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

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The role of PAPI-1 genes in virulence has been evaluated in other models of infection. Table 4-8 lists these highlighted in the literature as responsible for a reduction in virulence. To date only the model used during this project assesses the contribution of PAPI-1 in an acute respiratory model and therefore cannot be considered directly comparable. Gene Other names Function C. elegans Mouse burn-sepsis PA14_60130 RL003 Hypothetical protein � uvrD (PA14_60080) RL008 DNA helicase � PA14_60090 RL009 Hypothetical protein � PA14_59960 RL020 Putative proteindisulfide isomerase � PA14_59870 RL029 Hypothetical protein � PA14_59840 RL033 Hypothetical protein � PA14_59800 RL036 Kinase sensor protein � pvrR RL037 Two � (PA14_59790) component response regulator rcsC RL038 Putative � (PA14_59780) kinase sensor protein rcsB RL039 Putative two � (PA14_59770) component response regulator PA14_59490 RL068 Hypothetical protein � PA14_59150 RL095 Single-strand DNA-binding protein � PA14_59090 RL101 Hypothetical protein � PA14_59070 RL102 Hypothetical protein � PA14_59010 RL107 Hypothetical protein � PA14_58950 RL112 Hypothetical protein � Table 4-8 The genes that have identified in other models within PAPI-1 that contribute to virulence of PA14. C. elegans data generated from Lee et al, 2006 (Lee et al. 2006) Mouse burns-sepsis generated by He et al, 2004 (He et al. 2004) 120
Unfortunately, 65% of the ORFs within PAPI-1 have no known function and are labelled as hypothetical genes (He et al. 2004). These that have been assigned function and have sufficient literature are evaluated in the subsection. There are two, two component regulatory systems (TCS) within PAPI-1 that have been highlighted through previous infection studies as important for general virulence of the strain. Two component regulatory systems act to change the gene expression within a cell to favour changes in the external environment. They consists of two parts, one the sensor kinase, which senses the changes in the environment by being bound to the cellular membrane and two, the response regulator, which mediates the intracellular response to the changes in environment. The published data in conjugation with the data generated during this project, suggests that the two TCS are important for mammalian infection, as the differences in virulence are currently only noted in mice models of infection. The TCS involving RcsC and RcsB described in the literature is involved in the adaptation of the cell surface by changing its structure and integrity (Huang, Ferrieres & Clarke 2006). In E. coli there are three components involved in this system, RcsC, the sensor kinase, which activates RcsD, an intermediate, which in turn activates RcsB, the response regulator. A RcsD homologue has not been discovered in P. aeruginosa PA14, but the literature suggest that the Yersinia spp. genome does not encode a functional RcsD and the TCS has taken on a different role (Huang, Ferrieres & Clarke 2006). As this TCS, is now found on a pathogenicity island rather than the core genome and does not encode the intermediate protein RcsD, suggests the TCS is now performing some auxiliary role crucial to PAPI-1. This is supported by the fact that mutagenesis of either RcsB or RcsC results in attenuation in other models of infection (He et al. 2004), demonstrating that they are functional. In the circumstances that the rcs TCS system maintained its usual functional activities the loss of RcsB or RcsC would result in cells that have a reduced ability to create a capsule and to form biofilms. A capsule is a highly noted protection of bacteria from the host innate immunity. This phenomenon could contribute to the 121
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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
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
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: interesting result is that the ∆P
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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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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