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

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tRNA site. This suggests that two different CRISPR systems arouse independently within E. coli. The data presented does not differentiate between two scenarios for the island. The first scenario being that the genomic island was acquired via horizontal transfer by an ancestral E. coli to E. coli E106 and E. coli APEC O1, B7A and UTI89. The second scenario is that the genomic island is still mobile and has been horizontal transferred between these E. coli strains. The genomic region does contain any known mobility genes, but the literature suggests that the functional role of some CRISPR-associated proteins is to aid horizontal transfer of the CRISPR system (Haft et al. 2005). CRISPR systems have been suggested in the literature to be horizontally transferred, as Jansen et al. (2002) discovered in a larger scale study that there was no general correlation between the phylogenetic relationship of bacteria species and the CRISPR system present. Despite the method of acquiring the genomic island, the maintenance of a genomic island containing a CRISPR system would be beneficial to the bacteria as it plays a role in bacterial defence against bacteriophage (Sorek, Kunin & Hugenholtz 2008). In conclusion, E106-serW genomic island has a functional CRISPR system that appears to target serine tRNA sites within E. coli strains (APEC O1, B7A and UTI89) and closely related species such as Enterobacter spp. 638. The system appears to be closely related to the CRISPR system subtype usually restricted to Yersinia species than those previously identified and highlighted in the literature for other E. coli strains. Analysis highlighted that the Yersinia CRISPR system is found within the core genome within the Yersinia strains, but when found in the other strains highlighted during this project it is found on a genomic island targeting a serine tRNA site. The data have also demonstrated the usefulness of the technique to compare the genomic islands to those previously identified. 3.4 E105-leuX E. coli E105 is a clinical isolate recovered from a human septicaemia infection. The strain was originally part of a strain collection used to evaluate if a particular genome profile correlated with disease (Thani A, PhD thesis, University of 70
Leicester). E105-leuX was the first genomic island captured from a clinical isolate using the yeast-based genomic island capture technique. This section will proceed by analysing the preliminary data generated for the genomic island. This is followed by analysis to determine that the insertion at the leuX tRNA locus is a genomic island (islet). This is followed by analysis of the genomic structure of E105-leuX and concludes with the possible evolution of the genomic island. Preliminary experiments were preformed on the genomic island capture clone to verify the correct recombination event had occurred. Firstly, PCR was performed on the bacterial plasmid preparation to confirm that the plasmid contained TS1 and TS2 as well the positive screening region. The plasmid was also subjected to restriction digestion with I-SceI to get an approximation of the size of the captured insert (the genomic island plus the conserved flanks). As the captured insert was only approximately 11kb, it was within the range that could be amplified using long-range PCR. The capture insert was amplified from the genomic DNA of E. coli E105 using the leuX TS1 U primer and the leuX TS2 D primer. The size of the insert from long-range PCR and the I-SceI restriction digest are shown in Figure 3-6. The SGSP data provided by Ali Thani were re-analysed; construction of the SGSP library, screening and sequencing was performed by Ali Thani. The following analysis was performed independently. A 1.4kb SGSP amplicon was generated from the PstI genomic library of E. coli E105 using the leuX U primer and the T3 universal primer. The amplicon was sent for sequencing with the KS universal primer. Results of the analysis are depicted in Figure 3-13. 71
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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
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
Page 77 and 78: elated genomic islands and this res
Page 79: All the Yersinia species available
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 and 130: 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
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VAN HEECKEREN, A.M. and SCHLUCHTER,
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