Targeting the cell surface molecules responsible for the African sleeping sickness parasite survival and infectivity

Targeting the cell surface molecules responsible for the African sleeping sickness parasite survival and infectivity

Speaker: João A. Rodrigues

Affiliation: Centre for Malaria & Tropical Diseases-IHMT/UNL

Host: Jaime Mota- Infection Biology Laboratory

Abstract:

The African trypanosome Trypanosoma brucei is the causative agent of human African sleeping sickness (HAT) and the cattle disease nagana. These parasites are dependent on glycoproteins for their survival and infectivity. N-Glycosylation of proteins is a ubiquitous and generally essential process in eukaryotic organisms. It is involved in key functions such as the exit of glycoproteins from the endoplasmatic reticulum (ER), and quality control of protein folding. Typically, the dolichol-pyrophosphate-linked oligosaccharide precursor is transferred co-translationally to N-X-S/T protein acceptor sequons in the ER through the action of the Oligosaccharyltransferase (OST). OST is generally a complex of up to eight proteins associated with the Sec61 translocon complex. However, the T.brucei genome lacks homologues of known OST complex subunits, except for the catalytic subunit (STT3) for which it has three different isoforms STT3A, B and C. By using RNAi, as a functional genomics approach, we have induced knockdowns of the first two subunit STT3A & B in bloodstream form T. brucei. Knockdown results in altered glycosylation as identified by the

characterisation of the corresponding most abundant glycoprotein in T. brucei, the variant surface glycoprotein (VSG) and reduced infectivity.

This functional genomics approach has been complemented with a comprehensive analysis of the glycoproteome of T. brucei using a serial lectin affinity capture approach to enrich for glycosylated proteins and subsequent sequential enzymatic deglycosylation steps prior to geLC-MS/MS analysis using reversed-phase nanoflow liquid chromatography sampled into the hybrid LTQ-Orbitrap mass spectrometer and MASCOT database search. These results are crucial in the attempt to define a consensus sequence pattern with which to predict sites modified by the different T. brucei STT3 isoforms. Our goal of establishing the molecular basis of this phenomenon is already highlighting what we believe to be therapeutically exploitable host/parasite differences.