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

More documents

Recommendations

Info

generated from the subcloning experiment does not verify the presence of E106serW_1. But the gene is found within the other three related genomic islands and the size of the captured insert is conserved thereby strongly supporting its presence. The DNA sequence data generated does not verify the presence of E106_Cas-Csy1. The EcoRV RFLP is consistent with the other related serW- associated regions and therefore the restriction site that is found within Cas_Csy1 is maintained thereby supporting its presence. Figure 3-10 shows the DNA structure of E106-serW (~14kb). The text in grey represents data taken from SGSP data generated by Ali Thani. Restriction sites are represented by inverted triangles: PstI = red, KpnI = purple and EcoRV = orange. In conclusion, this data shows that the insertion at the serW tRNA site within E. coli E106 is a CRISPR system. The results demonstrated that the E106-serW genomic island is closely related to APEC O1 and UTI89 serW–associated genomic islands. The E106-serW genomic island contains a number of CRISPR-associated genes and also two hypothetical genes. E106serW_1 and E106serW_2 are completely hypothetical proteins, containing no known conserved proteins domains but have counterparts in all three related serW-associated genomic islands. The data highlights the limitations of SGSP as restriction sites assumed to be presence within the genomic island are in reality not. The data show that there is a single PstI site found within E106-serW (Figure 3-10). The SGSP data suggests there is an additional restriction site 2.4kb away. The subcloning data generated a 4kb fragment in the same direction. The SGSP data also suggests there is an additional EcoRV site that generates a 2kb fragment that includes E106serW_Cas- Csy4 and infA (Figure 3-10). There is no EcoRV site proximal to infA in any of the 66
elated genomic islands and this restriction fragment was not presence in the E106- serW EcoRV RFLP. 3.3.2 Analysis of E106-serW CRISPR system This subsection aims to provide a brief overview of CRISPR systems. This is followed by discussion and comparison of the E106-serW CRISPR system to other CRISPR systems identified in the literature and databases. The CRISPR system is found in a wide range of bacterial species, it is currently found among 24 individual sequenced species ranging from P. aeruginosa PA14, Enterobacter spp 638 to Yersinia pestis. A CRISPR system consists of two components; the CRISPR array and the CRISPR-associated proteins. The CRISPR array consists of a number of palindromic repeat DNA sequences separated by DNA spacers of species-defined length. These CRISPR array are found in close proximity to a set of proteins known as CRISPR-associated proteins (Cas proteins). The CRISPR-associated proteins are divided into two categories. The first category is the core set of cas genes of which there are 6 (Peterson et al. 2001) named cas1 to cas6. All genomes containing a CRISPR system contain cas1 and one or more of the additional core cas genes (Jansen et al. 2002). The second category of genes associated with CRISPR systems define the subtype which are usually species related, there are 7 to 8 subtypes (Sorek, Kunin & Hugenholtz 2008). The role of CRISPR systems has only recently been elucidated and is shown in Figure 3-11. The CRISPR system is suggested to work in a RNA interference dependent manner (Sorek, Kunin & Hugenholtz 2008). The DNA spacers between the CRISPR repeats have identified as bacteriophage DNA. The CRISPR system acts to generate small RNAs that bind to complementary bacteriophage DNA which triggers degradation of the bacteriophage DNA. This acts to protect the bacterium from infection. 67
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: they both produced three fragments
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?