The identity and function of host factors required for efficient phagocytosis and intracellular maintenance of the protozoan parasite Leishmania donovani are poorly understood. Utilising the phagocytic capability of Drosophila S2 cells, together with available tools for modulating gene expression by RNAi, we have developed an experimental system in which to identify host proteins of this type on a genome-wide scale. We have shown that L. donovani amastigotes can be phagocytosed by S2 cells, in which they replicate and are maintained in a compartment with features characteristic of mammalian phagolysosomes. Screening with dsRNAs from 1920 conserved metazoan genes has identified transcripts that, when reduced in expression, cause either increased or decreased phagocytosis. Focussing on genes in the latter class, RNAi-mediated knockdown of the small GTPase Rab5, the prenylated SNARE protein YKT6, one sub-unit of serine palmitoyltransferase (spt2/lace), the Rac1-associated protein Sra1 and the actin cytoskeleton regulatory protein, SCAR, all lead to a significant reduction in parasite phagocytosis. A role for the lace mammalian homologue in amastigote uptake by mammalian macrophages has been verified using the serine palmitoyltransferase inhibitor, myriocin. These observations suggest that this experimental approach has the potential to identify a large number of host effectors required for efficient parasite uptake and maintenance.
|Number of pages||12|
|Publication status||Published - 13 Dec 2012|
- WIDE RNAI ANALYSIS
- AMASTIGOTE STAGES