Trypanosoma brucei

Trypanosoma brucei is parasitic protist species that causes African trypanosomiasis (or sleeping sickness) in humans and animals in Africa. There are 3 sub-species of T.brucei; T.b.brucei, T.b.gambiense and T.b.rhodesiense.

The entirely parasitic species has two hosts - its insect vector and mammalian host. Because of the large difference between these hosts the cell undergoes complex changes to facilitate its survival in the insect gut and the mammalian bloodstream. It also features a unique and notable variable surface glycoprotein (VSG) coat in order to avoid the host's immune system. There is an urgent need for the development of new drug therapies as current treatments can prove fatal to the patient as well as the trypanosomes.

The trypanosome cytoskeleton is the subject of considerable research. The cytoskeleton, as the structure behind mitosis, locomotion and surface binding, is vital for viability and so is a target of interest for drug development. Much research on Trypanosoma brucei is first done on Crithidia fasciculata a highly similar organism that is not dangerous to humans.

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The infection: Trypanosomiasis

Main article: African Trypanosomiasis

The insect vector for T.brucei is the tsetse fly. The parasite lives in the gut of the fly (procyclic form), until it migrates to the salivary glands for injection to the mammalian host on binding. The parasite lives within the bloodstream (bloodstream form) where it can reinfect the fly vector after biting. Later during a T.brucei infection the parisite may migrate to other areas of the host. A T.brucei infection may be transferred human to human via bodily fluid exchange, primarily blood transfer.

There are three different sub-species of T.brucei cause different variants of Trypanosomiasis.

• T.b.gambiense - Causes slow onset chronic trypanosomiasis.
• T.b.rhodesiense - Causes fast onset acute trypanosomiasis.
• T.b.brucei - Causes animal African trypanosomiasis (or Nagana), along with several other parasites.

The cell structure

The structure of the cell is fairly typical of eukaryotes, see eukaryotic cell. All major organelles are seen, including the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus etc. Main unusual features include the single large mitochondria with a condensed mitochondrial DNA structure, and its association with the basal body of the flagellum, unusually the cytoskeleton organisation mechanism of the cell. The cell also features a dense coat of variable surface glycoproteins (VSGs).

  Trypanosomatids show specific cellular forms:

• Amastigote - Basal body anterior of nucleus, with a short, essentially non-functional, flagellum.
• Promastigote - Basal body anterior of nucleus, with a long detached flagellum.
• Epimastigote - Basal body anterior of nucleus, with a long flagellum attached along the cell body.
• Trypomastigote - Basal body posterior of nucleus, with a long flagellum attached along the cell body.

T.brucei can be found in any of these forms, with the typical procyclic and bloodstream forms in the trypomastigote structure.

The genome

The genome of T.brucei is made up of:

• 11 large chromosomes of 1 to 6 megabase pairs.
• 6 intermediate chromosomes of 300 to 600 kilobase pairs.
• Around 100 mini chromosomes of around 50 to 100 kilobase pairs. These may be present in multiple copies per haploid genome.

The large chromosomes contain most genes, while the small chromosomes tend to carry genes involved in antigenic variation, including the VSG genes. The genome has been sequenced and is available online www.genedb.org.

The mitochondrial genome is found condensed into the kinetoplast, an unusual feature unique to the kinetoplastea class. It and the basal body of the flagellum are strongly associated via a cytoskeletal structure.

The VSG coat

The surface of the trypanosome is made up from a uniform coat of variable surface glycoproteins (VSGs). This coat has two roles:

• A physical barrier blocking recognition of the cell by the non-specific immune response of the mammalian host and hiding invariant surface proteins (such as ion chanels, receptors, etc.) from specific immune system recognition.
• A variable surface to the cell, allowing variation and adaptation to avoid the specific immune system.

The coat is highly variable - there are thought to be many hundred alternative copies of the gene in the genome. In each cell, and throughout a population of cells within the host, the same coat protein is expressed, but this expression is unstable and is likely to change with the next generation. Rates of switching of up to 1 in 50 cells per generation have been seen.

The protein is made up of a highly variable N terminal domain of around 300 to 350 amino acids, and a more conserved C terminal domain of around 100 amino acids. The c terminal domain forms a structural bundle of 4 alpha helices, while the N teminal domain forms a 'halo' around the helices. The tertiary structure of this halo is well conserved (surprisingly, given that the actual amino acid sequences vary widely), allowing close packing into the physical barrier the VSGs are required to form. The VSGs are anchored to the cell membrane via a GPI anchor - a covalent linkage from the c terminus, through around 4 sugars, to a phosphatidyl-inositol phospholipid acid which lies in the cell membrane.

The genome contains many copies of possible VSG genes. Around 20 are found on the large and intermediate chromosomes which are active and potentially transcribed (although only one per cell ever will be). Around 100 are found near the telomeres of the mini chromosomes. These are not active, but if moved via recombination to an active transcription site will produce a functional VSG protein. Finally around 1000 are found in repeated sections in the interior of the chromosomes. These are generally inactive, typically with omitted sections or premature stop codons, but are important in the evolution of new VSG genes. It is estimated up to 10% of the T.brucei genome may be made up of VSG genes or pseudogenes.

Upon infection the trypanosome originally expresses a particular VSG. As the host's immune system generates a specific response to this coat protein this selects against expression of that VSG, lowering the population of trypanosomes. At this point a cell with an alternative VSG will be strongly selected for, which will then go on to repopulate the infection. The overall effect of this boom and bust population cycle due to the predator/prey relationship with the hosts immune system leads to a succession of bouts of infection, each with a different VSG coat protein being expressed.

The cytoskeleton

The cytoskeleton is predominantly made up of microtubules, forming a subpellicular corset. The microtubules lie parallel to each other along the long axis of the cell, with the number of microtubules at any point roughly proportional to the circumference of the cell at that point. As the cell grows (including for mitosis) additional microtubules grow between the existing tubules, leading to semiconservative inheritance of the cytoskeleton. The microtubules are orientated + at the posterior and - at the anterior.

Microfilament and intermediate filaments also play an important role in the cytoskeleton, but these generally overlooked.

Flagellar structure

  The trypanosome flagellum has two main structures. It is made up of a typical flagellar axoneme which lies parallel to the paraflagellar rod, a lattice structure of proteins unique to the kinetoplastida, euglenoids and dinoflagellates.

The microtubules of the flagellar axoneme lie in the normal 9+2 arrangement, orientated with the + at the anterior end and the - in the basal body. The a cytoskeletal structure extends from the basal body to the kinetoplast. The flagellum is bound to the cytoskeleton of the main cell body by four specialised microtubules, which run parallel and in the same direction to the flagellar tubulin.

The flagellar function is twofold - locomotion via oscilations along the attached flagellum and cell body, and attachment to the fly gut during the procyclic phase.

Mitotic process

  The mitotic division of T.brucei is unusual in terms of the cytoskeletal process. The basal body, unlike a centrosome of most eukaryotic cells, plays an important role in the organisation of the spindle.

Stages of mitosis:

1. The basal body replicates, both remaining associated with the kinetoplast.
2. The kinetoplast undergoes replication, and the daughter kinetoplasts are separated by the basal bodies.
3. The second flagellum grows while the nucleus undergoes replication.
4. The mitochondria divides, and cytokinesis progresses from the anterior to posterior end.
5. The division resolves. The daughter cells may stay connected for a significant length of time after cytokinesis is complete.

References

• K. Gull (1999). "The Cytoskeleton of Trypanosomatid Parasites". Annual Review of Microbiology 53: 629-655. PMID 10547703.