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

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5.3.1 Over-expression of quorum-sensing products as a marker for virulence in an acute respiratory model Several LES isolates have been highlighted to exhibit an over-expression of quorum-sensing products labelled as the hypervirulence phenotype (Fothergill et al. 2007, Salunkhe et al. 2005). An increased virulence of LES isolates with the hypervirulence phenotype was first noted in a Drosophila fly model of infection (Salunkhe et al. 2005); when comparing over-expressing quorum-sensing products isolate LES431 to that of a quorum-sensing deficient isolate LES400. These findings were later replicated in a C. elegans model of infection (Winstanley C, University of Liverpool, unpublished), where isolates with the hypervirulence phenotype (including LESB58 and LESB65) had a lower LT50 in the slow killing assay. Previously published work has shown that a deficiency in the quorum-sensing system can reduce virulence in a number of acute murine infection models. The use of quorum-sensing deficient mutants in a murine burns injury model led to a reduction in mortality of the mice and the ability of the bacteria to disseminate (Rumbaugh et al. 1999). In a murine interperiontal infection model they noted that clearance of the bacteria was more rapid in quorum-sensing deficient mutants (Christensen et al. 2007). There have also been two previous studies (Pearson et al. 2000, Tang et al. 1996) that have shown that in a neonatal murine acute respiratory model the loss of quorum-sensing related genes resulted in a reduction in morbidity and mortality. The models involved using 7 to 10 day old Balb/c mice inoculating them with either 5x10 6 CFU (Tang et al. 1996) or 1 x10 9 CFU (Pearson et al. 2000) per mice. Pearson et al, constructed a PAO1 mutant which lacked two quorum- sensing associated genes (lasI and rhlI), termed PAO_JP2. Using this mutant in the neonatal respiratory model increased the survival of the mice (21% versus 5%), as well reducing the chance of developing pneumonia (56% versus 10%) and bacteraemia (~50% versus ~15%) 18 hours post-infection when compared with the wild-type, PAO1. Tang et al, presented similar results using a lasR mutant of PAO1. 154
Tang et al. (1996) performed additional in vitro experiments with the lasR mutant. One experiment involved an adherence assay on nasal polyp cells and the results showed that the LasR mutant had 26% binding in contrast to wild-type PAO1 which had 58%. The authors suggested that the difference in binding was due to LasR- dependent exoproducts which aid attachment to the cell surface. The data presented in this thesis supports this finding. LES400, which is a quorum-sensing deficient due to a mutation in the lasR gene, exhibited a poor recovery of CFU from the nasopharynx. In contrast, over-expressing quorum-sensing isolates, LES431 and LESB65 had significantly higher (P
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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
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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
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(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
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The genomic island capture method c
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unpublished). This was achieved by
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Pseudomonas aeruginosa PA14 (UCBPP-
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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
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: post-infection to that of healthy m
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,
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