If you live in sub-Saharan Africa, the bite of a fly can have life-threatening consequences. Trypanosomes, transmitted to the blood stream by the bite of the tsetse fly, will proliferate, invade the central nervous system and kill an affected individual if left untreated. For all the hardship it causes, African trypanosomiasis remains an orphan disease with few effective drugs. The only targeted compound, initially developed around 30 years ago for cancer chemotherapy, is eflornithine, which is less toxic but also more expensive than arsenic-based compounds that have a drug-induced fatality rate of 5–10% in recipients.

A better understanding of trypanosome biology and its genetic makeup is key for identifying targets for new compounds. Genome sequencing revealed ∼7,500 genes in Trypanosome brucei, but the function of 64% of them remains unknown.

In the bloodstream of human hosts, trypanosomes coat themselves in glycoproteins, which are expressed from subtelomeric sequences with rapid switches in expression, thus making the parasites poor targets for antibodies. This antigenic variation first lured David Horn of the London School of Hygiene and Tropical Medicine into the study of trypanosome biology. “But as the genome sequencing projects progressed,” he says, “we became very interested in developing technologies to exploit the genome sequences.” Starting in 2001 his group began a collaborative project to systematically look at the approximately 200 genes on chromosome 1 using RNA interference (RNAi) screens. This labor-intensive project took 3 years to complete.

It made Horn realize that if they wanted to do a genome-wide screen, a pooled approach would be needed, and the idea of RNAi target sequencing (RIT-seq) was born. His team used an inducible RNAi plasmid library made up of sheared genomic fragments with an average length of 600 base pairs. To ensure tightly inducible, efficient expression, they targeted the RNAi cassettes to a single genomic locus and examined the effects of gene knockdown in four different samples. They isolated genomic DNA from the bloodstream stage of the trypanosome life cycle 3 or 6 days after induction, from the procyclic form at the insect stage and from cells that were induced during the bloodstream stage and allowed to differentiate all the way through to the insect stage. Horn then worked with Matt Berriman's team at The Wellcome Trust Sanger Institute to amplify the RNAi library cassette inserts and to perform high-throughput sequencing. The comparison of aligned sequence reads between the different induced stages and an uninduced control showed them 'cold spots'—genes that were associated with a loss of fitness in various samples.

Although Horn acknowledges that the datasets with loss of fitness in all four conditions are more robust, he sees the differentiation-specific genes also as potential drug targets. “A drug could trigger the cells in the patient to differentiate,” he says, “and in the process they lose their glycoprotein coat and become subject to complement mediated lysis.”

It goes without saying that all these results require experimental follow-up. Horn has released all the screening data on TriTrypDB, a site for kinetoplastid genomics resources. His team is now doing selective drug-resistance screens to get at the pathogen-specific uptake and metabolism of certain compounds.

“We need more resources to move the field forward more rapidly,” Horn says. Helpful resources would include a collection of defined RNAi lines to do gain-of-function screens and more precise follow-up.

Horn also points out that the principle behind RIT-seq could be applied to other parasites as well, as long as they have an RNAi machinery.

For the time being, the rich data resource Horn's team created will help with drug target triage and assist in the first steps toward more effective disease treatment. Go to http://www.youtube.com/watch?v=WhdYbrv3YCs to see for yourself how sorely treatment is needed.