Trypanosoma brucei
Encyclopedia
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 parasite
s 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 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.
is fairly typical of eukaryote
s, 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:
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.
of T. brucei is made up of:
The large chromosomes contain most gene
s, while the small chromosomes tend to carry genes involved in antigen
ic variation, including the VSG genes. The genome has been sequenced and is available online http://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 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.
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 filament
s also play an important role in the cytoskeleton, but these are generally overlooked.
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 dinoflagellate
s.
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.
(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:
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).
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.
. Some of these are full-length, intact genes
; others are pseudogenes) typically with omitted sections or premature stop codon
s. Expression of an antigen
ically 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.
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.
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:
Protist
Protists are a diverse group of eukaryotic microorganisms. Historically, protists were treated as the kingdom Protista, which includes mostly unicellular organisms that do not fit into the other kingdoms, but this group is contested in modern taxonomy...
species that causes African trypanosomiasis (or sleeping sickness) in humans and nagana
Nagana
Nagana, also known as nagana pest or animal African trypanosomiasis, is a disease of vertebrate animals. The disease is caused by trypanosomes of several species in the genus Trypanosoma. The trypanosomes infect the blood of the vertebrate host, causing fever, weakness, and lethargy, which lead to...
in animals in Africa
Africa
Africa is the world's second largest and second most populous continent, after Asia. At about 30.2 million km² including adjacent islands, it covers 6% of the Earth's total surface area and 20.4% of the total land area...
. There are 3 sub-species of T. brucei: T. b. brucei, T. b. gambiense and T. b. rhodesiense.
These obligate parasite
Obligate parasite
An obligate parasite is a parasitic organism that cannot complete its life cycle without dependence on its host.-See also:*Obligate intracellular parasite*Parasitism*Parasitic plant*Facultative parasite...
s have two hosts - an insect
Insect
Insects are a class of living creatures within the arthropods that have a chitinous exoskeleton, a three-part body , three pairs of jointed legs, compound eyes, and two antennae...
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 flyTsetse fly
Tsetse , sometimes spelled tzetze and also known as tik-tik flies, are large biting flies that inhabit much of mid-continental Africa between the Sahara and the Kalahari deserts. They live by feeding on the blood of vertebrate animals and are the primary biological vectors of trypanosomes, which...
. 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 reservoirNatural reservoirNatural reservoir or nidus, refers to the long-term host of the pathogen of an infectious disease. It is often the case that hosts do not get the disease carried by the pathogen or it is carried as a subclinical infection and so asymptomatic and non-lethal...
. - 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 reservoirNatural reservoirNatural reservoir or nidus, refers to the long-term host of the pathogen of an infectious disease. It is often the case that hosts do not get the disease carried by the pathogen or it is carried as a subclinical infection and so asymptomatic and non-lethal...
. - T. brucei brucei - Causes animal African trypanosomiasisNaganaNagana, also known as nagana pest or animal African trypanosomiasis, is a disease of vertebrate animals. The disease is caused by trypanosomes of several species in the genus Trypanosoma. The trypanosomes infect the blood of the vertebrate host, causing fever, weakness, and lethargy, which lead to...
, along with several other species of trypanosomaTrypanosomaTrypanosoma is a genus of kinetoplastids , a monophyletic group of unicellular parasitic flagellate protozoa. The name is derived from the Greek trypano and soma because of their corkscrew-like motion. All trypanosomes are heteroxenous and are transmitted via a vector...
. T. b. brucei is not human infective due to its susceptibility to lysis by human apolipoprotein L1APOL1APOL1 is the gene, which in humans encodes the Apolipoprotein L1 protein.APOL1 is the gene, which in humans encodes the Apolipoprotein L1 protein....
. 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 laboratoryCell cultureCell culture is the complex process by which cells are grown under controlled conditions. In practice, the term "cell culture" has come to refer to the culturing of cells derived from singlecellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes,...
and animal studiesAnimal testingAnimal testing, also known as animal experimentation, animal research, and in vivo testing, is the use of non-human animals in experiments. Worldwide it is estimated that the number of vertebrate animals—from zebrafish to non-human primates—ranges from the tens of millions to more than 100 million...
.
The cell structure
The structure of the cellCell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
is fairly typical of eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
s, see eukaryotic cell
Eukaryotic Cell
Eukaryotic Cell is an academic journal published by the American Society for Microbiology. The title is commonly abbreviated EC and the ISSN is 1535-9778 for the print version, and 1535-9786 for the electronic version....
. All major organelles are seen, including the nucleus
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
, mitochondria, endoplasmic reticulum
Endoplasmic reticulum
The endoplasmic reticulum is an organelle of cells in eukaryotic organisms that forms an interconnected network of tubules, vesicles, and cisternae...
, Golgi apparatus
Golgi apparatus
The Golgi apparatus is an organelle found in most eukaryotic cells. It was identified in 1898 by the Italian physician Camillo Golgi, after whom the Golgi apparatus is named....
etc. Unusual features include the single large mitochondria with the mitochondrial DNA
Mitochondrial DNA
Mitochondrial DNA is the DNA located in organelles called mitochondria, structures within eukaryotic cells that convert the chemical energy from food into a form that cells can use, adenosine triphosphate...
structure known as the kinetoplast
Kinetoplast
A kinetoplast is a disk-shaped mass of circular DNA inside a large mitochondrion that contains many copies of the mitochondrial genome. Kinetoplasts are only found in protozoa of the class Kinetoplastida...
, and its association with the basal body
Basal body
A basal body is an organelle formed from a centriole, and a short cylindrical array of microtubules. It is found at the base of a eukaryotic undulipodium and serves as a nucleation site for the growth of the axoneme microtubules...
of the flagellum
Flagellum
A flagellum is a tail-like projection that protrudes from the cell body of certain prokaryotic and eukaryotic cells, and plays the dual role of locomotion and sense organ, being sensitive to chemicals and temperatures outside the cell. There are some notable differences between prokaryotic and...
. The cytoskeleton
Cytoskeleton
The cytoskeleton is a cellular "scaffolding" or "skeleton" contained within a cell's cytoplasm and is made out of protein. The cytoskeleton is present in all cells; it was once thought to be unique to eukaryotes, but recent research has identified the prokaryotic 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.
- AmastigoteAmastigoteAn amastigote is a cell that does not have a visible external flagella or cilia. The term is used mainly to describe a certain phase in the life-cycle of trypanosome protozoans. It is also called the leishmanial stage, since in Leishmania it is the form the parasite takes in the vertebrate host,...
- 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
Ancient Greek
Ancient Greek is the stage of the Greek language in the periods spanning the times c. 9th–6th centuries BC, , c. 5th–4th centuries BC , and the c. 3rd century BC – 6th century AD of ancient Greece and the ancient world; being predated in the 2nd millennium BC by Mycenaean Greek...
mastig- meaning whip
Whip
A whip is a tool traditionally used by humans to exert control over animals or other people, through pain compliance or fear of pain, although in some activities whips can be used without use of pain, such as an additional pressure aid in dressage...
, 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 genomeGenome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
of T. brucei is made up of:
- 11 pairs of large chromosomeChromosomeA chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
s 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 large chromosomes contain most gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s, while the small chromosomes tend to carry genes involved in antigen
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
ic variation, including the VSG genes. The genome has been sequenced and is available online http://www.genedb.org.
The mitochondrial genome is found condensed into the kinetoplast
Kinetoplast
A kinetoplast is a disk-shaped mass of circular DNA inside a large mitochondrion that contains many copies of the mitochondrial genome. Kinetoplasts are only found in protozoa of the class Kinetoplastida...
, an unusual feature unique to the kinetoplastea class. It and the basal body
Basal body
A basal body is an organelle formed from a centriole, and a short cylindrical array of microtubules. It is found at the base of a eukaryotic undulipodium and serves as a nucleation site for the growth of the axoneme microtubules...
of the flagellum
Flagellum
A flagellum is a tail-like projection that protrudes from the cell body of certain prokaryotic and eukaryotic cells, and plays the dual role of locomotion and sense organ, being sensitive to chemicals and temperatures outside the cell. There are some notable differences between prokaryotic and...
are strongly associated via a cytoskeletal structure.
VSG surface coat
Main section: The VSG coatThe 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 cytoskeletonCytoskeleton
The cytoskeleton is a cellular "scaffolding" or "skeleton" contained within a cell's cytoplasm and is made out of protein. The cytoskeleton is present in all cells; it was once thought to be unique to eukaryotes, but recent research has identified the prokaryotic 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
Microfilament
Microfilaments are the thinnest filaments of the cytoskeleton, a structure found in the cytoplasm of all eukaryotic cells. These linear polymers of actin subunits are flexible and relatively strong, resisting buckling by multi-piconewton compressive forces and filament fracture by nanonewton...
and intermediate filament
Intermediate filament
Intermediate filaments are a family of related proteins that share common structural and sequence features. Intermediate filaments have an average diameter of 10 nanometers, which is between that of 7 nm actin , and that of 25 nm microtubules, although they were initially designated...
s also play an important role in the cytoskeleton, but these are generally overlooked.
Flagellar structure
Dinoflagellate
The dinoflagellates are a large group of flagellate protists. Most are marine plankton, but they are common in fresh water habitats as well. Their populations are distributed depending on temperature, salinity, or depth...
s.
The microtubules of the flagellar axoneme
Axoneme
Numerous eukaryotic cells carry whip-like appendages whose inner core consists of a cytoskeletal structure called the 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 GlycoproteinGlycoprotein
Glycoproteins are proteins that contain oligosaccharide chains covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation. In proteins that have segments extending...
(VSG). This coat enables an infecting T. brucei population to persistently evade the host's immune system
Immune system
An immune system is a system of biological structures and processes within an organism that protects against disease by identifying and killing pathogens and tumor cells. It detects a wide variety of agents, from viruses to parasitic worms, and needs to distinguish them from the organism's own...
, 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 membraneCell membraneThe cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
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 loopsSecondary structureIn biochemistry and structural biology, secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids...
of the VSG that make up the coat. - Periodic antigenic variationAntigenic variationAntigenic variation refers to the mechanism by which an infectious organism such as a protozoan, bacterium or virus alters its surface proteins in order to evade a host immune response. Immune evasion is particularly important for organisms that target long-lived hosts, repeatedly infect a single...
- the VSG coat undergoes frequent stochasticStochasticStochastic refers to systems whose behaviour is intrinsically non-deterministic. A stochastic process is one whose behavior is non-deterministic, in that a system's subsequent state is determined both by the process's predictable actions and by a random element. However, according to M. Kac and E...
genetic modification - 'switching' - allowing variants expressing a new VSG coat to escape the specific immune response raised against the previous coat.
Antigenic variation
SequencingSequencing
In genetics and biochemistry, sequencing means to determine the primary structure of an unbranched biopolymer...
of the T. brucei genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
has revealed a huge VSG gene archive, made up of thousands of different VSG genes
Gênes
Gênes is the name of a département of the First French Empire in present Italy, named after the city of Genoa. It was formed in 1805, when Napoleon Bonaparte occupied the Republic of Genoa. Its capital was Genoa, and it was divided in the arrondissements of Genoa, Bobbio, Novi Ligure, Tortona and...
. All but one of these are 'silent' VSGs, as each trypanosome expresses
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
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
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
by a complement-mediated
Complement system
The complement system helps or “complements” the ability of antibodies and phagocytic cells to clear pathogens from an organism. It is part of the immune system called the innate immune system that is not adaptable and does not change over the course of an individual's lifetime...
lysis
Lysis
Lysis refers to the breaking down of a cell, often by viral, enzymic, or osmotic mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a "lysate"....
assay
Assay
An assay is a procedure in molecular biology for testing or measuring the activity of a drug or biochemical in an organism or organic sample. A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized...
. However, with each cell division
Cell division
Cell division is the process by which a parent cell divides into two or more daughter cells . Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort...
there is a possibility that one or both of the progeny
Offspring
In biology, offspring is the product of reproduction, of a new organism produced by one or more parents.Collective offspring may be known as a brood or progeny in a more general way...
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 sequenceDNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
level. However, for them to fulfil their shielding function, different VSGs have strongly conserved structural
Protein structure
Proteins are an important class of biological macromolecules present in all organisms. Proteins are polymers of amino acids. Classified by their physical size, proteins are nanoparticles . Each protein polymer – also known as a polypeptide – consists of a sequence formed from 20 possible L-α-amino...
features. VSGs are made up of a highly variable N terminal domain
Protein domain
A protein domain is a part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded. Many proteins consist of several structural...
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
Alpha helix
A common motif in the secondary structure of proteins, the alpha helix is a right-handed coiled or spiral conformation, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier...
, while the N terminal domain forms a 'halo' around the helices. The tertiary structure
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary 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
Glycophosphatidylinositol
Glycosylphosphatidylinositol is a glycolipid that can be attached to the C-terminus of a protein during posttranslational modification...
- a covalent linkage from the C terminus, to approximately four sugars, to a phosphatidylinositol
Phosphatidylinositol
Phosphatidylinositol is a negatively charged phospholipid and a minor component in the cytosolic side of eukaryotic cell membranes....
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 genomeGenome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
. Some of these are full-length, intact genes
Gênes
Gênes is the name of a département of the First French Empire in present Italy, named after the city of Genoa. It was formed in 1805, when Napoleon Bonaparte occupied the Republic of Genoa. Its capital was Genoa, and it was divided in the arrondissements of Genoa, Bobbio, Novi Ligure, Tortona and...
; others are pseudogenes) typically with omitted sections or premature stop codon
Stop codon
In the genetic code, a stop codon is a nucleotide triplet within messenger RNA that signals a termination of translation. Proteins are based on polypeptides, which are unique sequences of amino acids. Most codons in messenger RNA correspond to the addition of an amino acid to a growing polypeptide...
s. Expression of an antigen
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
ically 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
Protein structure
Proteins are an important class of biological macromolecules present in all organisms. Proteins are polymers of amino acids. Classified by their physical size, proteins are nanoparticles . Each protein polymer – also known as a polypeptide – consists of a sequence formed from 20 possible L-α-amino...
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
Vaccine
A vaccine is a biological preparation that improves immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism, and is often made from weakened or killed forms of the microbe or its toxins...
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
Genetic recombination
Genetic recombination is a process by which a molecule of nucleic acid is broken and then joined to a different one. Recombination can occur between similar molecules of DNA, as in homologous recombination, or dissimilar molecules, as in non-homologous end joining. Recombination is a common method...
for expression. Again, the exact mechanisms that control this are still only partially known.
Cell division
Centrosome
In cell biology, the centrosome is an organelle that serves as the main microtubule organizing center of the animal cell as well as a regulator of cell-cycle progression. It was discovered by Edouard Van Beneden in 1883...
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:
- The basal body duplicates and both remaining associated with the kinetoplast.
- Kinetoplast DNA undergoes synthesis then the kinetoplast divides coupled with separation of the two basal bodies.
- Nuclear DNA undergoes synthesis while a new flagellum extends from the younger, more posterior, basal body.
- The nucleus undergoes mitosis.
- CytokinesisCytokinesisCytokinesis is the process in which the cytoplasm of a single eukaryotic cell is divided to form two daughter cells. It usually initiates during the late stages of mitosis, and sometimes meiosis, splitting a binucleate cell in two, to ensure that chromosome number is maintained from one generation...
progresses from the anterior to posterior. - Division completes with abscission.