post-infection Left column represents from top to bottom a) LES400 b) LESB58 c) LESB65 at magnification x100; Middle and Right column represents the same stains respectively from top to bottom at magnification x200. Key: LCI-MCI-HCI represents low/medium/high cellular infiltration; LGC-MGC-HGC represents low/medium/high general consolidation; B represents bronchi; V represents blood vessel 5.2.5 Summary of acute respiratory infection outcomes In the survival experiments, LES431-infected mice did not survive beyond 24 hours post-infection. Of the mice infected with LESB65, 60% survived the experiment, whereas all the mice infected with LES400 or LESB58 survived the experiment. The symptom scores of LESB58-infected mice improved continually from 18 hours
post-infection to that of healthy mice (symptom score = 0) by 96 hours post- infection. The symptoms scores coincides with the lung CFU that peak at 18 hours and decreased to undetectable levels by 48 hours post-infection. This coincides with the lung damage that is consistent through 18 to 55 hours post-infection and improves by 96 hours post-infection. In contrast LES400-infected mice had their symptom scores peak at 48 hours post-infection and improved to 96 hours post- infection; this also correlates with the lung CFU which peaked at 48 hours post- infection and decreased steadily till 96 hours post-infection. This also coincides with the lung histopathology that peaks at 55 hours post-infection and improved until 96 hours post-infection. The LESB65-infected mice maintained a consistent median symptom score throughout the experiment. The CFU numbers recovered from the nasopharynx and lungs increased steadily from 18 hours to 96 hours post- infection. This correlates with the increase in histopathology score between 18 and 55 hours post-infection (4 > 4.5) and this high score was maintained until 96 hours post-infection. Based on the data generated during this project, the most virulent to the least virulent LES isolate is LES431, to LESB65, to LES400 and finally LESB58. 5.3 Discussion The aim of this study was to determine whether the over-expression of quorum- sensing products is a reliable marker for increased virulence exhibited by LES isolates. This hypothesis was tested in an acute respiratory infection model. The over-expressing isolates were represented by LES431, LESB58 and LESB65. The deficient isolates were represented by LES400. This section proceeds by discussing quorum-sensing over-expression as a marker of virulence of P. aeruginosa within a murine acute respiratory model. This is followed by discussions on the differences in virulence between LES isolates. The final discussion point is to compare the virulence of the LES isolates and the reference strains, PAO1 and PA14, within a murine acute infection model. This is followed by the conclusions and future work. 153
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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
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 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: Score: 4 HB Score: 4 Score: 4 B HCI
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
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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