Transposon Mutagenesis of Trypanosoma brucei Identifies Glycosylation Mutants Resistant to Concanavalin A

Simone Leal,Alvaro Acosta-Serrano,James Morris,George A.M Cross

doi:10.1074/jbc.m403479200

Abstract

We have engineered Trypanosoma brucei with a novel mariner transposition system that allows large populations of mutant cells to be generated and screened. As a proof of principle, we isolated and characterized two independent clones that were resistant to the cytotoxic action of concanavalin A. In both clones, the transposon had integrated into the locus encoding a homologue of human ALG12, which encodes a dolichyl-P-Man: Man(7)GlcNAc(2)-PP-dolichyl-alpha6-mannosyltransferase. Conventional knock-out of ALG12 in a wild-type background gave an identical phenotype to the mariner mutants, and biochemical analysis confirmed that they have the same defect in the N-linked oligosaccharide synthesis pathway. To our surprise, both mariner mutants were homozygous; the second allele appeared to have undergone gene conversion by the mariner-targeted allele. Subsequent experiments showed that the frequency of gene conversion at the ALG12 locus, in the absence of selection, was 0.25%. As we approach the completion of the trypanosome genome project, transposon mutagenesis provides an important addition to the repertoire of genetic tools for T. brucei.

Highlights

The medical and economic relevance of Trypanosoma brucei and the many peculiarities of this differently evolved parasite (1–3) make it an interesting model eukaryote
Transposon mutagenesis could potentially provide a valuable tool for forward genetics in T. brucei
Transposition in T. brucei—To circumvent the low transfection efficiency of T. brucei, we started with the objective of establishing permanent cell lines that could be amplified prior to selecting for transposition and screening or selecting for specific phenotypes

Summary

Introduction

The medical and economic relevance of Trypanosoma brucei and the many peculiarities of this differently evolved parasite (1–3) make it an interesting model eukaryote. Among the many important adaptations that occur during the life cycle of T. brucei, the change of its surface coat is crucial. Upon differentiation in the tsetse (Glossina), variant surface glycoprotein is shed and replaced by members of the procyclin family of GPI1-anchored glycoproteins. Present address: Wellcome Centre for Molecular Parasitology, Anderson College, University of Glasgow, Glasgow G11 6NU, Scotland, UK. In T. brucei Lister 427, the EP-procyclin isoforms are encoded by the EP1, EP2, and EP3 genes (4, 8). Procyclins are important for survival in the tsetse (10 –12) but can be dispensed with in culture, after a period of adaptation that involves increased expression of free GPIs on the parasite surface (13). Despite the major advances in reverse genetics approaches to study T. brucei (14, 15), tools that would allow large scale functional analysis of its genome are urgently needed. Transposition is independent of host-specific factors (20), which has allowed mariner to be used for gene disruption in a variety of organisms, including Leishmania (21), nematodes (22), and vertebrates (23, 24)

Objectives

Methods

Results

Conclusion