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Gene identification in the encystation pathway of the Dictyostelid Polysphondylium pallidum

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.