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Using new genetic tools to elucidate the importance of exported proteins in intra-erythrocytic survival of the malaria parasite Plasmodium falciparum

Diehl, Mathias

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Propagation of Plasmodium parasites within erythrocytes is responsible for the majority of the symptoms associated with the human malaria disease. Survival of these parasites relies on remodelling of their host cells via exported proteins. These proteins are trafficked to various locations within the host cell in order to fulfil their function. In particular, the causative agent of the most virulent form of the human malaria disease –P. falciparum– is known to introduce drastic changes of their infected host cells. Investigation of parasite genes implicated in host cell remodelling and protein trafficking is critical for our understanding of the disease and might open up novel ways to combat malaria. However, analysis of essential genes involved in these processes requires the use of conditional knockout or knockdown system. Despite this there was a prolonged lack of genetic systems suitable to interrogate essential P. falciparum genes until recently, when several new systems were introduced. This includes the promising new glmS system which enables knockdown of a modified gene through the self-cleaving action of the glmS ribozyme triggered by addition of glucosamine. Here we apply glmS for conditional downregulation of four P. falciparum genes: PFA0660w, GEXP18, CBP1 and PfJ23. PFA0660w was modified using conventional single-crossover recombination, while modification of the other three genes was executed via selection-linked-integration, a new system for integration into the P. falciparum genome. PFA0660w, CBP1 and PfJ23 were successfully modified with a glmS-sequence and enabled reliable glucosamine-dependent downregulation. Modification of GEXP18 coincided with retention of the wild-type GEXP18 locus, which meant that these cell lines could not be used in further experiments. The cell lines generated by selection-linked-integration also displayed signs indicative of malfunction of the SKIP peptide. Addition of glucosamine to the glmS-modified PFA0660w, CBP1 and PfJ23 cell lines enabled downregulation of protein abundance to ~10% of the non-treated control. This had no effects on parasite morphology and viability. Furthermore, no differences in knob-morphology upon downregulation of PFA0660w or CBP1 were observed. Also downregulation of PFA0660w did not induce changes in parasite cytoadhesion. However, inactivation of PFA0660w via severe truncation caused drastically deformed, elongated knob morphologies. This demonstrates that the downregulation of PFA0660w achieved via glmS was not strong enough to cause the observed mutant phenotype upon truncation. Overall effectivity of the glmS system seemed to be highly gene-specific and should therefore be restricted to high-priority targets. Also alternative systems should be considered for the further dissection of essential P. falciparum genes. Truncation of PFA0660w induced fascinating changes in infected red blood cell morphology which could be further dissected using high-resolution microscopy methods.

Item Type: Dissertation
Supervisor: Lanzer, Prof. Dr. Michael
Place of Publication: Heidelberg
Date of thesis defense: 10 September 2019
Date Deposited: 30 Sep 2019 12:23
Date: 2020
Faculties / Institutes: Medizinische Fakultät Heidelberg > Department for Infectiology
Subjects: 570 Life sciences
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