United front

Tritryp parasites are insidious enemies. To them, a host is not merely a safe place to live, it is also an energy supply to be subverted and consumed. It is a malevolent yet highly successful strategy. Many scientists want to understand and tackle these microscopic pests. During the past couple of years, their job has been made much easier with the sequencing of the tritryp genomes. With all of a parasite's genes unveiled, researchers can examine them to understand its strengths and to search for potential weaknesses.

In the late 1990s, three collaborations set out to sequence the genomes of the trypanosomatids: Trypanosoma brucei, T. cruzi and Leishmania major. Independent genome projects at first, they have come together into a unified 'tritryp team', with annual meetings and all data stored in a single database called GeneDB (view the online database).

As the projects have progressed, genome sequence data have been freely released onto the web, providing an invaluable tool for researchers studying the parasites, as the following articles indicate. But there are other benefits in the unified approach: "A specialist in Leishmania will be able to examine equivalent genes from T. brucei and T. cruzi," says Dr Matt Berriman, Tritryp Project Manager at the Wellcome Trust Sanger Institute, which has played a pivotal role in the L. major and T. brucei projects and hosts GeneDB. "Researchers who have spent their lives looking at one organism can expand their remit into two closely related organisms."

In the genome

Superficially, the parasites look similar. But their chromosomes are by no means the same, most obviously in number: T. brucei has 11 large chromosomes (along with a selection of intermediate-sized and mini chromosomes), T. brucei 36 and T. cruzi about 56 (it is hard to tell). The chromosome pairs of T. brucei can vary in size by about 15 per cent, and the two copies can have different variants of certain genes. Differences in sizes between the chromosome pairs might also give clues to the amount of variation in the genomes.

And T. cruzi? "The key characteristic of T. cruzi is that it is so hard to work on because of its huge genetic diversity," says Dr Berriman. "Assembling the sequence is extraordinarily difficult: the chromosome pairs differ so much it's as though you're looking at two different genomes." Only since the data from the other two genomes became available have the sequencers in the USA and Sweden been able to look at similarities between the species and start to line up the T. cruzi DNA.

Having cracked these difficult nuts, the Sanger team is already working on the genomes of several other related parasites that infect humans and animals. Joining L. major, an 'Old World Leishmania' that causes a skin infection, are the genomes of L. infantum, a species that causes visceral disease, and the New World species L. braziliensis, which causes a very disfiguring disease (see Fighting back). Three other trypanosome species are being sequenced: T. brucei gambiense (the big killer of humans), and T. vivax and T. congolese, which infect cattle. "The comparative projects are going really well," says Dr Berriman.

"The really polished L. major sequence is allowing us to make rapid progress on the other leishmanias – it saves in sequencing costs as we need fewer sequences and can organise them on a template."

Using the genome

Dr Sara Melville is a vocal advocate of genome sequencing projects. "Having the genome available changes the way you do research," she asserts. "It suggests experiments that you may not have thought of otherwise, and it speeds up what you can do.

Instead of it taking most of a PhD project to find and clone a gene, the work is already done. You can go straight on to investigating what genes actually do, which is far more interesting (especially for the PhD students)."

In her research into T. brucei at the University of Cambridge, Dr Melville has been using the genome sequence to build resources for postgenomic studies – research that can examine thousands of genes and their proteins at once. Such large-scale studies are essential, given that the role of so many genes remains mysterious. "Even at sequencing centres such as the Sanger, where both humans and computers attempt to identify what genes might be, or what they might do, for only about 40 per cent of the genes can you make some kind of prediction of their role in the parasite. However, the genes that are still hypothetical may be the most interesting in the end; a gene found only in African trypanosomes might be very interesting indeed."

Scientists have already been using the genome data – even when incomplete – for microarray experiments. With tiny spots of DNA spots printed onto a chip, the arrays can examine hundreds or thousands of genes at once to see if they are expressed all the time, or only at certain stages in the parasite's life cycle (in the human bloodstream, for example). Professor Jennie Blackwell's research on Leishmania (see Fighting back) and Dr Melville's research on T. brucei show that some genes are indeed switched on at certain stages and off in others.

"Even using these early arrays, it appears that about 20 per cent of genes vary in their expression between stages", says Dr Melville. "This number is likely to go up as more stages are analysed, and a new array has been released that contains portions of every T. brucei gene found by the sequencers, so the experiments will become even more comprehensive." With the availability of a complete genome sequence, proteomic studies have also become more feasible. These technically demanding experiments examine the proteins present in a parasite, and can again be used to examine differences between lifecycle stages.

Even though her own research focuses on T. brucei, Dr Melville is particularly excited by the sequencing of the T. vivax and T. congolense genomes as the parasites infect cattle and other livestock, and prevent meat and milk production and animal-powered ploughing in a wide swathe of sub-Saharan Africa . "T. vivax and T. congolense are huge problems in Africa, but not many people have been investigating them," she says. "Hopefully the genome sequences will spark new research: it's an open field, and a real opportunity for young researchers."

Genes and drug resistance

Professor Andy Tait, Professor Mike Turner and Dr Annette MacLeod at the Wellcome Trust Centre for Molecular Parasitology in Glasgow have not just been examining the T. brucei genome sequence on the internet. They downloaded the entire unfinished sequence from GeneDB to identify a series of landmarks across the parasite's chromosomes in order to generate a genetic map from crosses between different strains of the parasite. This is helping the University of Glasgow team to find genes involved in traits such as drug resistance; it also proved useful to the Sanger sequencing team when it was finishing the sequence.

"We're interested in the genetics of traits involved in the treatment, transmission and pathogenesis of trypanosomiasis," says Professor Tait. "We want to find the genes that explain why some strains of parasites are resistant to drugs, why some infect humans and others are virulent and cause anaemia. We are interested in how the parasite causes disease and is transmitted."

The need to understand drug resistance is pressing, with confirmed reports of resistance to the few drugs available: to melarsoprol, a toxic arsenic-based drug, the only effective treatment for last stage disease (as it can cross the blood–brain barrier); and potential resistance to pentamidine and suramin, used for earlier stages of infection. There has also been a worrying, but less clear-cut, increase in reports of treatment failures, in some cases in up to 30 per cent of cases.

Trypanosomes that infect cattle have become resistant to drugs – about a third of parasite strains in Zambia, Kenya and Uganda. Indeed, Professor Tait suggests that the treatment of cattle trypanosomiais with drugs related to those used in humans may be a key issue if the strains that infect humans acquire resistance when they are living in cattle. The availability of the genome sequences of the cattle trypanosomes will allow researchers to examine whether common mechanisms of drug resistance occur.

To find the genes involved in resistance, Professor Tait is using a 'classical genetics' approach, breeding strains of trypanomes to understand the inheritance of the trait and then using the genetic map to identify the approximate region of a chromosome involved. Here, the genome sequence is proving invaluable: "From GeneDB you know which genes are in that region and can immediately start looking to see which of the genes are involved in the trait. It's the same approach as with human genetics – mapping human traits – only it's much easier in tryps as the genome is so small."

The advantage of this approach, he argues, is that it makes no preconceptions about what the gene might be. "The change could be in a gene involved in the drug's target, its uptake, its metabolism or degradation, none of these or several; there are likely to be multiple mechanisms of resistance. But once you've identified genes and mutations, you also need to go to Africa and see if they really are relevant to drug resistance in the field."

Some such genes may well be useful targets for future drug development, but diagnosis is likely to be a more immediate use: "At the moment it's very difficult to confirm that a parasite strain is resistant to a certain drug – you have to test them in rodents. What is needed is a test that can diagnose resistance very quickly – a doctor or veterinarian needs to be able to decide immediately whether or not to use a drug."

Image: Dr Sara Melville, courtesy of the Wellcome Trust Medical Photographic Library.

Related links

• Three of a kind: History and legacy of tritryp parasites (Feature: 15 July 2005)
• Moving forward: The trypanosome flagellum (Feature: 15 July 2005)
• Puppy dogs tales: Breaking the transmission of the tritryps (Feature: 15 July 2005)
• Fighting back: The immune response to the Leishmania and vaccine development (Feature: 15 July 2005)
• Tackling tritryps: The biology and business of drug development (Feature: 15 July 2005)
• Research: Neglected diseases sequenced (News 15 July 2005)