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

More documents

Recommendations

Info

shows the success of the genomic island capture technique for the use in characterisation of novel genomic islands. In conjugation with other data generated within this project, the technique has been successfully used with bacterial species other than P. aeruginosa. 3.5 Discussion This project explored the characterisation of pathogenicity islands in vitro. The aim of the project was to identify, capture and characterise genomic islands. To achieve this aim involved development of the genomic island capture technique used by Wolfgang et al. (2003) and the production of a generic system by targeting bacterial species other than P. aeruginosa. To achieve these aims, the development of the methodology was required to expand the use of the technique and to streamline the recovery of the genomic islands. 3.5.1 Genomic island capture as a yeast-based technology In terms of history of the genomic island capture technique, the current application was piloted by Raymond et al. (2002a). Their first contribution to this application was the introduction of a counterselectable marker, cycloheximide into the capture vector (Raymond, Sims & Olson 2002b). They suggested (Raymond, Sims & Olson 2002b) and proved (Raymond et al. 2002a) that the use of a counterselectable markers allowed capturing directly from genomic DNA by increasing the percentage of clones that harboured a capture vector that had undergone homologous recombination as opposed to those that harboured only the capture vector. Raymond et al. (2002a) also piloted the first use of the technique to capture variable genomic regions (O-antigen LPS region) from P. aeruginosa and were the first to report using bacterial DNA in the capture process. Wolfgang et al. (2003) adapted this technique further by modifying the structure of the capture vector. Wolfgang et al. (2003) also used the method to target a variable region within the P. aeruginosa genome and highlighted its use as a technique to capture genomic islands. They constructed and used a capture vector to target the lys10 tRNA site (p0975-0989capture) and captured genomic islands from a number of P. aeruginosa strains. The technique as proposed by Wolfgang et al, 2003 is the basis of this project. 86
The capture vectors used during this project were constructed using the Wolfgang et al, 2003 methodology. Six capture vectors were constructed targeting tRNA sites in both P. aeruginosa and E. coli. Five of which were designed during this project, the additional capture vector lys10 is described in Wolfgang et al. (2003). Wolfgang et al, 2003 designed their capture vectors by only specifying the requirement for TS1 and TS2 to be 1kb DNA regions upstream and downstream of the target region. During this project, the process was streamlined by utilising the MobilomeFinder bioinformatics tools (Ou et al. 2007). MobilomeFinder was able to define a 2kb conserved region flanking upstream and downstream of the tRNA site, of all (majority) of strains within a species with a GenBank available genome. This ensures the capture vector usefulness in a generic system, as it could be used against all strains within the target species and not solely the reference genome strain. Admittedly, this would have difficult at the time for Wolfgang et al, as there was only one P. aeruginosa genome sequence publicly available, P. aeruginosa PAO1. This could be considered as a limitation of the technique. The designing of the capture vectors requires at least one genome sequence being available and further genomes to increase the usefulness of the capture vector. The capture vector design protocol for this project also explicitly states that either TS1 or TS2 should contain a partial gene as this aids the design of the screening primers (Section 2.2.3). The assembly of the capture vector was also streamlined, as the recovery of the capture vector was not explicitly explained. A protocol was devised based on a research paper by Raymond et al. (2002b) to pool the colonies and perform a single yeast plasmid preparation (Section 2.2.7). Another technique that was intended to streamline the recovery of the capture vector was direct electroporation. This involved transferring the capture vector directly from the yeast colonies to E. coli by placing them together within an electroporation cuvette. These experiments were not as efficient as published in the literature (Marcil & Higgins 1992) and the technique was not included as part of the final methodology. The current structure of the capture vector, as a low copy yeast vector seems to impede this. This could be investigated further in future work. The methods and protocols in this thesis should streamline the process of design, construction and recovery of capture vectors. Recent literature has suggested techniques to improve the efficiency of transformation-associated recombination (TAR) cloning by re-structuring the 87
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 and 12:
1 Introduction 1 INTRODUCTION 1 1.1
Page 13 and 14:
Cell-surface Proteases Haemolysins
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
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 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: having multiple copies of itself. A
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
Page 131 and 132: Unfortunately, 65% of the ORFs with
Page 133 and 134: morphology. ∆PAPI-1∆PAPI-2 leav
Page 135 and 136: The intravenous sepsis model data s
Page 137 and 138: in PAO1(Lee et al. 2006). They show
Page 139 and 140: shown that deletion of the exoU gen
Page 141 and 142: 5 The role of quorum-sensing in the
Page 143 and 144: Figure 5-1 shows overnight growth o
Page 145 and 146: Quorum-sensing is bacterial cell de
Page 147 and 148:
Interestingly, the quorum-sensing d
Page 149 and 150:
Symptom score Symptom score Symptom
Page 151 and 152:
LES431 LESB58 LESB65 LES400 � �
Page 153 and 154:
Additional comparisons of the CFU r
Page 155 and 156:
log CFU/mg log CFU/mg log CFU/ml 5
Page 157 and 158:
For LESB58-infected mice the histop
Page 159 and 160:
Strain Mouse Timepoint HB CI GC Sco
Page 161 and 162:
Score: 4 HB Score: 4 Score: 4 B HCI
Page 163 and 164:
post-infection to that of healthy m
Page 165 and 166:
Tang et al. (1996) performed additi
Page 167 and 168:
Figure 5-14 PFGE comparison of the
Page 169 and 170:
isolates have a similar virulence i
Page 171 and 172:
The data presented in this thesis s
Page 173 and 174:
than PAO1. Despite the bacterial lu
Page 175 and 176:
6 Thesis conclusion The underlying
Page 177 and 178:
Hopefully the methodologies describ
Page 179 and 180:
1 2 3 4 5 6 7 8 9 10 11 12 13 λHin
Page 181 and 182:
AQ: 1 AQ: 2 AQ: 3 AQ: 4 AQ: 5 AQ: 6
Page 183 and 184:
AQ: 18 AQ: 19 AQ: 20 F1 AQ: 21 tapr
Page 185 and 186:
AQ: 32 AQ: 33 AQ: 34 tapraid5/z9d-j
Page 187 and 188:
AQ: 40 AQ: 41 DISCUSSION tapraid5/z
Page 189 and 190:
AQ: 48 tapraid5/z9d-jid/z9d-jid/z9d
Page 191 and 192:
tapraid5/z9d-jid/z9d-jid/z9d01009/z
Page 193 and 194:
8 Bibliography AL-ALOUL, M., CRAWLE
Page 195 and 196:
D'ARGENIO, D.A., WU, M., HOFFMAN, L
Page 197 and 198:
genomic islands in Pseudomonas aeru
Page 199 and 200:
MURRAY, A.W. and SZOSTAK, J.W., 198
Page 201 and 202:
specificity of chaperone-usher mach
Page 203 and 204:
VAN HEECKEREN, A.M. and SCHLUCHTER,
show all

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

Create successful ePaper yourself

Delete template?

Save as template?