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

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3.2 Capture experiments involving clinical bacterial strains After standardising the technique, putative genomic islands within bacterial strains with un-sequenced genomes (clinical isolates) were targeted. This section proceeds by describing how the suspected genomic islands were identified. This is followed by a summary of the genomic islands targeted within the clinical isolates and brief descriptions of three targets. Two targets are excluded from this section (E106-serW and E105-leuX), as they are discussed and analysed within their own section. The clinical isolate genomic island targets were determined by tRIP PCR (tRNA site interrogation for pathogenicity islands, prophages and other genomic islands) and SGSP PCR (single genome specific primer). The tRIP PCR and SGSP data were gathered by Ali Thani (Thani A, PhD thesis, University of Leicester) and Ewan Harrison (Harrison E, PhD thesis, University of Leicester, unpublished) for E. coli and P. aeruginosa respectively. The tRIP PCR technique is particularly usually for screening a large number of bacterial strains for genomic islands. The technique involves using a set of primers, one upstream and the other downstream of the tRNA site within the conserved flanks. If the site is empty or contains an islet of ≤5kb, it will lead to a successful amplification of DNA, if there is an insert present then the PCR will fail. The PCR negative strains are then analysed further by SGSP PCR to get a snapshot of the structure of the genomic island and then analysed to determine whether there are similar islands already present within sequenced strains. The clinical isolate targets and the capture vectors used are listed in Table 3-4. The captured insert size is estimated from restriction digestion with I-SceI resolved on a 0.6% agarose gel. Number Organism Capture Vector Size of insert 1 E. coli E105 leuX ~11kb 2 E. coli E106 serW ~14kb 3 E. coli E105 serT Unsuccessful 4 P. aeruginosa KR115 lys10 ~9kb 5 P. aeruginosa KR159 lys10 ~9kb Table 3-4 shows clinical isolate strains targeted for genomic island capture. The sizes of the captured inserts were estimated from I-SceI restriction digest resolved on a 0.6% agarose gel. 58
3.2.1 KR115-lys10 and KR159-lys10 Analysis of the lys10 loci of the two clinical strains of P. aeruginosa KR115 and KR159 suggested a presence of a genomic island. Restriction digestion with I-SceI showed that the genomic islands were similar in size to one another and to that of the PAO1-lys10 genomic island. A panel of restriction enzymes were selected to construct an RFLP for each genomic island. The results are shown in Figure 3-4. This demonstrates the ability of the technique to allow rapid comparison between genomic islands without the need for sequencing. Figure 3-4 shows a 0.6% agarose gel with the restriction profile of PAO1-lys10 and colonies of KR115-lys10 and KR159-lys10 with I-SceI, HindIII and BamHI Lane 1 λHindIII 500ng; Lane 2 -6 shows I-SceI, Lane 7 -11 shows HindIII and Lane 12 -16 shows BamHI, the clones represented are PAO1-lys10, three individual positive colonies of KR115-lys10 and one colony of KR159-lys10 respectively across the different digests 3.2.2 E105-serT 23kb 9.4kb 6.8kb 4.4kb 2.3kb 2.0kb 500b p E105-serT positive clones were recovered, restriction digestion with I-SceI always resulted in a 2.4kb insert (Figure 3-5) even after multiple capture experiments. This result was unusual as tRIP PCR was negative, which suggests the insert was larger than 5kb. SGSP data provided by Ali Thani suggested an interesting genomic island was present. To investigate the problem, the 2.4kb insert was sequenced. The resulting sequence generated aligned to the expected sequence of an empty tRNA site as denoted by E. coli K12 MG1655. The data suggest that the genomic island was too unstable for the capture technique. 1 2 3 4 5 6 7 8 9 10 11 12 13 1 15 59
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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
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
Page 63 and 64: 3.1 Capture experiments involving g
Page 65 and 66: DNA No of colonies Control No DNA 0
Page 67: were designed based on the assumpti
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 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
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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
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VAN HEECKEREN, A.M. and SCHLUCHTER,
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