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

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1.1 Pseudomonas aeruginosa and its clinical relevance Pseudomonas aeruginosa is a Gram-negative, free-living, aerobic bacillus that is commonly isolated from soil and water. P. aeruginosa is an opportunistic pathogen to both humans and plants. It usually targets immunocompromised patients such as burn victims and patients with AIDS, cancer or cystic fibrosis (CF). It is one of the top three hospital-acquired pathogens (Spencer 1996) and is most commonly isolated from patients hospitalised for more than a week (Qarah S & Cunha BA). Infection can results in urinary tract infection, pneumonia and septicaemia. Acquisition of this organism is associated with high mortality and can cause fatality within 24 hours. The high mortality rate is partially explained by P. aeruginosa being intrinsically resistance to a broad range of antibiotics and its ability to rapidly acquire resistance genes via horizontal gene transfer. A restricted number of antibiotics are effective against P. aeruginosa these include fluoroquinolones, gentamicin and imipenem and susceptibility to these can vary between strains. Table 1-1 shows common virulence factors identified within P. aeruginosa. 2
Cell-surface Proteases Haemolysins Other factors Virulence Factor Role Pili Adhesion to surfaces and Flagella Motility Type Three Secretion System (including toxins ExoS, ExoT, ExoU) 3 motility (type IV) Translocates numerous effectors into host cells Alginate Biofilm Adhesion to surfaces and LasA & LasB Alkaline Protease Phospholipase C Rhamnolipid defence against the environment Aid tissue invasion Aid tissue invasion Pyocyanin Antimicrobial ExoA Inhibits protein biosynthesis Table 1-1 Summary of the common virulence factors within P. aeruginosa The work described in this thesis investigates P. aeruginosa virulence in a murine respiratory model of infection. Therefore the focus of this introduction is to provide a background of P. aeruginosa as a respiratory pathogen. P. aeruginosa is associated with two types of respiratory infection within humans; acute and chronic. Acute infection usually occurs in hospitalised patients using a ventilator to aid breathing. P. aeruginosa is one of the leading causes of hospital-acquired pneumonia, causing significant morbidity and mortality. Ventilator-associated pneumonia caused by infection with P. aeruginosa is reported to have a high mortality rate of 70-80% (Chastre & Fagon 2002). Research into the virulence of P. aeruginosa in ventilator-associated pneumonia has focused on the type III secretion system (TTSS), which secretes four exotoxins known as ExoS, ExoT, ExoU and ExoY. ExoU is a potent effector of the TTSS and in ventilator-associated pneumonia it is correlated with more severe disease in terms of morbidity and mortality. It has also been reported that 40% of the isolates from these cases harbour
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: 1 Introduction 1 INTRODUCTION 1 1.1
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 and 26: Figure 1-5 depicts the principles o
Page 27 and 28: Balb/c mice. The models described b
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
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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
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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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