Rapid control of protein level in the apicomplexan Toxoplasma gondii

Abstract

Analysis of gene function in apicomplexan parasites is limited by the absence of reverse genetic tools that allow easy and rapid modulation of protein levels. The fusion of a ligand-controlled destabilization domain (ddFKBP) to a protein of interest enables rapid and reversible protein stabilization in T. gondii. This allows an efficient functional analysis of proteins that have a dual role during host cell invasion and/or intracellular growth of the parasite.

What new cell biology findings could bring to therapeutics: is it time for a phenome-project in Toxoplasma gondii?

“…The Tet-inducible transactivator system is currently the most robust method to generate conditional mutants for essential genes in a targeted approach and appears to be especially well suited for the characterisation of factors involved in invasion and apicoplast biology (Meissner et al 2002, Mital et al 2005, Huynh & Carruthers 2006, Mazumdar et al 2006, Kessler et al 2008, Plattner et al 2008) whereas employment of the system for other essential GOIs might result only in weak phenotypes (Fleige et al 2008) or might not be possible at all (unpublished observations). A major advantage of the Tet-inducible system is that virtually any GOI can be regulated whereas regulation at the protein level using ddFKBP might not work for proteins targeted to the secretory system (Herm-Gotz et al 2007, and unpublished observations). However, apart from certain inefficiency problems, the application of the Tet-inducible system is very work intense.…”

“…However, in case the GOI is essential during the asexual life cycle, the generation of mutant parasites is impossible. In order to characterise essential factors in detail, three strategies are currently being employed with great success: (i) generation of temperature sensitive mutants followed by complementation cloning to identify the mutated gene (Gubbels et al 2008); (ii) employment of a tetracycline inducible (Tet-inducible) transactivator system (Meissner et al 2002) and (iii) a recently established degradation system (ddFKBP-system) that allows the rapid regulation of a protein of interest (Herm-Gotz et al 2007). However, as mentioned above, the absence of a "real" high throughput system, such as siRNA based methods (Ullu et al 2004), is seriously hampering a genome wide approach as performed in other eukaryotes.…”

“…Here a single stable transfection is sufficient to generate the respective parasite mutant. Other advantages of this system are the rapid induction kinetics and the possibility to tune the level of protein level simply via adding different amounts of inducer (Herm Gotz et al 2007). …”

“…However, the limitation of expressing trans-dominant versions of a protein in transient transfections is that the resulting phenotype might be non-specific and is in general hard to study in detail. Employment of the ddFKBP-system now gives us the chance to generate a clonal parasite line expressing each Rab-proteins in a regulated and tuneable manner, which results in a population of parasites that behaves identical, as shown for Rab1 and Rab11A (Herm-Gotz et al 2007). Given the reduced core set of proteins involved in vesicular trafficking, we are optimistic that this approach will allow a systematic dissection of protein trafficking, organellar biogenesis and endocytosis in a relatively fast and straight forward approach.…”

Advantages and disadvantages of conditional systems for characterization of essential genes in Toxoplasma gondii

“…Residual expression of MyoA was Meissner et al 2002;Herm-Gotz et al 2007;Andenmatten et al 2013). Manipulation at the genomic level using the DiCre system (A)/(B).…”