Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2012 The use of non-mammalian infection models to study the pathogenicity of Pseudomonas aeruginosa and members of the genus Burkholderia Schwager, S Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-74401 Dissertation Originally published at: Schwager, S. The use of non-mammalian infection models to study the pathogenicity of Pseudomonas aeruginosa and members of the genus Burkholderia. 2012, University of Zurich, Faculty of Science. The Use of Non–Mammalian Infection Models to Study the Pathogenicity of Members of the Genus Burkholderia and Pseudomonas aeruginosa Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Stephan Alois Michael Schwager aus Frauenfeld (TG) Promotionskomitee Prof. Dr. Leo Eberl (Vorsitz) Prof. Dr. Kathrin Riedel Prof. Dr. Jakob Pernthaler Zürich, 2012 Meiner Familie Table of Contents I. Summary ..................................................................................................................................... I II. Zusammenfassung ......................................................................................................................III III. Abbreviations ............................................................................................................................. V 1 Introduction ................................................................................................................................6 1.1 Burkholderia ................................................................................................................................6 1.2 Bcc infections and cystic fibrosis ..................................................................................................7 1.3 Pathogenicity models for memebers of the genera Burkholderia and Pseudomonas. ...................8 1.3.1 Caenorhabditis elegans ................................................................................................9 1.3.2 Galleria mellonella ..................................................................................................... 11 1.3.3 Drosophila melanogaster ........................................................................................... 13 1.4 The use of multiple hosts to study bacterial virulence................................................................ 13 1.5 P. aeruginosa ............................................................................................................................ 15 1.6 Quorum sensing: A cell-to-cell communication system .............................................................. 16 1.7 Goals of this work ...................................................................................................................... 18 2 Material and Methods ............................................................................................................... 19 2.1 Table 1: Strains plasmids and animals used in this study. ........................................................... 19 2.2 Table 2: Primers used in this study............................................................................................. 23 2.3 Non-mammalian pathogenicity models ..................................................................................... 23 2.3.1 C. elegans infection model ......................................................................................... 23 2.3.2 Media and buffer for C. elegans ................................................................................. 23 2.3.3 Cultivation and stock conservation of C. elegans ........................................................ 24 2.3.4 Egg preparation ......................................................................................................... 24 2.3.5 Pathogenicity assay .................................................................................................... 25 2.3.6 G. mellonella pathogenicity assay .............................................................................. 25 2.3.7 D. melanogaster pathogenicity assays ........................................................................ 26 2.3.8 Media for D. melanogaster ........................................................................................ 26 2.3.9 Stock conservation of D. melanogaster ...................................................................... 26 2.3.10 D. melanogaster feeding assay ................................................................................... 26 2.3.11 D. melanogaster pricking assay .................................................................................. 27 2.4 Construction of a cosmid library of B. cenocepacia H111 by using the pRG930 cosmid .............. 27 2.4.1 Total DNA extraction of B. cenocepacia ...................................................................... 27 2.4.2 Partial digestion of the extracted H111 DNA .............................................................. 28 2.4.3 Pooling of the partial digested H111 DNA .................................................................. 28 2.4.4 Extraction of the cosmid vector.................................................................................. 28 2.4.5 Ligation of the pRG930 with the partial digested DNA ................................................ 29 2.4.6 Packaging and tittering the cosmid packaging reaction .............................................. 29 3 Results and Discussion ............................................................................................................... 30 3.1 Investigations on the pathogenicity of P. aeruginosa and Bcc strains using multiple infection hosts ................................................................................................................................ 30 3.1.1 C. elegans pathogenicity assay ................................................................................... 30 3.1.2 G. mellonella pathogenicity assay .............................................................................. 31 3.1.3 D. melanogaster pathogenicity assay ......................................................................... 31 3.2 Novel drug targets from the B. cenocepacia mutant screen ....................................................... 34 3.3 Identification of Burkholderia cenocepacia H111 virulence factors using non-mammalian infection hosts. .......................................................................................................................... 36 3.4 Chromosome three of Bcc strains is essential for virulence in C. elegans and G. mellonella ......... 48 3.4.1 A conserved role of pC3 in pathogenicity of Bcc ................................................................ 49 3.5 LasI/R and RhlI/R quorum sensing system of P. aeruginosa PUPa3............................................. 54 3.6 Cystic fibrosis-niche adaptation of P. aeruginosa reduces virulence in multiple infection hosts.. 55 4 Future perspectives ................................................................................................................... 56 4.1 The genes purD, purF, purL, aroK and pyrD as potential drug targets ......................................... 56 4.2 Future work on the role of pC3 in pathogenicity of Bcc strains................................................... 58 5 Contribution to publications included in my thesis .................................................................... 59 5.1 Identification of Burkholderia cenocepacia H111 virulence factors using non-mammalian infection hosts. .......................................................................................................................... 59 5.2 Cystic fibrosis-niche adaptation of Pseudomonas aeruginosa reduces virulence in multiple infection hosts. .......................................................................................................................... 59 5.3 Exposing the third chromosome of Burkholderia cepacia complex strains as a virulence plasmid. ................................................................................................................................ 59 5.4 Identification of specific and universal virulence factors in Burkholderia cenocepacia strains by using multiple infection hosts. .................................................................................................. 60 5.5 LasI/R and RhlI/R quorum sensing in a strain of Pseudomonas aeruginosa beneficial to plants. 60 5.6 Burkholderia cenocepacia J2315 acyl carrier protein: A potential target for antimicrobials' development? .......................................................................................................................... 60 5.7 The genetic basis of cadmium resistance of Burkholderia cenocepacia. ..................................... 60 6 References ................................................................................................................................ 61 7 Appendix ................................................................................................................................... 78 Appendix 1 Exposing the third chromosome of Burkholderia cepacia complex strains as a virulence plasmid ....................................................................................................