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Fatty Acid-Based Signalling in the Genus Burkholderia: Distribution, Mechanism, Cross-Linkage

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Fatty Acid-Based Signalling in the Genus Burkholderia: Distribution, Mechanism, Cross-Linkage Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2016 Fatty Acid-Based Signalling in the Genus Burkholderia: Distribution, Mechanism, Cross-Linkage Suppiger, Angela Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-129350 Dissertation Published Version Originally published at: Suppiger, Angela. Fatty Acid-Based Signalling in the Genus Burkholderia: Distribution, Mechanism, Cross-Linkage. 2016, University of Zurich, Faculty of Science. Fatty Acid-Based Signalling in the Genus Burkholderia: Distribution, Mechanism, Cross-Linkage Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Angela Suppiger aus Buttisholz, LU Promotionskommission Prof. Dr. Leo Eberl (Vorsitz) Prof. Dr. Jakob Pernthaler Dr. Gabriella Pessi Zürich, 2016 Acknowledgements Numerous people contributed to this work by supporting me with their knowledge, experience, encouragement and their time and energy. I would like to express my gratitude to everyone who supported me during my time as a PhD student. In particular, I would like to thank: My thesis supervisor Prof. Dr. Leo Eberl. Thank you for offering me this exciting and challenging research project. You have provided an ideal balance of constant support, guidance and freedom. The possibility of finishing my thesis in the UK was a great motivation and I appreciate it a lot. Thank you for all feedback and corrections you have given me and thanks for all the discussions we have had; these have allowed me to develop my skills as a scientist. My committee members Prof. Dr. Jakob Pernthaler and Dr. Gabriella Pessi. Thank you for taking time to review my thesis and providing me with interesting comments. A critical feedback is always required for successful work. Dr. Gabriella Pessi, I especially would like to thank you for your warmth, kindliness and all your important suggestions. I am also very grateful that you always helped me with the analysis of complex data sets. The collaborators Dr. Angelika Lehner, Prof. Dr. med. vet. Roger Stephan and Athmanya Eshwar. I thank you for giving me the chance to be part of the very interesting Cronobacter project. I am grateful to Prof. Dr. Volkhard Kaever and Annette Garbe for the very efficient and excellent service of c-di-GMP quantification. I thank Margrith Meier and Prof. Dr. Seeger for the opportunity to work on the establishment on a potential novel water filter. The lab technicians and friends Isabell Scholl and Victoria Widrig. Thank you for all your help with the experiments; all the conjugations and cloning you have performed. I am so grateful for your support, but especially for the enormous energy you provided to the lab. The PhD student Martina Lardi and Dr. Kirsty Agnoli-Antkowiak. Martina, thank you for introducing me to the work with RNA and your help with analysing the data. iii Kirsty, thank you for proofreading my thesis and all your important suggestions. It was very enjoyable indeed to work with both of you. My supervisor Dr. Claudio Aguilar. Thank you so much for your support and encouragement. You have shown me how to work in a lab and how to perform experiments. Your experience, knowledge and ideas were not only supportive, but also motivating for pushing the project in different but interconnected directions. Thank you for your support that you have provided even when you were not in the lab. The Eberl Group, present and former members. Thank you for sharing all the emotional moments, both intensive and exciting. Antri Georgiou, Anugraha Mathew, Alessia Gandolfi, Alessandra Vitale, Aspasia Mitropoulou, Aurelien Bailly, Carlotta Fabbri, Christian Jenul, Elisabeth Steiner, Gabriela Purtschert, Gabriela Marinova, Kirsty Agnoli-Antkowiak, Marta Pinto, Martina Lardi, Masanori Toyofuku, Olga Mannweiler, Samanta Bolzan de Campos, Stefano Gualdi, Steven Higgins, Yilei Liu and Amber Gardiner, Aurelien Carlier, Carmen Frauenknecht, Dirk Blom, Daniel Janser, Gerardo Carcamo-Oyarce, Jessica Toller, Laure Weisskopf, Mario Juhas, Martina Stöckli, Maria Sanchez-Contreras, Nadine Schmid, Nejc Stopnisek, Putthapoom Lumjiaktase, Rita Baumgartner, Roman Freitag, Rubina Braunwalder, Stefanie Heller, Stephan Schwager and Thomas Kost. My family and friends. Thank you for your constant support, love and patience. You always have supported me and cheered me up, even though so much was going on in each of your own lives. I will always remember it. Thomas, thank you for being my friend, a critical discussion partner and in keeping me grounded in many activities also other than my PhD studies. It is wonderful to have you by my side. iv Everything becomes a little different as soon as it is spoken out loud. Hermann Hesse. v Summary The genus Burkholderia comprises more than 90 species, which inhabit diverse ecological niches. They occur in water, soil, the plant rhizosphere and in association with fungi, insects and animals. Some Burkholderia show beneficial traits including biocontrol, bioremediation and plant growth-promoting properties. However, several Burkholderia species are also associated with plant, animal and human diseases. Members of the Burkholderia pseudomallei group are primary human and animal pathogens, causative agents of glanders and melioidosis. Members of the Burkholderia cepacia complex (Bcc) are pathogens in immunocompromised patients including those with cystic fibrosis and chronic granulomatous disease. The expression of pathogenic traits is in many bacteria at least partially coordinated by specific cell-to-cell communication systems, a phenomenon commonly referred to as quorum sensing (QS). QS is a generic regulatory mechanism that allows bacteria to sense and respond to various self-generated signal molecules as a function of the population density. QS is very widespread among bacteria and it is a mechanism that allows intra- and interspecies, and even interkingdom communication. The clinical isolate Burkholderia cenocepacia H111, a member of the Bcc, employs two different QS systems. The first identified, and better characterized, QS system relies on N-acyl-homoserine lactones (AHLs). The second QS system utilizes the fatty acid derivative cis-2-dodecenoic acid (BDSF, Burkholderia diffusible signal factor). BDSF is synthesized by the enoyl-CoA- hydratase RpfFBc and binds its cognate receptor RpfR. RpfR is a bifunctional enzyme that consist of PAS, GGDEF and EAL domains. Upon binding of BDSF, the phosphodiesterase activity of RpfR is stimulated, the intracellular c-di-GMP level is lowered and gene expression is altered. Both QS systems control a specific and overlapping set of genes in parallel. This PhD project was designed to investigate i) the distribution of the BDSF-based QS system in the genus Burkholderia and other bacteria, ii) genomic factors relevant for BDSF signalling and iii) additional regulatory elements that affect the QS circuitry in B. cenocepacia H111. My investigations revealed that the BDSF-based QS system is present in various vii Burkholderia species, including both pathogenic and environmentally beneficial strains. In addition, it was shown that Cronobacter turicensis also employs an active RpfF/R QS system to control biofilm formation and virulence. These findings show for the first time the widespread distribution of the RpfF/R system within both β- and γ-Proteobacteria. For these investigations I constructed a cis-2 fatty acid specific biosensor, which is sensitive to BDSF and related molecules in the nM range. Furthermore, my investigations shed light on the underlying mechanism of BDSF-regulated gene expression in B. cenocepacia. In the case of the stringently BDSF-controlled gene bclA, regulation was shown to occur via the 5’ untranslated leader region. I could also provide evidence that BDSF- and AHL-dependent regulation is mediated via different promoter regions. Finally, a novel regulator, LepR (I35_4766), was identified, which was shown to be required for full expression of the lectins bclACB and for cepacian production. The LepR regulon partially overlaps the RpfFBc- and RpoN-regulon. This thesis contributes to our understanding how expression of virulence determinants is regulated in the opportunistic human pathogen B. cenocepacia. This knowledge will lead to a better understanding of the pathogens’ success in the environment and allows the identification of bacterial processes that might be targeted by anti-infective therapies. viii Zusammenfassung Der Genus Burkholderia umfasst mehr als 90 Spezies, die in verschiedensten ökologischen Nischen leben. Sie sind im Wasser, dem Erdboden, der Pflanzenrhizosphäre und in Assoziation mit Pilzen, Insekten und Tieren zu finden. Einige Burkholderia haben Merkmale, welche für den Menschen nützliche Eigenschaften sind; zur Bioremedation, der biologischen Bekämpfung von Pflanzenpathogenen und zur Förderung des Pflanzenwachstums. Allerdings werden auch mehrere Burkholderia Spezies mit Pflanzen-, Tier- und Humankrankheiten assoziiert. Mitglieder von der Burkholderia pseudomallei Gruppe sind primäre Human- und Tierpathogene, Verursachende von Malleus (Rotz) und Melioidose. Mitglieder vom Burkholderia cepacia Komplex (Bcc) können Krankheitserreger in immunsupprimierten Patienten, die z.B. an Zystischer Fibrose oder chronischer Granulomatose leiden, sein. Die Expression von Pathogenitätsmerkmalen ist in vielen Bakterien zumindest
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