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.

Giardia intestinalis is a unicellular protozoan parasite, causing giardiasis – a gastro-intestinal disease of variable outcome and severity, in a broad range of mammalian species. The parasite has a comparatively simple life cycle with two stages, proliferative trophozoites and infectious cysts, as well as a reduced set of eukaryotic-specific organelles. Its metabolic pathways are simplified if compared to non-parasitic organisms. Giardia compensates for this apparent simplicity with unique inventions and complex regulation of metabolic processes. Transition from a fragile trophozoite to a protected, compact cyst is called encystation. This process starts upon changes in the growing conditions: cholesterol deprivation and elevated pH, and leads to changes in membrane lipids, elevated cAMP, and induction of encystation-specific gene expression, starting with activation of Myb-like protein expression. Within hours postencystation induction, cyst wall components (GalNAc and CWPs) are produced and transported to the cell membrane, flagella and adhesive disc are disassembled and stored in cytoplasm, followed by DNA replication and diplomixis. One of the encystation-specific upregulated genes in Giardia (assemblage A, isolate WB) is GL50803_1470 (termed ORF1470). Its predicted protein product has an Alba_2 domain, binding nucleic acids, presumably DNA. To study its function in G. intestinalis, we created knockout mutants, using CRISPR/Cas9 technique, Cas9- HA cell line and pGdelp-BbsI-B00826, with integrated pac- and gRNA cassettes. Transfected Giardia Cas9-HA cells were PCR verified for the complete knockout of the gene, encysted, and effect of ORF1470 was studied using cyst counting, morphometric analysis, cell imagining and Western blot for detection of CWPs. We have found minor phenotypical differences between the parental strain Cas9-HA and wild-type WB and ORF1470 deficient cells. Future plans are further experiments with obtained ΔORF1470 strains including further KO verification, visualization of ORF1470 product during encystation and determination of its binding site in the genome using ChIP-seq technology.

Encystation of unicellular organisms is of considerable medical relevance since cysts are encapsulated byresilient cell walls, rendering them resistant to biocides and immune clearance. This survival strategymakes it complicated to produce effective treatment of diseases caused by many protozoan pathogens,e.g. species of Acanthamoeba which causes fatal granulomatous amebic encephalitis (GAE) and keratitis.Due to genetic limitations in most protists, the machinery of encystation is so far little understood.However, the signalling pathways can be investigated in the non-pathogenic social amoebas, Dictyostelia.In this master’s project, five genes in Polysphondylium pallidum were investigated, which might beinvolved in encystation. Research has demonstrated a relationship between encystation and the cyclicadenosine monophosphate (cAMP) signalling pathways in Dictyostelia spore formation. This indicates thatcysts are ancestral to spores, and hence are the sporulation genes: pkaC, yakA, regA, cudA and srfAselected for this study. The genes were individually knocked-out by a standard homologous recombinationapproach and the genes’ contribution to encystation was determined. Five knock-out constructs werecompleted and a preliminary analysis of the role of the intracellular cAMP phosphodiesterase RegA inPolysphondylium pallidum encystation process was performed.

Giardia intestinalis is an anaerobic parasite, that colonizes the small intestine of humans and other vertebrate hosts. This cosmopolitan parasite, which causes diarrhoea, is transmitted by contaminated water or food via a resistant stage, the cyst. The encystation process involves a number of events that lead to a complete reconstruction of the cell into the form of infectious cyst. The aim of this work was to visualize these modifications in vivo by means of enzymatic labelling of proteins. For the purposes of this work, enzymatic tags Y-FAST and HaloTag were chosen, as they enable visualizing live cells under anaerobic conditions. Chimeric protein constructs were created to visualize the dynamics of the encystation vesicles, the structures of endoplasmic reticulum, the adhesive disc and mitosis. Using the developed constructs, we successfully followed the dynamics of the encystation vesicles and the adhesive disc in vivo. Finally, this work has provided novel molecular tools, which will be used to follow the overall redesign of the parasite cell during encystation.

The resting resistant stage has several important roles for phytoplankton microorganisms. It protects them from hostile conditions, allows them to spread to new locations and is often part of their life cycle. Chrysophytes form large populations for only a short part of the year, and therefore the formation of a resting stage is key for them to re-establish a vegetative population the following year. Chrysophyte resting stages are called stomatocysts, which are silicified and have a unique species-specific morphology. They can be form after both sexual and asexual reproduction. This is an understudied area. Most described stomatocysts are not assigned to species and much is not known about the triggers of encystation and excystation. It is thought that a combination of external (temperature, light, nutrients) and internal (cell age and cell concentration) factors are involved. So far, only sexual encystation in Dinobryon cyindricum and Synura petersenii has been studied in detail. My diploma thesis is based on laboratory experiments with Ochromonas tuberculata, Synura uvella and two strains of Synura petersenii. The aim of the study was to investigating whether external conditions such as temperature and lack of nitrogen or phosphorus have the effect on asexual encystation and whether it is...

High osmolarity causes amoebae of the cellular slime mould Polysphondylium pallidum to individually encyst, forming microcysts. During microcyst differentiation, actin is tyrosine phosphorylated. Tyrosine phosphorylation of actin is independent of encystment conditions and occurs during the final stages of microcyst formation. During microcyst germination, actin undergoes dephosphorylation prior to amoebal emergence. Renewed phosphorylation of actin in germinating microcysts can be triggered by increasing the osmolarity of the medium which inhibits emergence. Immunofluorescence reveals that actin is dispersed throughout the cytoplasm in dormant microcysts. Following the onset of germination, actin is observed around vesicles where it co-localizes with phosphotyrosine. Prior to emergence, actin localizes to patches near the cell surface. Increasing osmolarity disrupts this localization and causes actin to redistribute throughout the cytoplasm, a situation similar to that observed in dormant microcysts. Together, these results indicate an association between actin tyrosine phosphorylation, organization of the actin cytoskeleton, and microcyst dormancy.

High osmolarity causes amoebae of the cellular slime mould Polysphondylium pallidum to individually encyst, forming microcysts. During microcyst differentiation, actin is tyrosine phosphorylated. Tyrosine phosphorylation of actin is independent of encystment conditions and occurs during the final stages of microcyst formation. During microcyst germination, actin undergoes dephosphorylation prior to amoebal emergence. Renewed phosphorylation of actin in germinating microcysts can be triggered by increasing the osmolarity of the medium which inhibits emergence. Immunofluorescence reveals that actin is dispersed throughout the cytoplasm in dormant microcysts. Following the onset of germination, actin is observed around vesicles where it co-localizes with phosphotyrosine. Prior to emergence, actin localizes to patches near the cell surface. Increasing osmolarity disrupts this localization and causes actin to redistribute throughout the cytoplasm, a situation similar to that observed in dormant microcysts. Together, these results indicate an association between actin tyrosine phosphorylation, organization of the actin cytoskeleton, and microcyst dormancy.

The diplomonads are a diverse group of eukaryotic flagellates found in microaerophilic and anaerobic environments. The most studied diplomonad is the intestinal parasite Giardia intestinalis, which infects a variety of mammals and cause diarrheal disease. Less is known about Spironucleus salmonicida, a parasite of salmonid fish, known to cause systemic infections with high mortality. We created a transfection system for S. salmonicida to study cellular functions and virulence in detail (Paper I). The system was applied to explore the mitochondrion-related organelle (MRO) in S. salmonicida. We showed that S. salmonicida possesses a hydrogenosome (Paper II) with a higher metabolic capacity than the corresponding MRO of Giardia, the mitosome. Evolutionary analysis of key hydrogenosomal proteins showed ancient origin, indicating their presence in the ancestral diplomonad and subsequent loss in Giardia. Annexins are of evolutionary interest since these proteins are found across all kingdoms. Annexin-like proteins are intriguingly expanded into multigene families in Giardia and Spironucleus. The annexins of S. salmonicida were characterized (Paper III) with distinct localizations to various cellular structures, including a putative adhesion structure anterior in the cell. The disease-causing Giardia trophozoites differentiate into infectious cysts, a process essential for transmission and virulence of the parasite. Cysts are often spread via contaminated water and exposed to environmental stressors, such as UV irradiation. We studied the survival and transcriptional response to this stress factor (Paper IV) and results showed the importance of active DNA replication machinery for parasite survival after DNA damage. In addition, we studied transcriptional changes along the trajectory of encystation (Paper V), which revealed a coordinated cascade of gene regulation. This was observed for the entire transcriptome as well as putative regulators. Large transcriptional changes appeared late in the process with the majority of differentially regulated genes encoding hypothetical proteins. We studied the localizations of several of these to gain information of their possible function. To conclude, the diplomonads are complex eukaryotic microbes with cellular processes adjusted to match their life styles. The work in this thesis has provided insight of their adaptations, differences and similarities, but also new interesting leads for future studies of diplomonad biology and virulence.

Giardia lamblia, a worldwide cause of diarrhoea, must differentiate into environmentally resistant cysts for dissemination and completion of its life cycle. Although G. lamblia is an early diverging eukaryote, encystation involves many complex cellular changes including formation of the cyst wall that contains at least two cyst wall proteins, cyst wall proteins 1 and 2. Cwp genes are transcribed only during encystation. In this study, we examine the regulatory elements for the encystation-specific gene cwp2. The 64 bp immediately upstream of the cwp2 open reading frame (-64 to -1 relative to ATG) was shown to be sufficient for the encystation-specific expression of luciferase. To determine which region(s) within this 64 bp contributed to encystation-specific expression in vivo, a series of deletions were cloned into a Giardia luciferase expression vector and their ability to control encystation-specific expression of luciferase was assessed. Deletion of elements in the -64 to -23 region of the cwp2 promoter significantly increased expression of luciferase in vegetative trophozoites, suggesting that this area contains a negative cis-acting element. Deletions of elements from -23 to -10 led to decreased expression in encysting cells, suggesting that this region may contain positive cis-acting elements. When the A/T-rich initiator was deleted but the cis-acting elements (-64 to -10) were retained, encystation-specific expression of luciferase was maintained but an aberrant transcriptional start site was utilised. These results indicate that Giardia has developed a classic repressor mechanism(s) that allows tight, encystation-specific control by the cwp2 promoter.

Giardia intestinalis is a protozoan parasite causing the diarrheal disease called giardiasis that infects millions of people worldwide each year. The life cycle of Giardia intestinalis is characterized by two stages; the sturdy, infectious cyst and the vegetative, motile trophozoite. This project sought to investigate the regulation of the encystation process where trophozoites transform into cysts. Twelve genes with interesting transcriptomic profiles were chosen to study as putative transcription factors and regulators of encystation. These genes were cloned onto a plasmid with a Strep-Tag and transfected into Giardia intestinalis. The protein expression and localization was studied using immunofluorescence microscopy with antibodies against the Strep-Tag at different time points after inducing encystation. Although the project did not fully characterize these genes, protein expression was seen in all cases except two. Some proteins were seen localized to the nuclei and others had a localization pattern similar to the localization of cyst wall protein. In addition, a dramatic phenotype resembling cells going through programmed cell death was observed in one of the transfectants early in encystation and would be interesting to study further. The transfectant strains generated from this project remain interesting candidates to investigate as putative transcription factors.