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

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genomic island capture as well as P. aeruginosa. The technique has previously only been reported with P. aeruginosa and expansion to other bacterial species would make it a valuable molecular biology tool. The project will also aim to characterise novel genomic islands captured using this technique. 1.4 In vivo analysis of pathogenicity island deletant mutants of Pseudomonas aeruginosa PA14 This section describes the respiratory models currently used to study P. aeruginosa virulence and provides a literature background to the strains used in this thesis project and finishes with the aims of the project. 1.4.1 Respiratory infection models There two types of respiratory infection caused by P. aeruginosa; acute and chronic. In a clinical setting, there are two major patient types to reflect these two types of infection; those that have extended periods of ventilation (acute) and those who suffer from cystic fibrosis (chronic). Through the use of acute murine respiratory models, a number of key virulence factors have been identified, using single gene knockout mutants of P. aeruginosa. The most studied of which is the TTSS proteins of which there are four, ExoS, ExoT, ExoU and ExoY. ExoS, ExoT and ExoU have been previously shown to contribute to mortality and morbidity in a murine acute respiratory model, demonstrated by intranasal infection of adult Balb/c females (Shaver & Hauser 2004). There is also a correlation in human clinical outcomes based on the presence or absence of components of the TTSS (Hauser et al. 2002). The production of pyocyanin has also been shown to cause significant damage to lungs during murine acute respiratory infection, demonstrated by intranasal infection of adult CD-1 mice (Lau et al. 2004). Pyocyanin is a blue pigment excreted by P. aeruginosa that has been shown to have anti-bacterial properties against other bacteria. The genes within the two P. aeruginosa quorum-sensing systems; LasR (Tang et al. 1996), LasI (Pearson et al. 2000) and RhlI (Pearson et al. 2000) has also been described as contributing to acute infection, all demonstrated by intranasal infection of neonatal 16
Balb/c mice. The models described by Allewelt et al. 2000, Laskowski, Kazmierczak 2006, Shaver, Hauser 2004 all use intranasal infection of adult female Balb/c mice, with analysis of bacterial loads in lungs, spleen and liver determined at 16-18 hours post-infection. The project described in this thesis will determine bacterial numbers in the lower respiratory tract and for the first time the upper respiratory tract by recovering nasopharyngeal tissue. The project will also investigate bacterial dissemination from the lungs into blood following respiratory challenge. Current modelling of chronic lung infection with mice is primarily achieved through the use of agarose beads laden with P. aeruginosa (van Heeckeren & Schluchter 2002). Infection with free P. aeruginosa into the lungs results either in acute infection or clearance in healthy mice (van Heeckeren & Schluchter 2002). The use of chronic infection model is principally to mimic disease patterns of infection of CF patients with P. aeruginosa in conjunction with the use of CF mice. Chronic infection with P. aeruginosa was first achieved in rats by infecting intratracheally with agarose beads laden with P. aeruginosa (Cash et al. 1979). The model was adapted by Starke et al (1987) for use in mice. The mouse model has also been modified to model CF infection by using mucoid strains of P. aeruginosa as it closely mimics pathogenesis seen in lungs of CF patients. The mucoid phenotype of P. aeruginosa is due to over-production of alginate and is found in 70–80% of CF patients (van Heeckeren & Schluchter 2002). This phenotypic change correlates with lung-tissue destruction and pulmonary decline. A second model to study chronic infection in relation to biofilms was developed by Yanagihara et al (2000). This model involves placing a plastic tube within the bronchus that is pre-coated with P. aeruginosa. They used the model to evaluate the effectiveness of two antibiotics on the biofilms created during chronic infection of P. aeruginosa. Chronic infection with P. aeruginosa in transgenic CF mice has been achieved by infecting the drinking water with P. aeruginosa (Coleman et al. 2003). This model first shows chronic infection of the oropharyngeal with subsequent translocation to the lungs. Importantly, mucoid isolates of P. aeruginosa were recovered from the lungs of the CF mice. 17
Page 1 and 2: Characterisation of pathogenicity i
Page 3 and 4: Statement of Originality This accom
Page 5 and 6: % Percent Abbreviations ºC Degrees
Page 7 and 8: Table of contents 1 INTRODUCTION 1
Page 9 and 10: 4 THE CONTRIBUTION OF PAPI-1 AND PA
Page 11 and 12: 1 Introduction 1 INTRODUCTION 1 1.1
Page 13 and 14: Cell-surface Proteases Haemolysins
Page 15 and 16: 1.2.1 Pathogenicity islands: a subg
Page 17 and 18: appear to be related and there have
Page 19 and 20: assessed in number of assays and mo
Page 21 and 22: TAR cloning is a method designed by
Page 23 and 24: Figure 1-3 Top plate shows colony g
Page 25: Figure 1-5 depicts the principles o
Page 29 and 30: urns-sepsis model, but not in the n
Page 31 and 32: ORF ID Gene name Gene function PA14
Page 33 and 34: functional activities it could be s
Page 35 and 36: Figure 1-6 Schematic showing the OR
Page 37 and 38: RhlI Autoinducer RhlR Rhl Regulon G
Page 39 and 40: 1.5.2 Pseudomonas aeruginosa LES Th
Page 41 and 42: 2 Material and Methods 2 MATERIAL A
Page 43 and 44: 2.1.2 Strains used Pseudomonas aeru
Page 45 and 46: Plasmids Plasmid Description Refere
Page 47 and 48: In the case of capture vector const
Page 49 and 50: Primer name Sequence Capture vector
Page 51 and 52: The relevant homology sequence was
Page 53 and 54: 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
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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
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mice exhibited visible symptoms at
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4.4.1 Survival times post-infection
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(P
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4.4.4 Intravenous infection A quest
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Symptom score Symptom score Symptom
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Fold increase Fold increase 15 10 5
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Figure 4-15 Histopathology lung sec
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4.4.7 Progression of disease post-i
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Figure 4-19 Leukocytes numbers moni
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interesting result is that the ∆P
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Unfortunately, 65% of the ORFs with
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morphology. ∆PAPI-1∆PAPI-2 leav
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The intravenous sepsis model data s
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in PAO1(Lee et al. 2006). They show
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shown that deletion of the exoU gen
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5 The role of quorum-sensing in the
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Figure 5-1 shows overnight growth o
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Quorum-sensing is bacterial cell de
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Interestingly, the quorum-sensing d
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Symptom score Symptom score Symptom
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LES431 LESB58 LESB65 LES400 � �
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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
Page 203 and 204:
VAN HEECKEREN, A.M. and SCHLUCHTER,
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