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

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SMITH, E.E., SIMS, E.H., SPENCER, D.H., KAUL, R. and OLSON, M.V., 2005. Evidence for diversifying selection at the pyoverdine locus of Pseudomonas aeruginosa. Journal of Bacteriology, 187(6), pp. 2138-2147. SMITH, E.E., BUCKLEY, D.G., WU, Z., SAENPHIMMACHAK, C., HOFFMAN, L.R., D'ARGENIO, D.A., MILLER, S.I., RAMSEY, B.W., SPEERT, D.P., MOSKOWITZ, S.M., BURNS, J.L., KAUL, R. and OLSON, M.V., 2006. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proceedings of the National Academy of Sciences of the United States of America, 103(22), pp. 8487-8492. SMITH, R.S., WOLFGANG, M.C. and LORY, S., 2004. An adenylate cyclasecontrolled signaling network regulates Pseudomonas aeruginosa virulence in a mouse model of acute pneumonia. Infection and immunity, 72(3), pp. 1677-1684. SOREK, R., KUNIN, V. and HUGENHOLTZ, P., 2008. CRISPR--a widespread system that provides acquired resistance against phages in bacteria and archaea. Nature reviews.Microbiology, 6(3), pp. 181-186. SPENCER, R.C., 1996. Predominant pathogens found in the European Prevalence of Infection in Intensive Care Study. European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology, 15(4), pp. 281-285. STARKE, J.R., EDWARDS, M.S., LANGSTON, C. and BAKER, C.J., 1987. A mouse model of chronic pulmonary infection with Pseudomonas aeruginosa and Pseudomonas cepacia. Pediatric research, 22(6), pp. 698-702. STOTLAND, P.K., RADZIOCH, D. and STEVENSON, M.M., 2000. Mouse models of chronic lung infection with Pseudomonas aeruginosa: models for the study of cystic fibrosis. Pediatric pulmonology, 30(5), pp. 413-424. STOVER, C.K., PHAM, X.Q., ERWIN, A.L., MIZOGUCHI, S.D., WARRENER, P., HICKEY, M.J., BRINKMAN, F.S., HUFNAGLE, W.O., KOWALIK, D.J., LAGROU, M., GARBER, R.L., GOLTRY, L., TOLENTINO, E., WESTBROCK- WADMAN, S., YUAN, Y., BRODY, L.L., COULTER, S.N., FOLGER, K.R., KAS, A., LARBIG, K., LIM, R., SMITH, K., SPENCER, D., WONG, G.K., WU, Z., PAULSEN, I.T., REIZER, J., SAIER, M.H., HANCOCK, R.E., LORY, S. and OLSON, M.V., 2000. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature, 406(6799), pp. 959-964. TAN, M.W., MAHAJAN-MIKLOS, S. and AUSUBEL, F.M., 1999. Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis. Proceedings of the National Academy of Sciences of the United States of America, 96(2), pp. 715-720. TANG, H.B., DIMANGO, E., BRYAN, R., GAMBELLO, M., IGLEWSKI, B.H., GOLDBERG, J.B. and PRINCE, A., 1996. Contribution of specific Pseudomonas aeruginosa virulence factors to pathogenesis of pneumonia in a neonatal mouse model of infection. Infection and immunity, 64(1), pp. 37-43. 180
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Page 1 and 2:
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
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assessed in number of assays and mo
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TAR cloning is a method designed by
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Figure 1-3 Top plate shows colony g
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Figure 1-5 depicts the principles o
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Balb/c mice. The models described b
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urns-sepsis model, but not in the n
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ORF ID Gene name Gene function PA14
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functional activities it could be s
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Figure 1-6 Schematic showing the OR
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RhlI Autoinducer RhlR Rhl Regulon G
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1.5.2 Pseudomonas aeruginosa LES Th
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2 Material and Methods 2 MATERIAL A
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2.1.2 Strains used Pseudomonas aeru
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Plasmids Plasmid Description Refere
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In the case of capture vector const
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Primer name Sequence Capture vector
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The relevant homology sequence was
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Important to the use of the capture
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2.3.4 Electroporation: Transfer of
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2.4.2 Infection dose preparation Th
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mould was then added to the metal c
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3 Analysis of genomic islands captu
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3.1 Capture experiments involving g
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DNA No of colonies Control No DNA 0
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were designed based on the assumpti
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3.2.1 KR115-lys10 and KR159-lys10 A
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estriction digestion with I-SceI to
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generated. The most significant nuc
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they both produced three fragments
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elated genomic islands and this res
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All the Yersinia species available
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Leicester). E105-leuX was the first
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The strains highlighted with alignm
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predict this protein to be a dnaJ-c
Page 87 and 88:
Figure 3-15 depicts the codon usage
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ORF Gene range Blastx Description y
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ecognition sequences are found with
Page 93 and 94:
(S: 150, E: 1e-37). This predicts t
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having multiple copies of itself. A
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The capture vectors used during thi
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cloning and their findings was inco
Page 101 and 102:
The genomic island capture method c
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unpublished). This was achieved by
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Pseudomonas aeruginosa PA14 (UCBPP-
Page 107 and 108:
stressful’ to the bacteria and th
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log CFU/mg 4 3 2 1 0 0 2 4 6 8 Days
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mice exhibited visible symptoms at
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4.4.1 Survival times post-infection
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(P
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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
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: specificity of chaperone-usher mach
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