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

More documents

Recommendations

Info

2.3.1 CAPTURING A GENOMIC ISLAND 43 2.3.1 PREPARATION OF HIGH MOLECULAR WEIGHT GENOMIC DNA 43 2.3.2 PREPARATION OF SPHEROPLASTS 43 2.3.3 SCREENING OF SPHEROPLAST COLONIES: COLONY PCR 44 2.3.4 ELECTROPORATION: TRANSFER OF SUSPECT PLASMIDS TO E. COLI 45 2.3.5 RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) 45 2.3.6 SUB-CLONING OF E106-SERW 45 2.4 PREPARATION OF PSEUDOMONAS AERUGINOSA FOR IN VIVO WORK 46 2.4.1 PREPARATION OF BACTERIAL STOCKS 46 2.4.2 INFECTION DOSE PREPARATION 47 2.5 IN VIVO MICE PROTOCOLS 47 2.5.1 ADMINISTRATION 47 2.5.2 ORGAN RETRIEVAL & CFU COUNTS 48 2.5.3 SYMPTOM SCORING 48 2.5.4 HISTOPATHOLOGY 48 2.5.5 LEUKOCYTE COUNTS 49 2.5.5 STATISTICS 50 3 ANALYSIS OF GENOMIC ISLANDS CAPTURED USING YEAST-BASED TECHNOLOGY: THE GENOMIC ISLAND CAPTURE TECHNIQUE 51 3.1 CAPTURE EXPERIMENTS INVOLVING GENOME SEQUENCED BACTERIAL STRAINS 53 3.1.1 K12 MG1655–SERW 54 3.1.2 K12 MG1655–LEUX 55 3.1.3 PAO1-LYS10 AND PA14-LYS10 56 3.1.4 PA14-LYS47 56 3.2 CAPTURE EXPERIMENTS INVOLVING CLINICAL BACTERIAL STRAINS 58 3.2.1 KR115-LYS10 AND KR159-LYS10 59 3.2.2 E105-SERT 59 3.3 E106-SERW 60 3.3.1 STRUCTURE OF THE E106-SERW 63 3.3.2 ANALYSIS OF E106-SERW CRISPR SYSTEM 67 3.4 E105-LEUX 70 3.4.1 DEFINING E105-LEUX AS A GENOMIC ISLAND (ISLET) 75 3.4.2 THE GENOMIC STRUCTURE OF THE E105-LEUX GENOMIC ISLET 78 3.4.3 EVOLUTION OF THE E105-LEUX GENOMIC ISLET 84 3.5 DISCUSSION 86 3.5.1 GENOMIC ISLAND CAPTURE AS A YEAST-BASED TECHNOLOGY 86 3.5.2 GENOMIC ISLAND CAPTURE AS A GENOMIC ISLAND DISCOVERY TOOL 90 3.6 CONCLUSION AND FURTHER WORK 92 viii
4 THE CONTRIBUTION OF PAPI-1 AND PAPI-2 TO THE VIRULENCE OF PSEUDOMONAS AERUGINOSA PA14 IN A MURINE ACUTE RESPIRATORY INFECTION MODEL 94 4.1 CONSTRUCTION OF THE DELETANT MUTANTS AND IN VITRO DATA 95 4.2 PRELIMINARY STUDIES OF CHRONIC RESPIRATORY INFECTION MODEL 97 4.3 PRELIMINARY STUDIES OF ACUTE RESPIRATORY INFECTION MODEL 100 4.4 THE CONTRIBUTION OF PAPI-1 AND PAPI-2 TO THE VIRULENCE OF PSEUDOMONAS AERUGINOSA PA14 102 4.4.1 SURVIVAL TIMES POST-INFECTION 103 4.4.2 SYMPTOMS SCORES POST-INFECTION 103 4.4.3 BACTERIAL NUMBERS IN NASOPHARYNX, LUNGS AND BLOOD POST-INFECTION 104 4.4.4 INTRAVENOUS INFECTION 107 4.4.5 IMMUNE PROFILE 110 4.4.6 LUNG HISTOPATHOLOGY POST-INFECTION 112 4.4.7 PROGRESSION OF DISEASE POST-INFECTION WITH PA14 ∆PAPI-1∆PAPI-2 115 4.5 SUMMARY OF ACUTE RESPIRATORY OUTCOMES 118 4.6 DISCUSSION 119 4.6.1 THE ROLE OF PAPI-1 DURING ACUTE RESPIRATORY INFECTION 119 4.6.2 THE ROLE OF PAPI-2 DURING ACUTE RESPIRATORY INFECTION 124 4.6.3 PAO1 AS VIRULENT AS PA14 ∆PAPI-2 IN AN ACUTE RESPIRATORY MODEL 125 4.6.4 THE IMMUNE RESPONSE DURING ACUTE RESPIRATORY INFECTION 128 4.7 CONCLUSIONS AND FURTHER WORK 129 5 THE ROLE OF QUORUM-SENSING IN THE VIRULENCE OF PSEUDOMONAS AERUGINOSA LES ISOLATES 131 5.1 IN VITRO ANALYSIS OF THE QUORUM-SENSING EXPRESSION OF THE LES ISOLATES 132 5.2 ACUTE RESPIRATORY INFECTION OUTCOMES 135 5.2.1 SURVIVAL TIMES POST-INFECTION 135 5.2.2 SYMPTOMS SCORES POST-INFECTION 136 5.2.3 BACTERIAL NUMBERS WITHIN THE NASOPHARYNX, LUNGS AND BLOOD POST- INFECTION 140 5.2.4 LUNG HISTOPATHOLOGY POST-INFECTION 146 5.2.5 SUMMARY OF ACUTE RESPIRATORY INFECTION OUTCOMES 152 5.3 DISCUSSION 153 5.3.1 OVER-EXPRESSION OF QUORUM-SENSING PRODUCTS AS A MARKER FOR VIRULENCE IN AN ACUTE RESPIRATORY MODEL 154 5.3.2 THE DIFFERENCES IN VIRULENCE BETWEEN THE LES ISOLATES 156 5.3.3 THE LES ISOLATES VERSUS THE REFERENCE STRAINS, PAO1 & PA14 162 5.4 CONCLUSIONS AND FURTHER WORK 164 6 THESIS CONCLUSION 165 ix
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: Table of contents 1 INTRODUCTION 1
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 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
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 ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?