SMITH, E.E., SIMS, E.H., SPENCER, D.H., KAUL, R. and OLSON, M.V., 2005. Evidence for diversify<strong>in</strong>g selection at <strong>the</strong> pyoverd<strong>in</strong>e locus <strong>of</strong> <strong>Pseudomonas</strong> aerug<strong>in</strong>osa. Journal <strong>of</strong> 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 <strong>Pseudomonas</strong> aerug<strong>in</strong>osa to <strong>the</strong> airways <strong>of</strong> cystic fibrosis patients. Proceed<strong>in</strong>gs <strong>of</strong> <strong>the</strong> National Academy <strong>of</strong> Sciences <strong>of</strong> <strong>the</strong> United States <strong>of</strong> America, 103(22), pp. 8487-8492. SMITH, R.S., WOLFGANG, M.C. and LORY, S., 2004. An adenylate cyclasecontrolled signal<strong>in</strong>g network regulates <strong>Pseudomonas</strong> aerug<strong>in</strong>osa <strong>virulence</strong> <strong>in</strong> a mouse model <strong>of</strong> 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 aga<strong>in</strong>st phages <strong>in</strong> bacteria and archaea. Nature reviews.Microbiology, 6(3), pp. 181-186. SPENCER, R.C., 1996. Predom<strong>in</strong>ant pathogens found <strong>in</strong> <strong>the</strong> European Prevalence <strong>of</strong> Infection <strong>in</strong> Intensive Care Study. European journal <strong>of</strong> cl<strong>in</strong>ical microbiology & <strong>in</strong>fectious diseases : <strong>of</strong>ficial publication <strong>of</strong> <strong>the</strong> European Society <strong>of</strong> Cl<strong>in</strong>ical Microbiology, 15(4), pp. 281-285. STARKE, J.R., EDWARDS, M.S., LANGSTON, C. and BAKER, C.J., 1987. A mouse model <strong>of</strong> chronic pulmonary <strong>in</strong>fection with <strong>Pseudomonas</strong> aerug<strong>in</strong>osa and <strong>Pseudomonas</strong> cepacia. Pediatric research, 22(6), pp. 698-702. STOTLAND, P.K., RADZIOCH, D. and STEVENSON, M.M., 2000. Mouse models <strong>of</strong> chronic lung <strong>in</strong>fection with <strong>Pseudomonas</strong> aerug<strong>in</strong>osa: models for <strong>the</strong> study <strong>of</strong> 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 <strong>of</strong> <strong>Pseudomonas</strong> aerug<strong>in</strong>osa PA01, an opportunistic pathogen. Nature, 406(6799), pp. 959-964. TAN, M.W., MAHAJAN-MIKLOS, S. and AUSUBEL, F.M., 1999. Kill<strong>in</strong>g <strong>of</strong> Caenorhabditis elegans by <strong>Pseudomonas</strong> aerug<strong>in</strong>osa used to model mammalian bacterial pathogenesis. Proceed<strong>in</strong>gs <strong>of</strong> <strong>the</strong> National Academy <strong>of</strong> Sciences <strong>of</strong> <strong>the</strong> United States <strong>of</strong> 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 <strong>of</strong> specific <strong>Pseudomonas</strong> aerug<strong>in</strong>osa <strong>virulence</strong> factors to pathogenesis <strong>of</strong> pneumonia <strong>in</strong> a neonatal mouse model <strong>of</strong> <strong>in</strong>fection. Infection and immunity, 64(1), pp. 37-43. 180
VAN HEECKEREN, A.M. and SCHLUCHTER, M.D., 2002. Mur<strong>in</strong>e models <strong>of</strong> chronic <strong>Pseudomonas</strong> aerug<strong>in</strong>osa lung <strong>in</strong>fection. Laboratory animals, 36(3), pp. 291-312. VINCZE, T., POSFAI, J. and ROBERTS, R.J., 2003. NEBcutter: A program to cleave DNA with restriction enzymes. Nucleic acids research, 31(13), pp. 3688- 3691. WALKER, G.M., 1998. Yeast physiology and biotechnology. Chichester: J. Wiley. WANG, R.F. and KUSHNER, S.R., 1991. Construction <strong>of</strong> versatile low-copynumber vectors for clon<strong>in</strong>g, sequenc<strong>in</strong>g and gene expression <strong>in</strong> Escherichia coli. Gene, 100, pp. 195-199. WILLIAMS, R.J., 2003. Restriction endonucleases: classification, properties, and applications. Molecular biotechnology, 23(3), pp. 225-243. WILSON, K., 2001. Preparation <strong>of</strong> genomic DNA from bacteria. Current protocols <strong>in</strong> molecular biology / edited by Frederick M.Ausubel ...[et al.], Chapter 2, pp. Unit 2.4. WINSTANLEY, C., LANGILLE, M.G., FOTHERGILL, J.L., KUKAVICA- IBRULJ, I., PARADIS-BLEAU, C., SANSCHAGRIN, F., THOMSON, N.R., WINSOR, G.L., QUAIL, M.A., LENNARD, N., BIGNELL, A., CLARKE, L., SEEGER, K., SAUNDERS, D., HARRIS, D., PARKHILL, J., HANCOCK, R.E., BRINKMAN, F.S. and LEVESQUE, R.C., 2009. Newly <strong>in</strong>troduced genomic prophage islands are critical determ<strong>in</strong>ants <strong>of</strong> <strong>in</strong> vivo competitiveness <strong>in</strong> <strong>the</strong> Liverpool Epidemic Stra<strong>in</strong> <strong>of</strong> <strong>Pseudomonas</strong> aerug<strong>in</strong>osa. Genome research, 19(1), pp. 12-23. WOLFGANG, M.C. and ET AL, 27 April 2007-last update, protocol for yeast recomb<strong>in</strong>ational clon<strong>in</strong>g. Available: http://mml.sjtu.edu.cn/MobilomeFINDER/YCV_data/Protocol_for_Yeast_Recombi national_clon<strong>in</strong>g.pdf. WOLFGANG, M.C., KULASEKARA, B.R., LIANG, X., BOYD, D., WU, K., YANG, Q., MIYADA, C.G. and LORY, S., 2003. Conservation <strong>of</strong> genome content and <strong>virulence</strong> determ<strong>in</strong>ants among cl<strong>in</strong>ical and environmental isolates <strong>of</strong> <strong>Pseudomonas</strong> aerug<strong>in</strong>osa. Proceed<strong>in</strong>gs <strong>of</strong> <strong>the</strong> National Academy <strong>of</strong> Sciences <strong>of</strong> <strong>the</strong> United States <strong>of</strong> America, 100(14), pp. 8484-9. YANAGIHARA, K., TOMONO, K., SAWAI, T., KUROKI, M., KANEKO, Y., OHNO, H., HIGASHIYAMA, Y., MIYAZAKI, Y., HIRAKATA, Y., MAESAKI, S., KADOTA, J., TASHIRO, T. and KOHNO, S., 2000. Comb<strong>in</strong>ation <strong>the</strong>rapy for chronic <strong>Pseudomonas</strong> aerug<strong>in</strong>osa respiratory <strong>in</strong>fection associated with bi<strong>of</strong>ilm formation. <strong>The</strong> Journal <strong>of</strong> antimicrobial chemo<strong>the</strong>rapy, 46(1), pp. 69-72. YOON, S.H., PARK, Y.K., LEE, S., CHOI, D., OH, T.K., HUR, C.G. and KIM, J.F., 2007. Towards pathogenomics: a web-based resource for pathogenicity islands. Nucleic acids research, 35(Database issue), pp. D395-400. 181
- 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 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: specificity of chaperone-usher mach