A pathway to mitigating infection and disease caused by microsporidia

Abstract

Microsporidiosis poses an ongoing global threat to human health and animal wellbeing, in immunocompromised populations. As obligate intracellular parasites, microsporidia possess a greatly reduced genome and metabolome, whilst exhibiting an extensive array of extracellular proteins for host invasion and adherence. The aim of the work described in this thesis is to provide a clearer understanding of the microsporidian infectious pathway and presents a three- step pipeline to aid the discovery of novel proteins and mechanisms within that infectious pathway. Throughout the study, a combination of in-silico and laboratory-based investigations were performed to place equal emphasis on the proteins of the parasite and their respective hosts. Firstly, an in-silico experiment was devised using hidden Markov modelling and orthology-based prediction to collate and expand current putative microsporidian spore wall and polar tube proteins. Next, a novel proteomic “SHIPP” method was designed to isolate and identify novel microsporidian surface proteins and their respective host target sites. Lastly, two transfection methods of microsporidia were attempted to validate spore-host interactions: namely, trial use of cell penetrating peptides; and an adaptation of the CHoP-In mutagenesis approach for the knockdown of host membrane proteins. The results produced a consensus database comprising 294 surface protein orthologues, split across 34 distinct protein types. As such it represents one of the most comprehensive databases of its kind, to date. A trial of the “SHIPP” assay was able to successfully select for the extracellular proteins of both microsporidia and host cells, whilst identifying six novel protein candidates in microsporidia for further characterisation. Finally, use of cell penetrating peptides was shown to be an ineffective method for microsporidian transfection, whilst the adaption of the CHoP-In protocol for the mutagenesis of host cells requires further work. CHoP-In components were successfully designed, but could not be fully implemented into a complete trial run. Ultimately, it is believed that the components of this pipeline will assist in the discovery of novel microsporidian surface proteins, with future adaptations potentially assisting with the design of novel microsporidian treatments and the generation of resistant livestock to minimise the impacts of microsporidiosis.