Encystment and Excystment in Acanthamoeba castellanii

Differentiation processes such as the encystment are common in numerous eukaryotic single cell organisms of which many are causative agents of human infections. The cyst enables these microorganisms to endure adverse environmental conditions and thus holds up the infectious cycle. In the ubiquitous genus Acanthamoeba, which comprises potential pathogens causing Acanthamoeba keratitis and granulomatous amoebic encephalitis, cysts are formed in response to unfavourable environmental conditions including also biocide treatment. Cysts surviving treatment lead to a more dramatic course of disease or to recurrent infection and are also important for the vector role of acanthamoebae. Although Acanthamoeba castellanii has, due to its rapid growth and easily inducible synchronous cell differentiation, been serving as a model organism for studies on a variety of problems in cell biology for many decades, the molecular mechanisms of Acanthamoeba encystment have not yet been elucidated. However, the ability to form cysts is not only crucial for the role of acanthamoebae as active and passive pathogens, but is of essential clinical and ecological importance also in other pathogenic protozoa. Thus, a deeper insight into this complex process is of extensive interest. It is the aim of our project to apply contemporary molecular biological methodology to study the encystment in A. castellanii and establish a model for protozoan cell differentiation. Proteome analysis with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and subsequent genetic analysis will be applied to elucidate the genetic and physiological key players involved in the encystment and excystment processes of this organism. First, proteins that are strongly expressed during differentiation will be isolated from 2D-gels, identified and checked for regulatory sequences on the genetic level. With regulatory sequences at hand, the A. castellanii genome will be searched for other differentiation specific genes, including low copy proteins such as transcriptional activators and repressors. This reverse genetic approach will provide us with data on a large number of structural and regulatory genes, thereby allowing the elucidation of A. castellanii differentiation. The Acanthamoeba model will include the temporal and hierarchical order of expressed genes that range from the induced early cyst to the mature cyst and will be the basis for comparative studies including other cyst forming protozoan pathogens as Entamoeba histolytica and Giardia lamblia. Moreover, the influence of bacterial endocytobionts on Acanthamoeba castellanii encystment will be assessed by RNA-profiling experiments based on the knowledge of the genes involved in differentiation.

The representatives of the genus Acanthamoeba are free-living amoebae which fulfil an important role as bacterial predators in a wide range of habitats worldwide. However, acanthamoebae are not only notable for their ecological role but also for being vectors for pathogenic bacteria, e.g. for Legionella pneumophila, and for being human pathogens in their own right, causing keratitis and encephalitis. The life cycle of Acanthamoeba consists of two stages: the feeding and replicating trophozoite, and the endurable cyst stage which does not divide but which is highly resistant to environmental stress and therapeutic drugs. In terms of medical treatment, encystment is highly problematic, as cysts surviving initially successful treatment can excyst again and cause relapses of disease. The prime objective of this project was to study the encystment process in Acanthamoeba and to shed more light on the molecular processes behind, mainly at the protein level. We found that in the initial phase of encystment protein expression is dispensable and that practically all changes in the protein composition observed are due to autolytic protein degradation exerted by cysteine proteases. The ability to synthesize protein is greatly impaired in encysting Acanthamoeba, as indicated by a steep decrease of expression levels of factors which are necessary for protein translation. Later in encystment, the original protein content is partly restored and also proteins which are specific for the cyst stage are synthesized. Interestingly, overall protease activity and encystment capability of Acanthamoeba were both found to be strongly diminished in strains which were grown in the laboratory for extended periods of time, thereby further underlining the strong dependence of encystment on protease activity. These changes were not due to gene mutations but due to epigenetic regulation of gene expression as both traits could be widely restored by growing Acanthamoeba in the presence of a human cell line or by adding chemicals which revert epigenetic changes in gene expression. Finally, we also found that the intracellular dwelling bacterium Parachlamydia acanthamoebae inhibits encystment in Acanthamoeba at a very early stage and prevents autolytic protein degradation as normally observed during the initial phase of encystment.