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

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from the opposite end using the KS universal primer and generated 364bp of DNA sequence. Based on blastn data APEC O1 and UTI89 (S: 567, E: 1e-158, I: 97%) gave full coverage of the DNA sequence 364/364 nucleotides. There were two additional bacterial species that aligned significantly Citrobacter koseri ATCC BAA 895 and Enterobacter spp 638. Enterobacter spp 536 give two partial alignments, one at nucleotides 1-69 (S: 68, E: 1e-06, I: 79%) and 221-255 (S: 44, E: 3.8, I: 85%) which come from the same ORF Ent638_1406 (255bp). This indicated that the core DNA sequence does not significantly match. Citrobacter koseri matches from 107-257 (S: 78, E: 2e-09, I: 69%) ORF called CKO_0219 (309bp). The nucleotide alignments identified the region as containing DNA sequence for the CRISPR-associated protein Csy4. Figure 3-7 summarises the significant nucleotide alignments of the SGSP amplicon. KS T3 Figure 3-7 depicts the SGSP amplicon generated using the serW U and T3 primers from the E. coli E106 EcoRV genomic library. The most significant alignments are depicted adjacent to the SGSP amplicon. 100bp 1.9kb SGSP amplicon 350bp 400bp Cas_Csy4 infA 1-364 APECO1_1206 1-364 UTI89_C0896 Escherichia coli APEC O1 (CP000468) 107-257 CKO_0219 Citrobacter koseri ATCC BAA-895 (CP000822) A 2.4kb SGSP amplicon was generated from the PstI genomic library of E106 using the serW D primer and the T3 universal primer. The amplicon was sent for sequencing with the KS universal primer and 609bp of DNA sequence was 62 Escherichia coli UTI89 (CP000243) serW U E106
generated. The most significant nucleotide alignment (blastn) was to E. coli strains APEC O1 and UTI89 which produced an identical score to one another (S: 1054, E: 0, I: 98%). Another significant match was Enterobacter spp 638 (S: 255, E: 2e-64, I: 69%). All the alignments mentioned align for the full 609bp of the DNA sequence and aligns to genes predicted to encode the CRISPR-associated protein Cas1. serW D E106 Figure 3-8 depicts the SGSP amplicon generated using the serW D and T3 primers from the E. coli E106 PstI genomic library. The most significant alignments are depicted adjacent to the SGSP amplicon. In conclusion, the preliminary data suggest that a similar genomic island has been found in previously sequenced strains of E. coli. The size and DNA sequence of the captured inserts for these E. coli strains were generated using the GenBank genome sequences. The results for the three E. coli strains were as follows: APEC O1 (13.3kb), B7A (16.6kb) and UTI89 (13.5kb). These captured insert sizes were similar to the estimated size of E106-serW (~14kb). The preliminary data also suggests that the genomic island, E106-serW contains a CRISPR system. The following subsection further investigates the similarity of E106-serW to those previously sequenced putative islands. 3.3.1 Structure of the E106-serW This subsection proceeds by describing the structure of the previously identified genomic islands. It is then followed by the structure of E106-serW as determined by the experimental data. 2.4kb SGSP amplicon Escherichia coli APEC O1 (CP000468) 63 Escherichia coli UTI89 (CP000243) Enterobacter spp 638 (CP000653) 1-609 1-609 1-609 600bp Cas1 APECO1_1211 UTI89_C0890 Ent638_1401 KS T3
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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
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
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: estriction digestion with I-SceI to
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 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
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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
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VAN HEECKEREN, A.M. and SCHLUCHTER,
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