Possible Mechanisms Of Antigenic Variation In Trypanosomes Biology Essay

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Several pathogens of humans and domestic animals depend on hematophagous arthropods to transmit them from one vertebrate reservoir host to another and maintain them in an environment. These pathogens use antigenic variation to prolong their circulation in the blood and thus increase the likelihood of transmission. Antigenic variation is of enormous importance to the African trypanosome. Whereas many eukaryotic parasites have evolved an intracellular habitat that helps conceal them from the host immune system, trypanosomes have obstinately remained extracellular throughout their entire life cycle. As a consequence, their cell surface is the primary target of vigorous, trypanocidal immune responses, and in turn presents the main line of defense against these responses. Defense like this is not a straightforward task, as a balance has had to be achieved between the accessibility of the parasite's surface to positively acting host molecules, such as nutrients and the protective nature of this interface with the host. Perhaps the most effective means of achieving the balance, because we can see that it has evolved independently in several other extracellular micropathogens living systemically in mammalian hosts, is antigenic variation. The trypanosome has invested very heavily in its antigenic-variation system [1].

Trypanosomes are members of the order Kinetoplastida, which comprises unicellular parasitic protozoa distinguished by a single flagellum and a kinetoplast. They are parasites of invertebrate and vertebrate hosts throughout the world. African trypanosomes are a more constant factor in animal husbandry (cattle, goats, sheep, pigs, horses, and dogs are constantly challenged) than for humans, but in many locations, human sleeping sickness remains a serious threat, and appears to be largely dependent on the range of their specific insect vector Glossina commonly known as the tsetse. Several developmental stages that trypanosomes obligatorily undergo in the tsetse, transmission can occur in the absence of this vector, whereby the presence of T. vivax and T. evansi in South America and Asia, are transmitted by biting flies and vampire bats. Trypanosoma brucei is the most widely studied of the African trypanosomes, because it is by far the most convenient laboratory model. The infection consists of rising and falling parasitemia resulting from the generation of subpopulations that have antigenically different forms of a major variant surface glycoprotein coat (VSG) at the cell surface [2].

Antigenic Variation

The basis of the trypanosome system of antigenic variation is the protective coat on the parasite. The entire cell surface of the bloodstream and metacyclic form trypanosomes, including the flagellum is covered with a coat that is thought to provide general protection against non-specific host resistance mechanisms. The coat is potent immunogen and elicits high titres of antibodies that are lytic to the parasite. Through antigenic variation, which operates simply by rare individuals change to another coat, some parasites survive and can produce a new wave of growth. Each variant is termed a distinct variable antigen type (VAT). The different VATs retain the general protectiveness of the coat, while providing the variation enabling avoidance of specific antibodies. Switching is preemptive, generating a minority of new VATs before the antibody assault on trypanosomes of the old VAT. Antigen switching is rapid, at an average rate of one switch in every 100 trypanosome doubling. Metacyclic stage in the salivary glands of the tsetse is the first stage to be introduced into the mammalian host. The metacyclic population is diverse in these coats, expressing up to 27 metacyclic VATs in T. brucei and 12 in T. conglense.[3].

The Variant Surface Glycoprotein Coat

Animal-infective bloodstream and tsetse salivary-gland 'metacyclic' trypanosomes are characterized by an electron-dense surface coat. The surface coat of an individual trypanosome consists of about 10 million molecules of a single molecular species of variant surface glycoprotein (VSG). Antigenic variation involves the sequential expression of coats composed of different VSGs. The VSG is a glycosyl-phosphatidyl inositol (GPI) anchored glycoprotein of around 60 kDa, with two domains. The N-terminal domain is a rod-shaped hairpin structure that exposes a few variable loops at the parasite surface, containing the only epitopes recognised by the host. N-terminal region is extremely variable between VSGs. The C-terminal domain is more conserved and attached to the plasma membrane by a GPI anchor, and is linked to the N-terminal domain through a hinge region that is very sensitive to proteolytic cleavage [4]. When trypanosomes pass from the mammalian bloodstream into the tsetse midgut, the VSG coat is shed and replaced by a similarly dense coat formed from a small family of midgut-stage proteins called procyclins, and may serve to protect the procyclic stage from proteases in the insect midgut [2].

The almost complete sequencing of the T. brucei genome has revealed the presence of as many as 1700 VSGs, most of which are pseudogenes. The vast majority of these sequences are clustered in subtelomeric arrays. Many VSGs are also found at the extremity of telomeres, particularly in minichromosomes, but also at the end of larger chromosomes. It is generally believed that the function of minichromosomes is to provide a large repertoire of telomeric VSGs [5].

VSG Expression

Individual trypanosomes express only one VSG, and this requires a very tight control mechanism, which is achieved by the use of special transcription units, located at telomeres. These bloodstream expression sites (BESs) are polycistronic transcription units composed of a strong promoter, several genes known as ESAGs (expression site associated genes), which encode proteins not known to have any direct involvement in antigenic variation, a very long array of 70 bp repeats and then the VSG. Immediately downstream of the BES, lies the telomere tract that constitutes the end of the chromosome. To become expressed, silent VSGs must be moved into the BES. One complication of the system is that there are an estimated 20-30 BESs in each trypanosome, requiring further control to ensure only one is active. In addition, the different BESs encode different isoforms of the transferrin receptor (in ESAG6 and ESAG7), which differ in their affinity for transferrin from different potential host species, thereby possibly expanding host range.

An abnormality of the transcription of VSGs is the use of RNA polymerase I, which is the classical transcribing enzyme for ribosomal RNA genes, but not for protein-coding genes. This enzyme can provide a high rate of transcription. Promoters of inactive sites are poised for activity but are missing a unique factor. The factor comes in the shape of a novel nuclear structure, the ESB (expression site body), which contains RNA polymerase I and the active BES, but not an inactive BES. The ESB is not present in the procyclic stage, where no VSG is synthesized. Trypanosomes can change their VSG coat during antigenic variation by switching off the actively transcribed BES, and fully activating one of the silent BESs. It is not known how one BES can displace another from the ESB during such in situ switches, but a degree of communication between the two sites is probably involved [1].

Mechanisms of VSG switching

Two basically distinct processes can lead to the change of the active VSG. Either a transcriptional switching occurs between different VSG ESs, or the VSG resident in the active site is changed by DNA recombination [6].

The transcriptional switching between VSG ESs is called ''in situ activation'', and its only characteristic is the absence of DNA rearrangement, at least within the limits of the two concerned ESs and in the vicinity of their promoters. This mechanism of antigenic variation is not understood, but is obviously related to the process that allows full and efficient transcription of a single VSG ES at a time. The switching between sites does not seem to occur totally at random, since the activation of some ESs appears to be preferred. Whether this reflects a selection based on the expression of ESAGs, or an intrinsic capacity of some telomeres to be activated more easily.

VSG switching by DNA recombination is better understood. The active VSG ES is in a fully open chromatin configuration that renders DNA particularly susceptible to cleavage by various endonucleases, triggering a high rate of recombination. Different processes of homologous recombination, such as gene conversion (replacement of a sequence by the copy of another one) or telomere exchange can insert one of many hundreds of silent VSG genes into an active VSG expression site, which have been found to change the VSG sequence within the active ES. As the regions of homology between the partner sequences dictate recombination, the frequency and size range of this process clearly depend on the relative importance and localization of homology between the VSG in the active ES on one hand and the particular sequence within and around the concerned VSG partners on the other hand. In this respect, many different situations exist, because the several hundred VSGs present in the genome do not share the same environment, and largely differ in sequence. Telomeric VSGs, especially those already present in ESs, exhibit a higher probability of recombination due to the extensive similarity of their environment with that of the active VSG. Non-telomeric VSGs can be flanked by variable extents of sequences immediately surrounding the VSG in the active site, in particular variable numbers of repeats, which can allow recombination but with a lower probability than when a full telomeric environment is present. VSGs devoid of these homology regions are unable to recombine unless the active gene shares internal homology with them. This is possible given both the organization of VSGs in families and the presence of some stretches conserved between many genes. Finally, VSG pseudogenes, which constitute the majority of the repertoire, need intragenic recombination to restore the coding sequence for a functional antigen. Thus, in these cases only segmental gene conversion can lead to antigenic variation. This process generates chimerical genes made of fragments from different donors, and constitutes therefore a mechanism for creating novel VSGs through rearrangement between different sequences, which result in prolonging the infection perhaps beyond the achievement of immunity against the products of all intact VSGs. It is interesting that, so far in the sequencing of the trypanosome genome, there are very few intact VSG genes, but many pseudogenes. This raises the distinct possibility that mosaic formation is, in fact, a predominant route to VSG switching [7].

Another possibility is that telomere exchange, which occurs regularly during infections, has an important role in the maintenance of newly generated VSG gene diversity. This mechanism of VSG switching would result in shuttling newly created VSG genes within the active VSG expression site into another telomere, including those of the multitude of nontranscribed minichromosomes. Because there is no selection pressure operating on minichromosome telomeres for VSG switching movement to this location would enable new chimeric VSG genes to be retained for future switch events. If this exchange is occurring frequently, one would expect that the VSG genes present at the telomeres of minichromosomes would be all or mainly intact. The completed sequence of the entire T. brucei genome, including all of the chromosome ends, will tell us if this is the case. If this scenario is correct, an important function of the minichromosomes, in addition to providing a large pool of recombinogenic telomeric VSG genes, would be as a reservoir preserving newly created mosaic VSGs. This would enable trypanosomes at the population level to undergo relatively rapid change of at least part of their VSG gene repertoire [8]. (Fig. 1).

Fig. 1: VSG genes and VSG switching in African trypanosomes. (a) Genomic location of VSG genes in T. brucei. VSG genes are indicated with coloured boxes. VSGs are present in subtelomeric VSG arrays, at telomeres or in one of the many VSG expression site transcription units. VSG expression sites are shown with flags indicating the promoters, and a red arrow indicating transcription at the active VSG expression site. An approximate estimate of the total size of the pool of VSGs in the different genomic locations is indicated above. (b) Different VSG switching mechanisms in African trypanosomes. The coloured rectangular outlines represent trypanosomes expressing a single VSG gene (filled coloured box) from a telomeric VSG expression site. The VSG expression site promoter is indicated with a flag, and transcription with an arrow. Silent VSG genes are located either in tandem arrays at subtelomeric locations or at telomeres, including within VSG expression sites. Switching the active VSG gene can be mediated by different switching mechanisms. Left: gene conversion results in the duplication of a previously silent VSG gene into the active VSG expression site. Centre: telomere exchange involves a DNA crossover within two chromosome ends. Right: in situ switch - transcriptional activation of a new VSG expression site concurrent with silencing of the old one.