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Protein secretion and encystation in Acanthamoeba Alvaro de Obeso Fernández del Valle Doctor of Philosophy The University of Edinburgh 2018 Abstract Free-living amoebae (FLA) are protists of ubiquitous distribution characterised by their changing morphology and their crawling movements. They have no common phylogenetic origin but can be found in most protist evolutionary branches. Acanthamoeba is a common FLA that can be found worldwide and is capable of infecting humans. The main disease is a life altering infection of the cornea named Acanthamoeba keratitis. Additionally, Acanthamoeba has a close relationship to bacteria. Acanthamoeba feeds on bacteria. At the same time, some bacteria have adapted to survive inside Acanthamoeba and use it as transport or protection to increase survival. When conditions are adverse, Acanthamoeba is capable of differentiating into a protective cyst. This study had three objectives. First, isolate and identify new FLA and Acanthamoeba strains. Second, identify encystation factors of Acanthamoeba . Third, identify and characterise new potential antimicrobial proteins produced by Acanthamoeba . The isolation of environmental amoebae was performed, and several strains of Acanthamoeba were identified from previously known genotypes. Also, two new species of FLA were identified: Allovahlkampfia minuta and Leptomyxa valladaresi . The dynamics of encystment were studied in different strains of Acanthamoeba . RNAseq was used to study gene expression during differentiation and identify differentially expressed genes. We identified different encystment factors including at least two encystment related proteases. A new antimicrobial zymogram was developed that identified antimicrobial proteins being secreted by Acanthamoeba . A 33 kDa protease was found to be able to lyse bacteria. We created DNA constructs encoding the protease and a lysozyme from Acanthamoeba for heterologous expression. The genes were successfully cloned. However, bacteria were not able to produce the proteins most probably due to their antimicrobial characteristics. Further studies are required regarding encystment and antimicrobial factors identified. Such experiments should help elucidate critical factors of Acanthamoeba ’s biology that could help treat several infections. Lay summary Free-living amoebae are microbes of ubiquitous distribution. Although they do not have a common evolutionary origin, all FLA are similar in their range of morphologies and locomotion. Some of these FLA are of particular importance as they are capable of producing infection. Acanthamoeba can produce a life-altering eye infection, among other less common diseases. In the environment, Acanthamoeba feeds on bacteria, and for this, it has developed different strategies to digest them. At the same time, some bacteria have adapted to be able to withstand Acanthamoeba , and survive and multiply inside of the amoeba. Bacteria that can survive inside Acanthamoeba can use it as a transport mechanism and as a haven from environmental challenges. Additionally, when conditions are adverse, Acanthamoeba can change its morphology and produce a protective cyst until conditions improve. This study had three objectives. First, isolate new amoebae. Second, identify critical factors in the production of the cyst by Acanthamoeba . Third, identify proteins used by Acanthamoeba to kill bacteria. Several Acanthamoeba organisms were isolated as well as two new species of FLA. In addition, different experiments to observe encystment dynamics were performed. From these, several factors for encystment were identified. Furthermore, two different proteins with potential to lyse bacteria were selected. One of the proteins was selected after the development of a new technique to observe bacterial lysis on a gel and the other protein through literature research. The genes for these proteins were isolated, but the proteins could not be produced in the laboratory. Although antimicrobial and encystment factors were identified, future studies are required to characterise them and have a deeper understanding of their functions and mechanisms. Such experiments should help elucidate critical factors of Acanthamoeba ’s biology that could help treat several infections. Own Work Declaration I declare that the thesis has been composed by myself and that the work has not be submitted for any other degree or professional qualification. I confirm that the work submitted is my own, except where explicitly stated otherwise or where work which has formed part of jointly-authored publications has been included. My contribution to these works have been explicitly indicated below. I confirm that appropriate credit has been given within this thesis where reference has been made to the work of others. Work presented in Chapter 5 and found in Appendix 1 was previously published in Experimental Parasitology as “Leptomyxa valladaresi n. sp. (Amoebozoa, Tubulinea, Leptomyxida), from Mount Teide, Tenerife, Spain” by Alvaro De Obeso Fernandez Del Valle, Jacob Lorenzo-Morales and Sutherland K. Maciver. This study was conceived by all of the authors. I carried out the molecular identification of the new organism. Work presented in Chapter 5 and found in Appendix 1 was previously published in Acta Protozoologica as “Allovahlkampfia minuta nov. sp., (Acrasidae, Heterolobosea, Excavata) a New Soil Amoeba at the Boundary of the Acrasid Cellular Slime Moulds” by Alvaro De Obeso Fernandez Del Valle and Sutherland K. Maciver. This study was conceived by all of the authors. I carried out the isolation and molecular identification of the new organism. Alvaro de Obeso Fernández del Valle January 2018 Acknowledgements ................................................................................................ i Abbreviations ......................................................................................................... ii Chapter 1 Introduction..................................................................................... 1 1.1 Amoebae .................................................................................................. 1 1.2 Free-living amoebae ................................................................................. 4 1.3 Acanthamoeba ......................................................................................... 4 Life cycle of Acanthamoeba .................................................................. 4 Morphology .......................................................................................... 6 Taxonomy ............................................................................................ 8 Isoenzyme profile .........................................................................10 Riboprinting .................................................................................10 18s rDNA .....................................................................................12 Cytochrome oxidase subunit I ......................................................13 Locomotion ..........................................................................................14 Feeding systems .................................................................................15 Ecology ...............................................................................................16 1.4 Bacteria-Amoebae interactions ................................................................17 Amoebae prey on bacteria ...................................................................17 Bacteria invade amoebae ....................................................................17 Mutualism ............................................................................................19 Bacterial infections aided by Acanthamoeba .......................................22 Legionella pneumophila .......................................................................22 1.5 Interactions between amoebae and other organisms ..............................23 Virus ....................................................................................................23 Fungi ...................................................................................................23 1.6 Diseases and importance ........................................................................24 Granulomatous amoebic encephalitis (GAE) .......................................24 Acanthamoeba rhinosinusitis ...............................................................26 Cutaneous acanthamebiasis or granulomatous dermatitis ...................26 Acanthamoeba keratitis (AK) ...............................................................26 Contact lens wear ........................................................................27