gms | German Medical Science

Objectives: An obligatory step during Plasmodium infection is the invasion of host hepatocytes and subsequent intracellular development of the parasite. The molecular mechanisms and the host contribution to Plasmodium liver infection remain largely unknown even though this stage is the most appealing for vaccine development and prophylactic approaches. We are applying RNA interference (RNAi) screening combined with high content analysis to identify novel host factors involved in parasitic invasion and intracellular growth.

Methods: We employ GFP expressing Plasmodium berghei for infection of cultured human hepatoma cells. By use of an automated high throughput platform, the cells are transfected with siRNAs targeting genes involved in intracellular trafficking, glycolysis, and autophagy. Two days after transfection, cells are infected with P. berghei and fixed 48h post infection. The samples are analysed by high throughput microscopy and automated image analysis. By evaluating infection rates as well as size of parasitophorous vacuoles (PV), we aim to identify host factors involved in invasion as well as intracellular growth of the parasites. Hits are selected on an algorithm based on hyper geometrical distribution (RSA; redundant siRNA activity) and confirmed by additional siRNAs. To further characterize the role of the identified factors, we apply molecular and cell biological approaches.

Results: After screening of roughly 500 genes represented by almost 1500 siRNAs, we now compiled a primary hit list of 44 trafficking factors. This hit list comprises all genes leading to either an increase or decrease of infection rate or PV size, with a statistical significance of P< 0.01, according to the RSA algorithm. Statistical analysis of neutral and inhibitory screen controls (Z-prime of 0.35) confirmed our setup to be relatively robust and the data obtained to be reliable. We are currently validating the primary hits with additional siRNAs.

Conclusions: We were able to set up a Plasmodium berghei infection system applicable to automated RNAi screening. We could already identify several host trafficking factors playing either a beneficial or detrimental role in Plasmodium invasion or intracellular growth. Follow up studies of these factors might lead to the development of new therapeutic strategies targeting host factors.