Characterizing protein function at the parasite-host interface during both liver and blood infection stages.

ALYSSA INGMUNDSON (HUB) in partnership with Melanie Rug (ANU)

Host cell remodelling by Plasmodium was historically associated with red blood cell (RBC) infection by human malaria species; however, membrane compartments in the cytoplasm of infected RBCs have now been found to be a common feature of various Plasmodium species and a membrane network containing parasite proteins is also generated in Plasmodium-infected liver cells [1,2]. Through studies of the P. berghei protein IBIS1 we discovered previously unrecognized intra-erythrocytic P. berghei-induced structures (IBIS) formed in the cytoplasm of P. berghei-infected RBCs and characterized the liver-stage tubovesicular network (TVN), which comprises membrane extensions and vesicles that protrude into the host cell [2,3]. Until now, no function is ascribed to most known liver-stage TVN resident proteins [4]. Furthermore, knowledge about the function of membrane structures generated in P. falciparum-infected RBCs is limited and the role of such structures in RBCs infected by other Plasmodium species has only been speculated.

Immunoprecipitation of IBIS1 has revealed three additional P. berghei proteins, which the group has termed IBIS2, IBIS3 and IBIS4, that localize to IBIS in P. berghei-infected RBCs. Furthermore, IBIS2 and IBIS3 colocalize with IBIS1 on the liver-stage TVN. The presence of these proteins on membranes at the interface with the host cell in both liver cells and RBCs suggests the possibility of shared functions occurring at these membranes in both infection stages. IBIS2, 3 and 4 have annotated orthologs in P. vivax and P. knowlesi, which indicates that functions carried out by proteins present within IBIS may be shared across Plasmodium species. Determining the functions of the P. berghei IBIS2, 3 and 4 will elucidate processes that occur at the liver-stage TVN and blood-stage IBIS that support parasite development.

To functionally characterize these proteins, the PhD student will complete the following aims:

1) Phenotypic characterization of P. berghei in which IBIS2, IBIS3 and IBIS4 are genetically targeted. In addition to assessing liver- and blood-stage growth, the liver-stage development will be assessed for recruitment of host factors known to be associated with the liver-stage parasitophorous vacuole and TVN. The fine structure of the IBIS-positive membranes in liver and blood stages will be assessed microscopically in the laboratory of Melanie Rug. 

2) Identification of protein interaction partners of IBIS2, 3 and 4. Potential binding partners will be identified using BioID from infected cells and GST-pulldowns using liver and blood cell extracts. Furthermore, protein interactions will be analysed by blue native PAGE in collaboration with Alexander Maier and high-resolution microscopy with Melanie Rug. P. knowlesi IBIS orthologs and any identified host interaction partners can be investigated in P. knowlesi-infected RBCs also in collaboration with the Rug group.

Interlinkages: Kai Matuschewski (HUB), Gaétan Burgio (ANU), Alexander Maier (ANU), Brendan McMorran (ANU)

References:

(1) Mueller, A.K. et al. (2005) Proc. Natl. Acad. Sci. USA 102:3022-3027

(2) Grützke, J. et al. (2014) Traffic 15:362-382

(3) Ingmundson, A. et al. (2012) Mol. Microbiol. 83:1229-1243

(4) Ingmundson, A. et al. (2013) Cell. Microbiol. 16:324-333