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

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any of its isogenic mutants under the conditions used within our laboratory. There data is in keeping with He et al. (2004) who reported no differences between wild- type PA14 in growth, production of pyocyanin, in vitro adhesion, swimming, twitching and swarming motilities and any of the PAPI-1 ORF mutants. Growth curves were generated for PA14 and its isogenic mutants by inoculating fresh liquid media with a set starting OD and then the OD was measured hourly for 24 hours time period. No differences in growth characteristics suggest that the resulting isogenic mutants are equally as fit as the wild-type, PA14. Swarming and twitching are two types of mobility used by bacteria. Swarming uses the bacterial flagella to move across solid surfaces. The test involved inoculating bacterial cells in the centre of a minimal media plate that has a low agar content (~0.5%), using a toothpick. The variable parameter is the distance the bacteria have moved across the plate after growth overnight. Twitching is a type of movement that uses the type IV pili. The test is similar to that of swarming except the use of LB media plates that has an agar content of 1%. PAPI-1 has genes that have been annotated as encoding a putative type IV B pilus. The results do not show a difference between PA14 and the two mutants without PAPI-1; ∆PAPI-1 and ∆PAPI-1∆PAPI-2. This result could be due to the presence of the additional type IV gene cluster within the core genome. Pyocyanin is a virulence factor of P. aeruginosa that is excreted into the extracellular environment. The genes involved in pyocyanin production and its regulatory factors are found within the core genome, therefore no difference is expected. Biofilm is an extracellular matrix of polysaccharide and bacteria that aids attachment to solid surfaces. The test to determine biofilm formation involves growing a set density of cells in liquid media within a 96-well PVC plate and analysing biofilm growth via straining with crystal violet and quantification by OD reading. PAPI-1 contains the gene PvrR that is involved in the phenotypic switching of colony morphology types that correlates with biofilm formation. There are two basic types of P. aeruginosa colony, normal and small-colony variant (SCV). Under stressful condition such as the presence of antibiotics, there is an increase in colonies exhibiting the SCV compared with normal colony morphology. SCV produce larger amount of biofilm and produce it more rapidly than the normal colonies. The protein, PvrR acts to switch the SCV to the normal phenotype. Conclusions are that the conditions used during this test could be considered ‘un- 96
stressful’ to the bacteria and therefore PA14 and the isogenic mutants are all growing as normal colonies. 4.2 Preliminary studies of chronic respiratory infection model An aim of the project was to develop a chronic infection model that more closely mimicked a natural infection and does not involve invasive procedures as previously published models. Previous models have involved using P. aeruginosa embedded in agarose beads and being infected intratracheally (Stotland, Radzioch & Stevenson 2000) or infecting drinking water with P. aeruginosa (Coleman et al. 2003). The first challenge dose attempted was 2x10 4 CFU/50µl with 4 mice (Figure 4-2). The mice exhibited no signs of illness 48 hours post-infection and two mice were culled and their tissues recovered. P. aeruginosa was recovered from the nasopharynx and lungs of both mice, so the residual mice were monitored till 7 days post-infection. The tissues were recovered and P. aeruginosa was detected. The results appeared to indicate that there was an increase in CFU within the nasopharynx and lungs from 48 hours to 7 days post-infection. Repeat experiments were conducted but similar results were not achieved (Figure 4-2, Figure 4-3). The first experiment used mice that were 8 weeks old. The second experiment used mice that were 12 weeks old. To determine if this was the variable factor, the third experiment was performed using 8 week old mice. 97
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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
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 and 104: unpublished). This was achieved by
Page 105: Pseudomonas aeruginosa PA14 (UCBPP-
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
Page 155 and 156: 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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