Abstract text

Introduction

Trypanosoma brucei is a eukaryotic parasite. Subspecies of the parasite can infect humans and livestock. The parasites are transmitted by the tsetse fly. After infection of a mammal, they inhabit the blood and diverse tissues, where the parasite features a dense coat of a glycosylphosphatidylinositol-anchored variant surface glycoprotein (VSG). The VSG coat shields invariant proteins and the plasma membrane from the host immune system. The density of the VSG coat is tightly controlled by the parasite.

Objectives

We want to deepen our understanding of cell surface processes and how they can function at the biophysical limit. Therefore, we are trying to artificially recreate the surface of the trypanosome. An artificial membrane will give us the option to control all aspects of the surface system to investigate the mechanisms behind the high density of the VSG coat, how both VSG structure and density effects diffusion on the parasite surface, the antigenic switch of one VSG type to another and the largely unknown behaviour of invariant surface proteins.

Materials and methods

We developed an artificial membrane system on glass. Phospholipids and natural VSG proteins are dissolved in detergents and reconstituted into protein-dense proteoliposomes, which are then fabricated into supported lipid bilayers. The diffusion behaviour of VSG in the bilayers is analysed with single-molecule fluorescence microscopy.

In the near future, we aim to replace the simple lipid bilayer with a chemically diverse and structurally functional membrane. Therefore, we purify and identify the surface lipidome of T. brucei and use the trypanosome-derived lipid composition to create an artificial, trypanosome-specific membrane system.

Results

We developed a process that routinely creates artificial membranes with a high protein density. The diffusion behaviour of VSG on our artificial membrane resembles the observed in-vivo diffusion coefficients of plasma-membrane anchored VSG. Early results show that we can separate the plasma membrane from inner membrane compartments to selectively purify membrane lipids for reconstitution into artificial lipid bilayers.

Conclusion

By combining our newly developed tools, we want to draw a detailed picture of the biophysical processes at the host-parasite interface.