Moving Forward

The trypanosome flagellum

The flagellum makes the trypanosome a speedy assassin. But it does more than just move the parasite. And understanding what it does is suggesting ways to combat the trypanosome.

Look at Trypanosoma brucei under the microscope and there will be a hyperactive cell, thrashing about for all its worth. Its great locomotory abilities stem from its flagellum, which can propel the parasite through the bloodstream or the gut of the tsetse fly.

But that is not the only string to the flagellum's bow: it also acts as an anchor and has a central role in cell division. And the specialised area at the base of the flagellum – the flagellar pocket – is turning out to have some very interesting properties indeed.

New tools

Research on the cell biology of trypanosomes has been revolutionised in recent years by two technological advances. The first has been the sequencing of the T. brucei genome. With this information, researchers have been able to identify genes involved in biological processes. Also, the gene sequences can be compared between T. brucei and its close relatives Leishmania and T. cruzi, giving more clues to their function.

The second advance is a new technique, RNA interference. This allows researchers to switch off genes very precisely in the living parasite.

"Being able to couple the genome sequence information with RNA interference technology is synergistic," suggests Dr Mark Field, a Wellcome-funded trypanosome cell biologist at the University of Cambridge. "I think over the next five years we are going to see an enormous amount of biology coming out that we wouldn't have had access to without this combination."

Studies of the flagellum illustrate his point. It is a sophisticated structure, with a complex base, composed mainly of microtubules, embedded within the cell. Professor Keith Gull, a Wellcome Trust Principal Research Fellow at the University of Oxford, is defining its component structures and wants to understand how they are put together to build the flagellum, and also how these structures influence the building of the flagellar pocket.

As well as being important in motility, the trypanosome flagellum is used by the parasite to attach itself to the tsetse salivary gland – without this, it would be quickly washed out. So it provides a specialised area of surface membrane, with different interactions with the outside world.

In addition, says Professor Gull, "it defines a third differentiated area of the surface membrane – the flagellar pocket – which is important for uptake and secretion of different molecules and in resistance to acquired and innate immune responses." And finally the flagellum is involved in defining the form and structure of the parasite cell (see below).

The cell's skeleton

By analysing the T. brucei genome, Dr Bill Wickstead in Professor Gull's lab has found that African trypanosomes have fewer genes coding for the actin components of the cell's internal scaffolding (cytoskeleton) than other parasites. Instead, it has many more genes coding for components of the microtubule cytoskeleton. Professor Gull believes this is associated with the parasite's unique cell biology. "The malarial parasite has a cell biology based onactin, and the actin cytoskeleton is mainly involved in invasion of its host's cells. But a parasite like the African trypanosome, which is not interested in being intracellular, is a different beast." In this case, microtubule-based processes defining shape and active motility are much more important.

But although the actin cytoskeleton is not important when the parasite is in the fly, it is essential for the survival of bloodstream forms of the parasite, where actin is found associated with the flagellar pocket. Dr Derek Nolan, a Wellcome Trust Senior Research Fellow at Trinity College Dublin, has used RNA interference to show that in the bloodstream trypomastigote, the filamentous form of actin (F-actin) is essential for uptake of nutrients and other material at the flagellar pocket – the trypanosome's 'mouth'.

"Using specific inhibitors, we have shown directly that the formation of F-actin is critical to maintaining the polarity of actin distribution and the organization of the endocytic pathway," he says. So, he suggests, it should be possible to design drugs targeted at the trypanosome-specific F-actin.

Picking the pocket

Unlike most other parasites, trypanosomes make no attempt to escape detection when in the bloodstream of their mammalian hosts. "Most pathogens hide in a particular cell or tissue space to try and avoid the host's immune system, or they attempt to modulate the immune system," says Dr Field. "But the African trypanosome doesn't seem to bother."

One of the reasons for this is that the trypanosome is sheathed in a dense protective coat of variant surface glycoproteins, VSGs (see Tackling tritryps). The only gap in this armour is the flagellar pocket, where nutrients are taken up and molecules are secreted. As well as presenting many fascinating biological puzzles, this pocket may also be, researchers hope, the parasite's Achilles heel.

The trypanosome takes up material in much the same way as most cells, by the invagination of the surface membrane and formation of vacuoles (endocytosis). Secretion is achieved by the reverse mechanism: vacuoles fuse with the surface membrane and discharge their contents into the outside world (exocytosis). But trypanosomes are different in one key respect.

"Processes such as endocytosis, exocytosis – and possibly surface membrane turnover in general – are highly polarised and restricted entirely to a small invagination of the plasma membrane at the base of the flagellum called the flagellar pocket," explains Dr Nolan.

The flagellar pocket membrane accounts for about 1 per cent of the surface area of the parasite, but in the bloodstream form at least, it exhibits extremely high levels of cross-membrane traffic, far higher than those seen in any multicellular organism. By stark contrast, uptake into the cell is almost completely shut down in the insect stages of the parasite. These striking differences have yet to be explained.

The parasite also uses the flagellar pocket to take up and degrade antibodies. Within minutes of antibody binding, the parasite cleans its surface by transferring the bound antibody to the flagellar pocket where it is rapidly internalised and degraded. If the parasite is prevented from grooming itself in this way, it will not survive. So, the flagellar pocket may be a point of weakness for the trypanosome.

Sorting problems

A second notable property of the flagellar pocket is its ability to route different proteins to different parts of the cell surface. VSGs, for example, account for 90 per cent of the parasite's surface protein. They are connected to the cell surface via a lipid-based anchor (a glycosyl-phosphatidylinositol or GPI anchor, see Tackling tritryps). But GPIs also anchor other proteins, such as the transferrin receptor, which is restricted to the flagellar pocket. How are some proteins kept in the pocket while others are free to diffuse over the rest of the parasite surface?

Usually, the subcellular destination of proteins depends on short 'sorting' sequences within them, but these are not present in the GPI-anchored proteins. "This [selective retention] raises the possibility that a luminal-based sorting mechanism might operate in the flagellar pocket," suggests Dr Nolan. And, he adds, the parasite might be using similar mechanisms to control the delivery of enzymes and other proteins to different parts of the cell.

He plans to exploit the genome sequence information to find out more about the sorting mechanisms involved. Crucially, with the entire genome sequenced, he can use whole-genome and proteomic approaches to identify all the genes that potentially code for proteins from the flagellar pocket endocytic pathway.

Complex complexes

Uptake and secretion are also being studied by Dr Mark Field and colleagues at Cambridge. They have identified a number of proteins, called Rabs, that control various steps in the trafficking system.

The Rab proteins are part of a large family of enzymes known as GTPases, which exist in two conformations depending on whether they are binding GTP or GDP. "We can think of them as a binary switch or like a transistor within a circuit," says Dr Field.

Typically, GTPases are responsible for bringing together (and dissolving) multicomponent complexes. "GTPases are often control elements that are at the heart of protein complexes and signal to say 'form a complex'," he says. "The complex is formed and then when the GTP is hydrolysed to GDP the complex will fall apart." In this case, the Rabs seem to be involved in controlling both the direction and speed of traffic into, out of and around the cell.

The Rabs are of particular interest as some of the family are found in Leishmania and T. cruzi, but not in mammals or yeasts, so again may provide therapeutic targets.

Poisoning the parasite

Other researchers are looking to exploit membrane transporters to develop new therapies. Trypanosomes do not have the enzymes necessary to make purines, essential components of RNA and DNA. Instead,

they must import them from their surrounding environment. Dr Harry de Koning at the University of Glasgow has been characterising the transporters involved in purine uptake in the hope that they might provide a novel therapeutic target.

Unfortunately, trypanosomes have at least five different purine transporters, so they would be difficult to target with specific inhibitors. Dr De Koning is therefore exploring an alternative approach – using the transporters to deliver toxic compounds into the cell. Purine analogues, for example, could disrupt nucleic acid synthesis and block cell reproduction.

"These parasites have the most efficient purine uptake system I am aware of," says Dr De Koning. "They take purines up very rapidly and accumulate them in the cell where they reach high concentrations." And as Dr De Koning says, this type of medication is already approved for use in humans. "A lot of similar compounds are used as anti-virals and in anti-cancer therapies, so purine-based drugs have a long history of excellent efficacy."

Exploring the third dimension

The trypanosome parasite moves through its insect and mammalian hosts like a corkscrew threading into a cork. This characteristic motion gives the parasite its name which comes from the Greek word ‘trypanon’, meaning ‘borer’ (which also gives us ‘trepanning’, the practice of drilling holes in the skull).

But the parasite’s flagellum, which facilitates this unusual motion, is more than just a means of locomotion: as Professor Keith Gull’s research shows, it is essential for the parasite’s survival.

The trypanosome flagellum is vital for motility, says Professor Gull. But, he adds, it does a lot more than that: “It defines the flagellar pocket and a whole cell biology associated with the shape and form of the parasite, and this is really important for pathogenicity.”

But that is not all. Like other kinetoplastids, trypanosomes have large amounts of mitochondrial DNA enclosed in a single organelle known as a kinetoplast. And for the parasite to survive, the kinetoplast and its DNA contents must be faithfully copied at each cell division and segregated into the two daughter cells. This complicated molecular choreography, it turns out, is orchestrated by the flagellum.

“The mitochondrial genome is segregated during cell division as a consequence of the movement apart of the new and old flagella,” explains Professor Gull. This all depends on the correct positioning of the flagellum. Professor Gull and his team have used a technique called EM tomography to produce computer-generated 3D images of organelle positioning during cell division.

He has recently found that as the cell divides, the new flagellum emerges from the cell and is guided down the helix of the old flagellum by a structure he calls the flagellar connector. This allows spatial information from the old cell to be mimicked in the daughter cell, and ensures accurate positioning of the new flagellum.

Professor Gull’s team is now using EM tomography to explore the role of proteins revealed by the genome-sequencing project, creating mutant forms in which the intricate process of cell division is disrupted.

“And in order to precisely define the phenotype of the mutants, and hence what the gene does, we need to understand the cellular changes in three dimensions.”

Image courtesy of the Wellcome Trust Medical Photographic Library.