Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

Functional biomolecular condensates implicated in parasite antigen trafficking of malaria-infected red blood cells (#144)

Olivia M. S. Carmo 1 , Yoon Hee Choi 1 , Kiersten M. Ruff 2 , Dezerae Cox 3 , Rohit V. Pappu 2 , Danny M. Hatters 1 , Leann Tilley 1 , Matthew W. A. Dixon 4
  1. Biochemistry and Pharmacology, Bio21 Institute, University of Melbourne, Melbourne, VIC, Australia
  2. Biomedical Engineering, Center for Science & Engineering of Living Systems, Washington University, St. Louis, MO, USA
  3. Chemistry, University of Cambridge, Cambridge, UK
  4. Infectious Diseases, Peter Doherty Institute, University of Melbourne,, Melbourne, VIC, Australia

The parasite Plasmodium falciparum is responsible for the most virulent form of malaria, killing ~500,000 people annually. The parasite’s virulence arises during the asexual blood stage, in which the parasite invades our red blood cells (RBCs). Interestingly, the mature RBC lacks endogenous organelles for the parasite to co-opt. To achieve host cell remodeling, virulence antigen presentation, and ultimately host immune evasion, the parasite exports ~500 proteins beyond its boundary into the host cell cytoplasm, more than a quarter of which contain predicted intrinsically disordered regions (IDRs). Deletion of some IDRs has been shown to have marked effects on virulence factor trafficking. Despite the identification of virulence factor trafficking partners, virulence factor transport to the host cell surface remains enigmatic, occurring in the absence of GTP-dependent or canonical trafficking machinery.

In this study, we demonstrate that regions from two P. falciparum proteins, both relevant to virulence factor trafficking, form droplets in a cytosolic environment using the optoDroplet technique. OptoDroplet relies on the expression of the IDR of interest fused to a fluorophore and a Cry2 photoactivatable domain, allowing for observation of activatable and tunable protein-droplet formation in living cells. We found that scrambling one protein sequence increased the likelihood of droplet formation, perhaps due to the shortened distribution of aromatic stickers. Droplet formation of the second protein tested is abrogated in a phospho-mimetic mutant, indicating that the charge status of 5 residues of the 250 aa sequence has a major effect on droplet propensity.

These preliminary results have shifted our focus towards biomolecular condensates as a prism to understand virulence factor trafficking. Studies underway probe the sequence basis of these changes in droplet propensity between wildtype and mutant proteins, in addition to assessing the functional consequence of the mutant sequences in the P. falciparum-infected RBC.