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

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AQ: 43 AQ: 44 AQ: 45 the focus of studies describing their desialylation of host cells and involvement in adhesion in vitro and in vivo [3, 26, 27, 42, 43, 46]. Deletion of either nanA or nanB was shown to have significant effects on nasopharyngeal colonization and pneumonia [34, 47], a finding that strongly matches the proposed role of these enzymes in the novel sialic acid–inducible carriage phenotype described in the present study. We now propose a new model for the pathogenesis of pneumococcal pneumonia in which a local, virus-induced increase in the free sialic acid concentration in the upper respiratory tract would serve as a signal for colonizing pneumococci to increase in numbers, possibly by promoting the formation of biofilm. In this context, the up-regulation of the pneumococcal neuraminidases would provide a positive feedback loop. The higher colonization density in the nasopharynx would eventually lead to passive shedding of pneumococci to the lower respiratory tract, which, worsened by virus-induced damage to bronchoalveolar cells [48], would finally initiate development of an acute pulmonary infection. In conclusion, the data from the present study demonstrate that free sialic acid (1) is essential for biofilm formation in vitro at concentrations with physiologic relevance, (2) is able to induce the expression of neuraminidase genes and a virulence regulator, and (3) leads to an increase in nasopharyngeal pneumococcal loads and instigates the spread of pneumococci to the lower respiratory tract. These data support the hypothesis that free sialic acid is a trigger that converts a harmless colonizing pneumococcus to an invasive pathogen. This finding, in addition to adding substantial knowledge to the pathogenesis of pneumococcal disease, points to novel strategies for the prevention of invasive disease, similar to those proposed for the prevention of disease due to nontypeable H. influenzae [15], by treating carriage or even overt disease through interventions affecting free sialic acid on human mucosae and, thus, preventing sialic acid from acting as an environmental cue to the pneumococcus. Acknowledgments We thank Velia Braione, Tiziana Braccini, and Anna Cuppone for help with laboratory procedures. References 1. Ispahani P, Slack RCB, Donald FE, Weston WC, Rutter N. Twenty year surveillance of invasive pneumococcal disease in Nottingham: serogroups responsible and implications for immunisation. Arch Dis Child 2004; 89:757–62. 2. Melegaro A, Edmunds WJ, Pebody R, Miller E, George R. The current burden of pneumococcal disease in England and Wales. J Infect 2006; 52:37– 48. 3. Kadioglu A, Weiser JN, Paton JC, Andrew PW. The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat Rev Microbiol 2008; 6:288–301. 4. Gray BM, Converse GM 3rd, Dillon HC Jr. Epidemiologic studies of Streptococcus pneumoniae in infants: acquisition, carriage, and infection during the first 24 months of life. J Infect Dis 1980; 142:923–33. 8 ● JID 2009:199 (15 May) ● Trappetti et al. tapraid5/z9d-jid/z9d-jid/z9d01009/z9d4168d09a sangreyj S�5 3/20/09 Art: 42180 5. Hogberg L, Geli P, Ringberg H, Melander E, Lipsitch M, Ekdahl K. Age- and serogroup-related differences in observed durations of nasopharyngeal carriage of penicillin-resistant pneumococci. J Clin Microbiol 2007; 45:948–52. 6. Mühlemann K, Uehlinger DE, Büchi W, Gorgievski M, Aebi C. The prevalence of penicillin-non-susceptible Streptococcus pneumoniae among children aged �5 years correlates with the biannual epidemic activity of respiratory syncytial virus. Clin Microbiol Infect 2006; 12: 873–9. 7. Iyer R, Camilli A. Sucrose metabolism contributes to in vivo fitness of Streptococcus pneumoniae. Mol Microbiol 2007; 66:1–13. 8. Polissi A, Pontiggia A, Feger G, et al. Large-scale identification of virulence genes from Streptococcus pneumoniae. Infect Immun 1998; 66:5620–9. 9. Lau GW, Haataja S, Lonetto M, et al. A functional genomic analysis of type 3 Streptococcus pneumoniae virulence. Mol Microbiol 2001; 4:555– 71. 10. Hava DL, Camilli A. Large-scale identification of serotype 4 Streptococcus pneumoniae virulence factors. Mol Microbiol 2002; 45:1389–406. 11. Shanks RM, Donegan NP, Graber ML, et al. Heparin stimulates Staphylococcus aureus biofilm formation. Infect Immun 2005; 73:4596–606. 12. Hooper LV, Xu J, Falk PG, Midtvedt T, Gordon JI. A molecular sensor that allows a gut commensal to control its nutrient foundation in a competitive ecosystem. Proc Natl Acad Sci USA 1999; 96:9833–8. 13. Sohanpal BK, El-Labany S, Lahooti M, Plumbridge JA, Blomfield IC. Integrated regulatory responses of fimB to N-acetylneuraminic (sialic) acid and GlcNAc in Escherichia coli K-12. Proc Natl Acad Sci USA 2004; 101:16322–7. 14. Greiner LL, Watanabe H, Phillips NJ, et al. Nontypeable Haemophilus influenzae strain 2019 produces a biofilm containing N-acetylneuraminic acid that may mimic sialylated O-linked glycans. Infect Immun 2004; 72:4249– 60. 15. Johnston JW, Apicella MA. Sialic acid metabolism and regulation by Haemophilus influenzae: potential novel antimicrobial therapies. Curr Infect Dis Rep 2008; 10::83–4. 16. Kilian M, Rölla G. Initial colonization of teeth in monkeys as related to diet. Infect Immun 1976; 14:1022–7. 17. Iannelli F, Pozzi G. Method for introducing specific and unmarked mutations into the chromosome of Streptococcus pneumoniae. Mol Biotechnol 2004; 26:81–6. 18. Oggioni MR, Trappetti C, Kadioglu A, et al. Switch from planktonic to sessile life: a major event in pneumococcal pathogenesis. Mol Microbiol 2006; 61:1196–210. 19. Oggioni MR, Iannelli F, Ricci S, et al. Antibacterial activity of a competence-stimulating peptide in experimental sepsis caused by Streptococcus pneumoniae. Antimicrob Agents Chemother 2004; 48:4725–32. 20. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008; 3:1101–8. 21. Alymova IV, Portner A, Takimoto T, Boyd KL, Sudhakara Babu Y, McCullers JA. The novel parainfluenza virus hemagglutinin-neuraminidase inhibitor BCX 2798 prevents lethal synergism between a paramyxovirus and Streptococcus pneumoniae. Antimicrob Agents Chemother 2005; 49:398– 405. 22. McCullers JA. Effect of antiviral treatment on the outcome of secondary bacterial pneumonia after influenza. J Infect Dis 2004; 190:519–26. 23. Tram TH, Brand Miller JC, McNeil Y, McVeagh P. Sialic acid content of infant saliva: comparison of breast fed with formula fed infants. Arch Dis Child 1997; 77:315–8. 24. Lee JY, Chung JW, Kim YK, Chung SC, Kho HS. Comparison of the composition of oral mucosal residual saliva with whole saliva. Oral Dis 2007; 13:550–4. 25. Siqueira WL, Siquiera MF, Mustacchi Z, de Oliveira E, Nicolau J. Salivary parameters in infants aged 12 to 60 months with Down syndrome. Spec Care Dentist 2007; 27:203–6. 26. Berry AM, Paton JC, Glare EM, Hansman D, Catcheside DE. Cloning and expression of the pneumococcal neuraminidase gene in Escherichia coli. Gene 1988; 71:299–305. AQ: 46 AQ: 47
AQ: 48 tapraid5/z9d-jid/z9d-jid/z9d01009/z9d4168d09a sangreyj S�5 3/20/09 Art: 42180 27. Camara M, Mitchell TJ, Andrew PW, Boulnois GJ. Streptococcus pneumoniae produces at least two distinct enzymes with neuraminidase activity: cloning and expression of a second neuraminidase gene in Escherichia coli. Infect Immun 1991; 59:2856–8. 28. Burnaugh AM, Frantz LJ, King SJ. Growth of Streptococcus pneumoniae on human glycoconjugates is dependent upon the sequential activity of bacterial exoglycosidases. J Bacteriol 2008; 190:221–30. 29. King SJ, Hippe KR, Gould JM, et al. Phase variable desialylation of host proteins that bind to Streptococcus pneumoniae in vivo and protect the airway. Mol Microbiol 2004; 54:159–71. 30. Orihuela CJ, Radin JN, Sublett JE, Gao G, Kaushal D, Tuomanen EI. Microarray analysis of pneumococcal gene expression during invasive disease. Infect Immun 2004; 72:5582–96. 31. LeMessuier KS, Ogunniyi DA, Paton JC. Differential expression of key pneumococcal virulence genes in vivo. Microbiology 2006; 152:305–11. 32. Deutscher J, Francke C, Pot B, Postma PW. How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria. Microbiol Mol Biol Rev 2006; 70:939–1031. 33. Hemsley CJ, Joyce E, Hava DL, Kawale A, Camilli A. MgrA, an orthologue of Mga, acts as a transcriptional repressor of the genes within the rlrA pathogenicity islet in Streptococcus pneumoniae. J Bacteriol 2003; 185:6640–7. 34. Manco S, Hernon F, Yesilkaya H, Paton JC, Andrew PW, Kadioglu A. Pneumococcal neuraminidases A and B both have essential roles during infection of the respiratory tract and sepsis. Infect Immun 2006; 74: 4014–20. 35. Kadioglu A, Taylor S, Iannelli F, Pozzi G, Mitchell TJ, Andrew PW. Upper and lower respiratory tract infection by Streptococcus pneumoniae is affected by deficiency of pneumolysin and by differences in serotype. Infect Immun 2002; 70:2886–90. 36. Kadioglu A, Gingles NA, Grattan K, Kerr A, Mitchell TJ, Andrew PW. Host cellular immune response to pneumococcal lung infection in mice. Infect Immun 2000; 68:492–501. 37. von Itzstein M. The war against influenza: discovery and development of sialidase inhibitors. Nat Rev Drug Discov 2007; 6:967–74. 38. Hall-Stoodley L, Hu FZ, Gieseke A, et al. Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media. JAMA 2006; 296:202–11. 39. Watson M, Gilmour R, Menzies R, Ferson M, McIntyre P. The association of respiratory viruses, temperature and other climatic parameters with the incidence of invasive pneumococcal disease in Sydney, Australia. Clin Infect Dis 2006; 42:211–5. 40. Streicher H. Inhibition of microbial sialidases—What happened beyond the influenza virus? Curr Med Chem- Anti-infective Agents 2004;3: 149–61. 41. McCullers JA, Rehg JE. Lethal synergism between influenza virus and Streptococcus pneumoniae: characterization of a mouse model and the role of platelet-activating factor receptor. J Infect Dis 2002; 186:341–50. 42. Tong HH, Blue LE, James MA, De Maria TF. Evaluation of the virulence of a Streptococcus pneumoniae neuraminidase-deficient mutant in nasopharyngeal colonization and development of otitis media in the chinchilla model. Infect Immun 2000; 68:921–4. 43. Grewal PK, Uchiyama S, Ditto D, et al. The Ashwell receptor mitigates the lethal coagulopathy of sepsis. Nat Med 2008; 14:648–55. 44. Tess BR, Kempf JE. Decrease of bound sialic acid and inhibitor in chorioallantoic membranes infected with influenza virus. J Bacteriol 1963; 86:239–45. 45. McCullers JA, Bartmess KC. Role of neuraminidase in lethal synergism between influenza virus and Streptococcus pneumoniae. J Infect Dis 2003; 187:1000–9. 46. King SJ, Hippe KR, Weiser JN. Deglycosylation of human glycoconjugates by the sequential activities of exoglycosidases expressed by Streptococcus pneumoniae. Mol Microbiol 2006; 59:961–74. 47. Orihuela CJ, Gao G, Francis KP, Yu J, Tuomanen EI. Tissue-specific contributions of pneumococcal virulence factors to pathogenesis. J Infect Dis 2004; 190:1661–9. 48. Matrosovich MN, Matrosovich TY, Gray T, Roberts NA, Klenk H. Human and avian influenza viruses target different cell types in cultures of human airway epithelium. Proc Natl Acad Sci USA 2004; 101:4620–4. Sialic Acid and Pneumococcal Virulence ● JID 2009:199 (15 May) ● 9 AQ: 49
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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
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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
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
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Page 183 and 184: AQ: 18 AQ: 19 AQ: 20 F1 AQ: 21 tapr
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Page 187: AQ: 40 AQ: 41 DISCUSSION tapraid5/z
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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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