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

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transferred to a pre-chilled falcon tube and incubated on ice for 20 minutes. The culture was then centrifuged at 1,500g, 4ºC for 15 minutes and the supernatant was discarded. The pellet was washed three times in 10% (v/v) ice-cold glycerol. After the final wash, the pellet was re-suspended in 5ml of 10% (v/v) ice-cold glycerol. The suspension was then separated into 100µl aliquots in pre-chilled microcentrifuge tubes. The aliquots were snap frozen in an ethanol/dry ice bath and stored at -70ºC. 2.1.4 Preparation of chemical competent cells A 5ml culture of E. coli DH10B was grown overnight 200rpm, 37ºC in LB. 500µl of the overnight culture was added to 400ml of LB and incubated at 200rpm, 37ºC until culture reached an OD600 of 0.3. The culture was centrifuged at 1,500g, 10 minutes, 4ºC. The supernatant was discarded and the pellet was washed once in 50ml 100mM magnesium chloride. The cells were then re-suspended in 50ml 100mM calcium chloride and incubated on ice for 10 minutes and then centrifuged at 1,500g, 4ºC for 15 minutes. After the final wash, the pellet was re-suspended in 4ml of 100mM calcium chloride/15% glycerol solution. The suspension was then separated into 50µl aliquots in pre-chilled microcentrifuge tubes and stored at -70ºC. 2.1.5 Bacteria plasmid preparation All plasmid preparations were performed using the QIAprep Spin Mini-prep kit (Qiagen, UK) as described in the manual for mini-preps. For larger plasmid preparations, HiSpeed Plasmid Midi Kit (Qiagen, UK) or HiSpeed Plasmid Maxi Kit (Qiagen, UK) was used. The protocols followed were as stated by the manufacturer. 2.1.6 Yeast plasmid preparation All plasmid preparations were performed using the Zymoprep Yeast Plasmid Mini- prep I kit (Zymoresearch, USA). The following modifications were made to the manufacturer’s protocol, each preparation used 5ml of overnight yeast culture and an additional wash was performed in 1ml 70% ethanol after the stated isopropanol wash and the final re-suspension was in 10µl nanopure water. 36
In the case of capture vector construction, 1ml of nanopure water was added to a single plate and scraped with a sterile spreader to produce a suspension. 100µl of the suspension was added to a microcentrifuge tube and then the protocol provided with the kit was followed with the modification as stated above. 2.1.7 Restriction digestion All restriction enzyme were performed using enzymes purchased from New England Biolabs (NEB, UK), using the Manufacturer’s instructions. All restriction digests were incubated at 37ºC overnight. Digestion of pLLX13 was performed in a final volume of 20µl digestion using 200ng of pLLX13 and 2U of MluI enzyme for 3hrs. Digestion of capture vectors with MluI were performed by using a complete Qiagen Midi plasmid preparation in a final volume of 200µl, using 3µl of enzyme. Digestion of a capture vector with insert was generally performed using 5µl of Qiagen plasmid preparation in a final digestion volume of 30µl, using 0.5µl of enzyme. 2.1.8 Gel electrophoresis Gel electrophoresis was used to estimate size and quantity of DNA. Agarose gels were produced using an appropriate w/v of agarose (Multipurpose agarose, Bioline, UK) in TAE buffer (40mM Tris-base, Glacial acetic acid, 1mM EDTA, pH 7). The mixture was heated until homogenised, cooled to 56ºC. Ethidium bromide was added to a final concentration of 0.5 µg/ml and poured into an appropriate size gel tray. Two different DNA ladders were generally used; λHindIII (Fermentas, UK) and GeneRuler® DNA ladder mix (Fermentas, UK), at a concentration of 500ng/6µl. 0.6% (w/v) agarose gels were used for capture clone RFLP digestion and quantifying capture vector after linearisation and 1% (w/v) agarose gels were used for colony PCR, otherwise a 0.8% (w/v) agarose gel was used. 37
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: Plasmids Plasmid Description Refere
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 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
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
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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,
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