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

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Figure 3-5 shows a 0.6% agarose gel with the restriction profile of E105-serT with I-SceI. Lane 1 λHindIII 500ng; Lane 2-7 E105-serT I-SceI; Lane 8 Generuler DNA ladder mix 500ng In conclusion, this section demonstrates that the genomic island capture technique can be used to compare genomic islands between strains. It can used to generate an RFLP of the complete island to highlight similarities as well as differences between strains. It also shows that there are still possible limitations to technique. The following two sections, will describe two genomic islands also captured from clinical isolates explored in detail. 3.3 E106-serW 23kb 9.4kb 6.8kb 4.4kb 2.3kb 2.0kb 500b p 1 2 3 4 5 6 7 8 E. coli E106 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 Leicester). E106-serW was the second genomic island captured from a clinical isolate. This section will proceed by analysing the preliminary data generated for the genomic island. This is followed by experiments designed to determine the genomic structure of E106-serW and concludes with analysis of the genomic island. Preliminary experiments were preformed on the genomic island capture colony 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 60 10kb 8kb 6kb 5kb 4kb 3.5kb 3kb 2.5kb 2kb 1.5kb 1.2kb 1kb 900bp 800bp 700bp 600bp 500bp 400bp 300bp 200bp 100bp
estriction 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 14kb, it was within the range that could be amplified using long-range PCR. The captured insert was amplified from the genomic DNA of E. coli E106 using the serW TS1 U primer and the serW TS2 D primer. The size of the insert from long-range PCR and the I-SceI restriction digest are shown in Figure 3-6. Figure 3-6 shows a 0.6% agarose gel with the restriction profile of E105-leuX and E106-serW with I-SceI and the long-range PCR product using the primer TS1 U and TS2 D Lane 1 λHindIII 500ng; Lane 2 E105-leuX long-range PCR product; Lane 3 E105-leuX I-SceI Lane 4 E106-serW I-SceI; Lane 5 E106-serW long-range PCR product The SGSP data provided by Ali Thani was re-analysed; construction of the SGSP library, screening and sequencing was performed by Ali Thani (Thani A, PhD thesis, University of Leicester). The following analysis was performed independently. The SGSP data provides a preview of the DNA sequence present at the genomic island flanks. 23kb 9.4kb 6.8kb 4.4kb 2.3kb 2.0kb 500b p 1 A 1.9kb SGSP amplicon was generated from the EcoRV genomic library of E. coli E106 using the serW U primer and the T3 universal primer. The amplicon was sent for sequencing with the serW U primer and 405bp of DNA sequence was produced. The most significant nucleotide alignment (blastn) was to the E. coli strains APEC O1 and UTI89 which produced identical score to one another (S: 719, E: 0, I: 99%). All other significant alignments were to the first 341bp of the amplicon sequence, only the E. coli strains APEC O1, B7A and UTI89 covered the first 401/405 nucleotides. The amplicon contained one gene, infA (58-277 nucleotides) and is part of the conserved U flank. The same SGSP amplicon was also sent for sequencing 61 2 3 4 5
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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
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
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: 3.2.1 KR115-lys10 and KR159-lys10 A
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 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
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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
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VAN HEECKEREN, A.M. and SCHLUCHTER,
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