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

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To reflect the two patient types, the project outlined in this thesis will attempt to develop two models of respiratory infection (acute and chronic) to explore the role of pathogenicity islands in virulence of P. aeruginosa. 1.4.2 Pseudomonas aeruginosa PA14 Pseudomonas aeruginosa PA14 (UCBPP-PA14) is a highly virulent wild-type reference strain that has been used extensively to test the contribution of virulence factors to disease. The P. aeruginosa strain PAO1, another wild-type reference strain considered the laboratory strain of P. aeruginosa, has been shown to be less virulent than P. aeruginosa PA14 in a number of models of infection (He et al. 2004, Rahme et al. 2000, Tan, Mahajan-Miklos & Ausubel 1999). It has been shown in mouse and plant infection models to be less virulent than P. aeruginosa PA14 despite having the same host specificity (He et al. 2004). In a mouse burns- sepsis model, where a thermal injury is inflicted and P. aeruginosa is applied to the wound, PAO1 causes 25% mortality compared with 77% mortality caused by PA14 (Rahme et al. 2000). In the leaf infiltration model, where the leaf is infected with P. aeruginosa and the bacterial load at 5 days post-infection is determined, PAO1 was recovered at 8.0 × 10 5 cfu/cm 2 of leaf area in comparison to 2.6 × 10 7 cfu/cm 2 in PA14 (Rahme et al. 2000). The difference in virulence is also supported by the C. elegans model of infection, where the LT50 in this slow killing model was 37.8 hours for PA14 and 68.2 hours for PAO1 (Tan, Mahajan-Miklos & Ausubel 1999). This difference in virulence has prompted studies to determine the genomic differences between the two strains. There have been two previous studies to analyse the genomic difference between PAO1 and PA14. The first study was in 2002 and it used representational difference analysis (RDA), to compare the genome of these two strains to look for specific differences (Choi et al. 2002). This method led to the discovery of 4 genes that differed significantly between the strains. One of the genes highlighted is found within PA14 pathogenicity island PAPI-1, uvrD. The authors also discovered a gene that was a homologue of ybtQ, an ABC transporter found in Yersinia pestis. They produced a PA14 mutant in ybtQ and tested its virulence in a number of infection models, and it attenuated PA14 to levels comparable with PAO1 in both the wax moth Galleria mellonella and mouse 18
urns-sepsis model, but not in the nematode worm Caenorhabditis elegans model. The second study in 2006 involved sequencing the genome of PA14 and directly analysed the two genomes together (Lee et al. 2006). Their aim was to discover if the difference in virulence between PAO1 and PA14 was due to the presence of pathogenicity islands present in PA14 but absent in PAO1. They discovered that PA14 has a slightly larger genome than PAO1, 6.5Mb versus 6.3Mb and that PA14 has 58 additional gene clusters than PAO1. They tried to analyse these 58 clusters in two ways. Firstly, they screened 20 P. aeruginosa strains (including PA14 and PAO1) and compared their virulence in a C. elegans model of infection and used this data to rank the strains. They then proceeded to analyse the strains for the presence of PA14-specific gene regions with respect to PAO1. The results demonstrated no correlation between virulence and the presence of PA14-specific gene regions. A second approach taken was to create a transposon insertion mutant library for PA14. They screened the library using the C. elegans model of infection for those in the PA14-specific gene regions for attenuation. They discovered 9 genes that caused attenuation. The presence of these regions within the other P. aeruginosa strains, showed no set trend as to presence of these PA14-specific regions and the ranked virulence. This led the group to conclude that the presence of these gene regions within another P. aeruginosa strain was not an indicator of its virulence within the C. elegans model of infection. This study did not take into account the individual contribution to virulence each gene cluster makes and therefore the ranking of virulence may not give a true reflection of PA14 virulence. Despite the extensive use of plants and invertebrate models of infection for P. aeruginosa, in order to evaluate how these virulence factors affect and interact in human infection they must be tested in a mammalian model of infection. The host immune response is more complex in mammals and therefore a murine model of infection such as described in this thesis will provide a more valuable insight into human disease. A recent study (He et al. 2004) suggested that differences in virulence between PAO1 and PA14 could be due to the presence of two pathogenicity islands found in PA14 that are absent in PAO1, named Pseudomonas aeruginosa pathogenicity island 1 (PAPI-1) and Pseudomonas aeruginosa pathogenicity island 2 (PAPI-2). 19
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: Balb/c mice. The models described b
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
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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
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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
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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
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genomic islands in Pseudomonas aeru
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MURRAY, A.W. and SZOSTAK, J.W., 198
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specificity of chaperone-usher mach
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VAN HEECKEREN, A.M. and SCHLUCHTER,
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