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

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4 The contribution of PAPI-1 and PAPI-2 to the virulence of Pseudomonas aeruginosa PA14 in a murine acute respiratory infection model 4 THE CONTRIBUTION OF PAPI-1 AND PAPI-2 TO THE VIRULENCE OF PSEUDOMONAS AERUGINOSA PA14 IN A MURINE ACUTE RESPIRATORY INFECTION MODEL 94 4.1 CONSTRUCTION OF THE DELETANT MUTANTS AND IN VITRO DATA 95 4.2 PRELIMINARY STUDIES OF CHRONIC RESPIRATORY INFECTION MODEL 97 4.3 PRELIMINARY STUDIES OF ACUTE RESPIRATORY INFECTION MODEL 100 4.4 THE CONTRIBUTION OF PAPI-1 AND PAPI-2 TO THE VIRULENCE OF PSEUDOMONAS AERUGINOSA PA14 102 4.4.1 SURVIVAL TIMES POST-INFECTION 103 4.4.2 SYMPTOMS SCORES POST-INFECTION 103 4.4.3 BACTERIAL NUMBERS IN NASOPHARYNX, LUNGS AND BLOOD POST-INFECTION 104 4.4.4 INTRAVENOUS INFECTION 107 4.4.5 IMMUNE PROFILE 110 4.4.6 LUNG HISTOPATHOLOGY POST-INFECTION 112 4.4.7 PROGRESSION OF DISEASE POST-INFECTION WITH PA14 ∆PAPI-1∆PAPI-2 115 4.5 SUMMARY OF ACUTE RESPIRATORY OUTCOMES 118 4.6 DISCUSSION 119 4.6.1 THE ROLE OF PAPI-1 DURING ACUTE RESPIRATORY INFECTION 119 4.6.2 THE ROLE OF PAPI-2 DURING ACUTE RESPIRATORY INFECTION 124 4.6.3 PAO1 AS VIRULENT AS PA14 ∆PAPI-2 IN AN ACUTE RESPIRATORY MODEL 125 4.6.4 THE IMMUNE RESPONSE DURING ACUTE RESPIRATORY INFECTION 128 4.7 CONCLUSIONS AND FURTHER WORK 129 94
Pseudomonas aeruginosa PA14 (UCBPP-PA14) is a wild-type reference strain that has been used extensively to test the contribution of virulence factors to disease. The P. aeruginosa strain PAO1, another wild-type reference strain, has been noted to be less virulent than P. aeruginosa PA14 in a number of models. A recent study (He et al. 2004) suggested the differences in virulence was due to the presence of two pathogencity islands found in PA14 that were absent in PAO1, named Pseudomonas aeruginosa pathogenicity island 1 (PAPI-1) and Pseudomonas aeruginosa pathogenicity island 2 (PAPI-2). The aim of the project is to compare the contribution of PAPI-1 and PAPI-2 to the virulence of P. aeruginosa PA14 within a murine acute pneumonia model. Importantly, this is the first project to look at the effects of pathogenicity islands on in vivo respiratory infection. To achieve this aim, three isogenic pathogenicity island deletant mutants were created in the strain P. aeruginosa PA14, a mutant with PAPI-1 deleted, another with PAPI-2 deleted and a third mutant with both PAPI-1 and PAPI-2 deleted. The PA14 mutants and wild-type PA14 were also compared against PAO1. Herein, the mutants will be referred to as ∆PAPI-1, ∆PAPI-2 and ∆PAPI-1∆PAPI-2. This results chapter proceeds by describing the mutants used, followed by results generated whilst developing the murine infection models, the next section describes the results generated using an acute respiratory model to compare virulence and ends with the discussion and conclusions generated from this project. 4.1 Construction of the deletant mutants and in vitro data Three isogenic pathogenicity island mutants were created by Ewan Harrison (University of Leicester, PhD, unpublished); ∆PAPI-1, ∆PAPI-2 and ∆PAPI- 1∆PAPI-2. The first mutants of its kind reported in P. aeruginosa. They were created using a mutagenesis system developed by Choi and Schweizer (2005) specifically targeting P. aeruginosa. A number of in vitro test were performed on PA14 and its isogenic mutants by Ewan Harrison, to assess swarming mobility, twitching mobility, pyocyanin production, biofilm production and growth characteristics. There were no significant differences reported between PA14 and 95
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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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Page 55 and 56: 2.3.4 Electroporation: Transfer of
Page 57 and 58: 2.4.2 Infection dose preparation Th
Page 59 and 60: mould was then added to the metal c
Page 61 and 62: 3 Analysis of genomic islands captu
Page 63 and 64: 3.1 Capture experiments involving g
Page 65 and 66: DNA No of colonies Control No DNA 0
Page 67 and 68: were designed based on the assumpti
Page 69 and 70: 3.2.1 KR115-lys10 and KR159-lys10 A
Page 71 and 72: estriction digestion with I-SceI to
Page 73 and 74: generated. The most significant nuc
Page 75 and 76: 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: unpublished). This was achieved by
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 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
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log CFU/mg log CFU/mg log CFU/ml 5
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For LESB58-infected mice the histop
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Strain Mouse Timepoint HB CI GC Sco
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Score: 4 HB Score: 4 Score: 4 B HCI
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post-infection to that of healthy m
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Tang et al. (1996) performed additi
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Figure 5-14 PFGE comparison of the
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isolates have a similar virulence i
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The data presented in this thesis s
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than PAO1. Despite the bacterial lu
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6 Thesis conclusion The underlying
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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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