Trypanosoma brucei



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

These obligate parasites have two hosts - an insect vector and mammalian host. Due to the large difference between these hosts the trypanosome undergoes complex changes during its life cycle 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 infection: Trypanosomiasis
The insect vector for T. brucei is the tsetse fly. The parasite lives in the midgut of the fly (procyclic form), whereupon it migrates to the salivary glands for injection to the mammalian host on biting. The parasite lives within the bloodstream (bloodstream form) where it can reinfect the fly vector after biting. Later during a T. brucei infection the parasite 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, which cause different variants of trypanosomiasis.
 * T. brucei gambiense - Causes slow onset chronic trypanosomiasis in humans. Most common in central and western Africa, where humans are thought to be the primary reservoir.
 * T. brucei rhodesiense - Causes fast onset acute trypanosomiasis in humans. Most common in southern and eastern Africa, where game animals and livestock are thought to be the primary reservoir.
 * T. brucei brucei - Causes animal African trypanosomiasis, along with several other species of trypanosoma. T. b. brucei is not human infective due to its susceptibility to lysis by human apolipoprotein L1.  However, as it shares many features with T. b. gambiense and T. b. rhodesiense (such as antigenic variation) it is used as a model for human infections in laboratory and animal studies.

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. Unusual features include the single large mitochondria with the mitochondrial DNA structure known as the kinetoplast, and its association with the basal body of the flagellum. The cytoskeleton is made up primarily of microtubules. The cell surface of the bloodstream form features a dense coat of variable surface glycoproteins (VSGs) which is replaced by an equally dense coat of procyclins when the parasite differentiates into the procylic in the tsetse fly midgut.

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.

These names are derived from the Greek mastig- meaning whip, referring to the trypanosome's whip-like flagellum.

T. brucei is found as a trypomastigote in the slender, stumpy, procyclic and metacyclic forms. The procylic form differentiates to the proliferitive epimastigote form in the salivary glands of the insect. Unlike some other trypanosomatids, the promastigote and the amastigote form do not form part of the T.brucei life cycle.

The genome
The genome of T. brucei is made up of : 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.
 * 11 pairs of large chromosomes of 1 to 6 megabase pairs.
 * 3-5 intermediate chromosomes of 200 to 500 kilobase pairs.
 * Around 100 mini chromosomes of around 50 to 100 kilobase pairs. These may be present in multiple copies per haploid genome.

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.

VSG surface coat
Main section: The VSG coat

The surface of the trypanosome is covered by a dense coat of Variable Surface Glycoprotein (VSG), which allows persistence of an infecting trypanosome population in the host. See below.

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 are 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.

The VSG coat
The surface of the trypanosome is covered by a dense coat of ~1x107 molecules of Variable Surface Glycoprotein (VSG). This coat enables an infecting T. brucei population to persistently evade the host's immune system, allowing chronic infection. The two properties of the VSG coat that allow immune evasion are:
 * Shielding - the dense nature of the VSG coat prevents the immune system of the mammalian host from accessing the plasma membrane or any other invariant surface epitopes (such as ion channels, transporters, receptors etc.) of the parasite. The coat is uniform, made up of millions of copies of the same molecule; therefore the only parts of the trypanosome the immune system can 'see' are the N-terminal loops of the VSG that make up the coat.
 * Periodic antigenic variation - the VSG coat undergoes frequent stochastic genetic modification - 'switching' - allowing variants expressing a new VSG coat to escape the specific immune response raised against the previous coat.

Antigenic variation
Sequencing of the T. brucei genome has revealed a huge VSG gene archive, made up of thousands of different VSG genes. All but one of these are 'silent' VSGs, as each trypanosome expresses only one VSG gene at a time. VSG is highly immunogenic, and an immune response raised against a specific VSG will rapidly kill trypanosomes expressing this VSG. This can also be observed in vitro by a complement-mediated lysis assay. However, with each cell division there is a possibility that one or both of the progeny will switch expression to a silent VSG from the archive (see below). The frequency of such a switch has been measured to be approximately 1:100. This new VSG will likely not be recognised by the specific immune responses raised against previously expressed VSGs. It takes several days for an immune response against a specific to develop, giving trypanosomes which have undergone VSG coat switching some time to reproduce (and undergo further VSG coat switching events) unhindered. Repetition of this process prevents extinction of the infecting trypanosome population, allowing chronic persistence of parasites in the host. The clinical effect of this cycle is successive 'waves' of parasitaemia (trypanosomes in the blood).

VSG structure
VSG genes are hugely variable at the sequence level. However, for them to fulfil their shielding function, different VSGs have strongly conserved structural features. VSGs are 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 four alpha helices, while the N terminal domain forms a 'halo' around the helices. The tertiary structure of this halo is well conserved between different VSGs (in spite of wide variation in amino acid sequence) allowing different VSGs to form the physical barrier required to shield the trypanosome's surface. VSG is anchored to the cell membrane via a glycophosphatidylinositol (GPI) anchor - a covalent linkage from the C terminus, to approximately four sugars, to a phosphatidylinositol phospholipid acid which lies in the cell membrane. VSGs form homodimers.

VSG archive structure
The VSG gene archive is the collection of silent VSGs in the T. brucei genome. Some of these are full-length, intact genes; others are pseudogenes) typically with omitted sections or premature stop codons. Expression of an antigenically novel VSG can occur by simply switching to a different full-length VSG gene.  However, only 5% of the archive is made up of such complete silent VSGs.  To utilise the rest of the silent VSG archive, ‘mosaic’ VSGs can be formed by replacing part of the expressed VSG with a structurally homologous region from the archive.  The combinatorial nature of mosaic formation in conjunction with the huge silent VSG archive gives the parasite a theoretically limitless VSG library, and is the major barrier to vaccine development.

VSG expression
One major focus in trypanosome research is how the majority of VSG genes are kept silent, and how these genes are switched. The expressed VSG is always located in an Expression Site - found at the telomeres of the large and intermediate chromosomes. Each is a polycistronic unit, containing a number of Expression Site-Associated Genes (ESAGs) all expressed along with the active VSG. While there are at least 20 known expression sites, only a single one is ever active at one time. A number of mechanisms appear to be involved in this process, but the exact nature of the silencing is still unclear.

The VSG can be switched either by changing the active expression (from the active to a previously silent site) or by changing the VSG gene in the active site. The genome contains many copies of possible VSG genes, both on minichromosomes and in repeated sections in the interior of the chromosomes. These are generally silent, 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. Any of these genes can be moved into the active site by recombination for expression. Again, the exact mechanisms that control this are still only partially known.

Cell division
The mitotic division of T.brucei is unusual compared to most eukaryotes. The nuclear membrane remains intact and the chromosomes do not condense during mitosis. The basal body, unlike the centrosome of most eukaryotic cells, does not play a role in the organisation of the spindle and instead is involved in division of the kinetoplast.

Stages of mitosis:
 * 1) The basal body duplicates and both remaining associated with the kinetoplast.
 * 2) Kinetoplast DNA undergoes synthesis then the kinetoplast divides coupled with separation of the two basal bodies.
 * 3) Nuclear DNA undergoes synthesis while a new flagellum extends from the younger, more posterior, basal body.
 * 4) The nucleus undergoes mitosis.
 * 5) Cytokinesis progresses from the anterior to posterior.
 * 6) Division completes with abscission.