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

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The authors generated transposon mutants and created in-frame deletions within PAPI-1 and demonstrated a reduction in mortality compared to wild-type PA14 infected mice within a number of ORFs using a mouse burns sepsis model. The work presented in this thesis aims to directly assess the contribution of PAPI-1 and PAPI-2 to the virulence of P. aeruginosa PA14 within a murine respiratory model by using complete pathogenicity island deletant mutants. 1.4.3 PAPI-1 PAPI-1 is a 108kb pathogenicity island with 108 ORFs that is associated with the lysine tRNA adjacent to PA4541 and is a member of the pKLC102 island family (Klockgether et al. 2007). It contains a number of virulence factors, including a type IV B pili, pyocin S5, cupD system, as well as PvrR, a two component response regulator involved in biofilm synthesis. The large majority of encoded proteins within this island have no assigned function. It contains many features that are exhibited by genomic islands (He et al. 2004): (1) associated with a tRNA site (2) G+C content different from that of the core genome (59.7% compared with 66.6% of core genome) (3) bound by direct repeats (4) containing mobility gene i.e. integrases and transposases. An interesting property of PAPI-1 is its ability to excise from the genome and be maintained as a plasmid (Qiu, Gurkar & Lory 2006). Table 1-4 contains all the ORFs within PAPI-1 that have been assigned a function. 20
ORF ID Gene name Gene function PA14_60140 xerC Integrase PA14_60120 dcd_2 Deoxycytidine deaminase PA14_60100 dtd Deoxycytidine triphosphate deaminase PA14_60050 parE Plasmid stabilization protein parE PA14_59960 dsbG Putative protein-disulfide isomerase PA14_59790 pvrR Regulator of two-component regulatory system; adhesion and antibiotic resistance PA14_59780 rcsC Sensor of two-component regulatory system PA14_59770 rcsB Regulator of two-component regulatory system PA14_59760 cupD5 Probable pili assembly chaperone / adhesion and protein secretion PA14_59750 cupD4 Adhesion and protein secretion PA14_59740 cupD3 Probable fimbrial biogenesis usher / adhesion and protein secretion PA14_59730 cupD2 Probable pili assembly chaperone / adhesion and protein secretion PA14_59720 cupD1 Probable fimbrial precursor / adhesion and protein secretion PA14_59390 pilM2 Type IV B pilus / adhesion and and protein secretion PA14_59380 pilV2 Type IV B pilus / adhesion and and protein secretion PA14_59370 pilT2 Type IV B pilus / putative peptidase / adhesion and and protein secretion PA14_59360 pilS2 Type IV B pilus / adhesion and and protein secretion PA14_59350 pilR2 Type IV B pilus / adhesion and and protein secretion PA14_59340 pilQ2 ATPase / Type IV B pilus / adhesion and and protein secretion PA14_59330 pilP2 Type IV B pilus / adhesion and and protein secretion PA14_59320 pilO2 Type IV B pilus / adhesion and and protein secretion PA14_59310 pilN2 Secretin / Type IV B pilus / adhesion and and protein secretion PA14_59300 pilL2 Type IV B pilus / adhesion and and protein secretion PA14_59290 Unassigned Colicin immunity protein PA14_59280 Unassigned Colicin-like toxin (pyocin S5) PA14_59270 Unassigned DNA Helicase PA14_59240 topA Topoisomerase I PA14_59210 ssb Single-stranded DNA binding protein PA14_59070 dnaB DNA replication and recombination PA14_59060 Unassigned Putative phage protein PA14_59010 soj Chromosome partitioning Table 1-4 Annotation of all ORFs that have assigned a function within the pathogenicity island, PAPI-1 in Pseudomonas aeruginosa PA14 (He et al. 2004) – Reproduced with modification by permission from PNAS 2004 101(8): 2530-2535. Copyright 2004 National Academy of Sciences, U.S.A. Based on the literature available the loss of PAPI-1 from PA14 should result in a reduction in virulence (Choi et al. 2002, He et al. 2004). The remainder of this section describes genes that have been previously highlighted in the literature to be involved in a reduction in virulence and their ORFs have been assigned a function. To date 65% of the ORFs within PAPI-1 have no known function and are labelled 21
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: urns-sepsis model, but not in the n
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
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Leicester). E105-leuX was the first
Page 83 and 84:
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
Page 97 and 98:
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
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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
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LES431 LESB58 LESB65 LES400 � �
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Additional comparisons of the CFU r
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log CFU/mg log CFU/mg log CFU/ml 5
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For LESB58-infected mice the histop
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Strain Mouse Timepoint HB CI GC Sco
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Score: 4 HB Score: 4 Score: 4 B HCI
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post-infection to that of healthy m
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Tang et al. (1996) performed additi
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Figure 5-14 PFGE comparison of the
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isolates have a similar virulence i
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The data presented in this thesis s
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than PAO1. Despite the bacterial lu
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6 Thesis conclusion The underlying
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Hopefully the methodologies describ
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1 2 3 4 5 6 7 8 9 10 11 12 13 λHin
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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
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AQ: 32 AQ: 33 AQ: 34 tapraid5/z9d-j
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AQ: 40 AQ: 41 DISCUSSION tapraid5/z
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AQ: 48 tapraid5/z9d-jid/z9d-jid/z9d
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tapraid5/z9d-jid/z9d-jid/z9d01009/z
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8 Bibliography AL-ALOUL, M., CRAWLE
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D'ARGENIO, D.A., WU, M., HOFFMAN, L
Page 197 and 198:
genomic islands in Pseudomonas aeru
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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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