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

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Score: 3 B Score: 5 HCI V Score: 3 MGC MCI HGC HB HB V MGC HB MCI MCI 118 HB V HCI HCI MCI HCI V CI B MCI Figure 4-20 Figure 4-21 Histopathology lung sections produced from mice infected with ∆PAPI-1∆PAPI-2 at 2x10 6 CFU/50µl Left column represents from top to bottom a) 18 hours b) 72 hours c) 96 hours 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 4.5 Summary of acute respiratory outcomes The data would indicate that wild-type PA14 and ∆PAPI-1 have no significant differences in virulence within a murine acute respiratory model. ∆PAPI-2 is less virulent than wild-type, PA14. The double deletant mutant, ∆PAPI-1∆PAPI-2 is severely attenuated compared to the wild-type and both single mutants. An
interesting result is that the ∆PAPI-1∆PAPI-2 is less virulent than PAO1, a strain that lacks both PAPI-1 and PAPI-2. In fact in terms of virulence PAO1 is similar to ∆PAPI-2. The differences in virulence are reflected in recovered CFU numbers from tissue, symptom scores, survival times and histopathology, but not readily apparent from the immune profile. 4.6 Discussion This project was to investigate the contribution of PAPI-1 and PAPI-2 to the virulence of Pseudomonas aeruginosa PA14 in a murine acute respiratory model by comparing wild-type PA14 and three isogenic mutants. The project shows that both pathogenicity islands contribute significantly to the virulence of PA14 and the loss of both pathogenicity islands results in a severely attenuated strain. Mice infected with the attenuated strain rather than the wild-type have an increased survival time from 18 hours to 72 hours (70%) to surviving the experiment (30%). Eighteen hours post-infection the CFU numbers recovered are drastically reduced in the lungs, nasopharynx and blood compared to wild-type, PA14, also a reduction in symptom score was only noted when both PAPI-1 and PAPI-2 are absent from PA14. This section is going to discuss the role PAPI-1 and PAPI-2 play in virulence with respect to the project data and the available literature. This is followed by evaluating the hypothesis raised by He et al. (2004) regarding the differences in virulence between PAO1 and PA14. The final discussion point will about the immune response during P. aeruginosa infection. 4.6.1 The role of PAPI-1 during acute respiratory infection The data generated during project demonstrated that PAPI-1 has a role in acute respiratory infection. ∆PAPI-1 did not show a reduction in virulence compared to PA14. The bacterial numbers recovered from the nasopharynx, lungs and blood were insignificantly different (P
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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
Page 77 and 78: elated genomic islands and this res
Page 79 and 80: All the Yersinia species available
Page 81 and 82: 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
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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: Figure 4-19 Leukocytes numbers moni
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 and 176: 6 Thesis conclusion The underlying
Page 177 and 178: Hopefully the methodologies describ
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1 2 3 4 5 6 7 8 9 10 11 12 13 λHin
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AQ: 1 AQ: 2 AQ: 3 AQ: 4 AQ: 5 AQ: 6
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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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