Cross-species RNA-protein interactions in the malaria-infected cell

Benedikt Beckmann (HUB) in partnership with Melanie Rug (ANU) and Brendan McMorran (ANU)


Malaria is caused by Apicomplexan parasites of the genus Plasmodium. In its human host, the parasite undergoes different stages of its life cycle; among those the parasite infects and remodels mature red blood cells (RBCs) cells, which lack organelles, protein synthesis and a trafficking machinery. Recently, a novel type of cell-to-cell communication between infected RBCs via exosomes was described [1].

The Beckmann lab focuses on understanding RNA-protein interactions during infection. The group recently started investigating the role of secreted, vesicle-borne RNA from pathogenic bacteria and its impact on the infection process as well as the potential to manipulate post-transcriptional regulation of gene expression in host cells as described before [2]. In the proposed project, the group will screen for secreted RNA in exosomes from Plasmodium-infected cells:

                  The group will purify exosomes from Plasmodium falciparum-infected RBCs using established ultracentrifugation techniques and screen for RNA of plasmodial origin using RNA-Seq. Exosome and RNA uptake in recipient, non-infected bystander RBCs will be tracked. This will also be cross-correlated with known expressed miRNAs/other RNAs from Plasmodium during infection of RBCs to search RBC-released exosomal RNA in a sequence-specific manner. The group will then test if exosome-exposed bystander cells are more susceptible to subsequent Plasmodium infection.

                  Next, the group will purify exosomes released from the parasite itself. Here, they will selectively lyse RBC membranes and the parasitophorous vacuolar membrane to be able to separate intracellular Plasmodium parasites and Plasmodium-secreted exosomes. If yields permit, the group will use RNA-Seq to identify vesicular parasite RNA analogous to exosomes from RBCs (see point 1).

            Identified parasite RNA will then be bioinformatically analysed for similarities with RBC miRNAs, hinting to potential RNA interaction partners. Of note, the limited set of RNA present in maturing RBCs [3] will ease the search of such target RNAs and the absence of the nucleus in RBCs will allow us to work with a defined landscape of potential interacting transcripts. We will then try to identify interacting proteins using an unbiased approach we have previously developed, involving UV crosslinking and purification of the crosslinked complexes.

            The candidate PhD student will start purification of exosomes and RNA-Seq experiments in the first year at HUB in the Beckmann lab. In the second year, the student will move to the ANU to the Rug lab using microscopy techniques to track uptake of the exosomes to recipient RBCs and to the McMorran lab for functional studies on identified interaction partners in RBCs, e.g. for manipulation of identified target genes (1/2 year stay each). The final year will be conducted at HUB again, characterizing the interaction using in vitro and in vivo methods and screening for potential protein interaction partners.

 

Interlinkages: Christian Schmitz-Linnweber (HUB), Nishith Gupta (HUB)

 

References:

(1) Regev-Rudzki, N. et al. (2013) Cell 153:1120-1133

(2) Buck, A.H. et al. (2014) Nat. Commun. 5:5488

(3) Chen, S.Y. et al. (2008) PLoS One 3:e2360