The 60 kDa Heat Shock Proteins of Leishmania donovani and their role on viability, stress tolerance and virulence ; Die 60 kDa Hitzeschockproteine von Leishmania donovani und ihre Rolle für die Lebensfähigkeit, Stresstoleranz und Virulenz

Leishmaniasis is a disease caused by the protozoan parasite Leishmania spp. and is a major neglected tropical disease. Individuals infected with Leishmania parasites suffer from ulcerating, mostly self healing skin lesion, infections of the mucous membranes, and systemic, visceral infections. The latter are fatal if left untreated. The parasite faces challenges with various stressors during its parasitic life, cycling between sand flies and vertebrate hosts. During these life stages the parasite expresses various Heat Shock Proteins. The Heat Shock Protein 60 kDa (chaperonin 60, CPN60) is present in four different isoforms and at least one is expressed throughout the parasitic life cycle. The major function of chaperones is to assist proper folding of newly synthesised, denatured, miss-folded or un-folded proteins into their correct tertiary structures. However, why the parasite maintains four different CPN60 isoforms, named CPN60.1, CPN60.2, CPN60.3 and CPN60.4, is not known. Therefore, the aim of this thesis was to analyse the role of the 4 different CPN60s in Leishmania donovani, the main causative agent of visceral leishmaniasis. Firstly, double allele gene replacement of the four different CPN60s was performed by homologous recombination and/or by CRISPR/Cas gene editing. For CPN60.1, CPN60.2 and CPN60.4 viable double allele replacement mutants were obtained while CPN60.3 is an essential gene in L. donovani of which only single allele replacement mutants could be generated. To verify any phenotypes, ectopic copies of the genes of interest (GOI) were introduced into the mutants and the wild type via episomal plasmids, to generate GOI over-expressing mutants. All mutants were analysed for phenotypic changes, such as growth under different conditions, morphology, infectivity and virulence. It was found that CPN60.1 plays a role in the virulence of the parasite, as a lack of CPN60.1 leads to a 50% reduction of the relative parasite load in murine macrophages. For CPN60.2 a slightly reduced cell body length and ...