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

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Quorum- sensing LES400 LES431 LESB58 LESB65 Deficient Over- expression 158 Over- expression Over- expression PAGI-1 � � � � PAGI-2 � � � � LES Genomic Island -5 � � � � Prophage 2 � � � � Prophage 3 Unconfirmed Unconfirmed � � Prophage 4 � � � � Prophage 5 Unconfirmed Unconfirmed � � TTSS ExoS ExoS ExoS ExoS Colony morphology Infection source SCV SCV SCV NM CF Non-CF CF CF Table 5-9 shows summary of the genomic, phenotype and source of each of the LES isolates Genomic information gathered by written communication with Winstanley, C, University of Liverpool. Key: SCV (Small colony variant) and NM (Normal morphology). Further work could elucidate the genomic differences between the over-expressing quorum-sensing isolates. It would be valuable to compare the two extremes, LES431 and LESB58. LES431-infected mice do not survive beyond 24 hours, the mice have high bacterial numbers within the lungs and the majority have bacteraemia. This is in contrast to LESB58-infected mice, which clear the bacteria within 48 hours and 100% of the mice survive the 96 hour survival experiment. The Caenorhabditis elegans model of infection does not differentiate between the isolates (Winstanley C, University of Liverpool, unpublished), implying that the
isolates have a similar virulence in the model tested. The work described in this thesis shows there is a larger continuum, supporting the notion that in vivo work within mammalian models of infection are important for assessing virulence and for detecting subtle differences in virulence. The acute respiratory model of infection has shown that over-expression of quorum-sensing products is not an over-riding factor and that other factors must also have a vital role in virulence. The results of further investigation between these two isolates could show the mutation or acquisitions that enable LES431 to cross from a CF individual to a non-CF individual. LES431 was isolated from a non-CF parent of a CF patient (McCallum et al. 2002). The isolate was originally labelled as an acute infection isolate (Salunkhe et al. 2005) but the non-CF patient was later re-admitted to hospital with respiratory symptoms in 2007 and LES was recovered from the sputum (Winstanley C, University of Liverpool, unpublished); suggesting a chronic infection. LESB65 represents a strain amidst the two extremes in terms of survival post- infection, as 60% of the mice challenged with LESB65 survived the 96 hour experiment. However, the lung and nasopharynx CFU numbers increased steadily over the same 96 hour period. At 18 hours post-infection, the LES431 and LESB65 have no significant differences (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
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4.4.1 Survival times post-infection
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(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: Figure 5-14 PFGE comparison of the
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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