Research Group:	Parasitology Unit
Address:	Humboldt-Universität zu Berlin, Department of Biology, Molecular Parasitology, Philippstr. 13, House 14, 10115 Berlin
Supervisor:	Dr. Alyssa Ingmundson
Doctoral Researcher:	Otto Netzel

Project Description

State of the art:
Liver-stage Plasmodium parasites reside intracellularly within a membrane-bound compartment known as the parasitophorous vacuole (PV). The related parasite, Toxoplasma gondii, also resides in a PV during infection, and the PV membranes of both of these pathogens are dynamic and undergo rapid expansion during parasite development. Host lipids contribute to these PV membranes, but knowledge about the extent to which the PV of either parasite fuses with host vesicles is limited. The Rab family of small GTPases regulate vesicle transport and membrane fusion and are attractive candidates to analyze host-parasite interactions because many pathogens usurp or subvert host Rabs to establish an intracellular replicative niche, and established tools allow functional interrogation of specific Rabs. Furthermore, host Rab proteins have been shown to influence susceptibility to infection. For example, expression levels of a regulator of Rab GTPases, RabGDIa, influences T. gondii infection, and particular Rabs are regulated by IFNg and IL-12, which are central during Plasmodium and Toxoplasma infection.

Previous own work:

We have localized a few Rabs during Plasmodium liver-stage infection and have observed Rab35-positive membranes surrounding the Plasmodium PV. Furthermore, our initial data indicate that siRNA-mediated knockdown of Rab35 restricts liver-stage Plasmodium growth.

Hypotheses and work plan:

1) Liver-stage Plasmodium, like T. gondii, exploits host vesicular traffic and localizing the host Rab GTPases in infected cells can indicate Rabs relevant for parasite infection.

2) Interfering with Rab function through the expression of dominant-interfering mutants, genetic targeting, or overexpression of negative regulators of Rabs reveal the importance of specific Rab-regulated processes for infection.

3) Naturally occurring polymorphisms in the genes encoding proteins regulating vesicular trafficking pathways may influence parasite intracellular development.

We will prioritize Rab GTPases expressed in hepatocytes and those involved in endocytic transport and autophagy. Host Rab GTPases that localize to the PV or change localization during infection will be investigated in both P. berghei and T. gondii infection. Furthermore, we will identify naturally occurring polymorphisms in these Rabs and their regulators in humans and in certain wild rodents and use CRISPR-Cas to generate comparable mutations in human and mouse cell lines, respectively. We will focus on non-synonymous SNPs, or SNPs close to splice sites or within predicted microRNA-binding sites. Infection phenotypes of P. berghei and T. gondii in these generated lines will subsequently be assessed. Exploring the influence of naturally occurring human polymorphisms on the liver-stage development of human-infecting Plasmodium is technically restricted because in vivo infection is not measurable in the liver and robust in vitro infection is limited to primary hepatocytes. Therefore, we are using P. berghei as a model for liver-stage Plasmodium development. We include rodent polymorphisms in our analyses since rodents are the natural hosts of P. berghei, and also a major reservoir of T. gondii. Completion of these aims will uncover host pathways that contribute to successful infection and reveal naturally occurring differences in how these pathways contribute to host susceptibility to infection.