Trypanosoma brucei

Early records Sleeping sickness in animals was described in ancient Egyptian writings. During the Middle Ages, Arabian traders noted the prevalence of sleeping sickness among Africans and their dogs. It was a major infectious disease in southern and eastern Africa in the 19th century. The Zulu Kingdom (now part of South Africa) was severely struck by the disease, which became known to the British as nagana, John Aktins, an English naval surgeon, gave the first medical description of human sleeping sickness in 1734. He attributed deaths, which he called "sleepy distemper," in Guinea to the infection. Another English physician, Thomas Masterman Winterbottom, gave a clearer description of the symptoms from Sierra Leone in 1803. Winterbottom described a key feature of the disease, swollen posterior cervical lymph nodes, and slaves who developed such swellings were ruled unfit for trade. Discovery of the parasite The Royal Army Medical Corps appointed David Bruce, who at the time was assistant professor of pathology at the Army Medical School in Netley with a rank of Captain in the army, in 1894 to investigate a disease known as nagana in South Africa. The disease caused severe problems among the local cattle and British Army horses. On the sixth day of investigation, Bruce identified parasites from the blood of diseased cows. He initially noted them as a kind of filaria (tiny roundworms), but by the end of the year established that the parasites were "haematozoa" (protozoan) and were the cause of nagana. The scientific name was created by British zoologists Henry George Plimmer and John Rose Bradford in 1899 as Trypanosoma brucii due to printer's error. The genus Trypanosoma was already introduced by Hungarian physician David Gruby in his description of T. sanguinis, a species he discovered in frogs in 1843. Outbreaks In Uganda, the first case of human infection was reported in 1898. By 1901, it became severe with death toll estimated to about 20,000. More than 250,000 people died in the epidemic that lasted for two decades. It was not known whether the human sleeping sickness and nagana were similar or the two disease were caused by similar parasites. Even the observations of Forde and Dutton did not indicate that the trypanosome was related to sleeping sickness. Sleeping Sickness Commission The Royal Society constituted a three-member Sleeping Sickness Commission on 10 May 1902 to investigate the epidemic in Uganda. The Commission comprised George Carmichael Low from the London School of Hygiene and Tropical Medicine as the leader, his colleague Aldo Castellani and Cuthbert Christy, a medical officer on duty in Bombay, India. At the time, a debate remained on the etiology, some favoured bacterial infection while some believed as helminth infection. The first investigation focussed on Filaria perstans (later renamed Mansonella perstans), a small roundworm transmitted by flies, and bacteria as possible causes, only to discover that the epidemic was not related to these pathogens. The team was described as an "ill-assorted group" and the expedition "a failure." In February 1902, the British War Office, following a request from the Royal Society, appointed David Bruce to lead the second Sleeping Sickness Commission. With David Nunes Nabarro (from the University College Hospital), Bruce and his wife joined Castellani and Christy on 16 March. By then the Royal Society had already published the report. By August 1903, Bruce and his team established that the disease was transmitted by the tsetse fly, Glossina palpalis. However, Bruce did not understand the trypanosoma life cycle and believed that the parasites were simply transmitted from one person to another. An open question, noted by Bruce at this stage, was how the trypanosome finds its way to the salivary glands. Muriel Robertson, in experiments carried out between 1911 and 1912, established how ingested trypanosomes finally reach the salivary glands of the fly. Discovery of human trypanosomes British Colonial Surgeon Robert Michael Forde was the first to find the parasite in human. He found it from an English steamboat captain who was admitted to a hospital at Bathurst, Gambia, in 1901. ==Species==

T. brucei is a species complex that includes: • T. brucei gambiense which causes slow onset chronic trypanosomiasis in humans. It is most common in central and western Africa, where humans are thought to be the primary reservoir. In 1973, David Hurst Molyneux was the first to find infection of this strain in wildlife and domestic animals. It is responsible for 98% of all human African trypanosomiasis, and is roughly 100% fatal when left untreated. • T. brucei rhodesiense which causes fast onset acute trypanosomiasis in humans. A highly zoonotic parasite, it is prevalent in southern and eastern Africa, where game animals and livestock are thought to be the primary reservoir. However, it is closely related to, and shares fundamental features with the human-infective subspecies. Only rarely can the T. b. brucei infect a human. The subspecies cannot be distinguished from their structure as they are all identical under microscopes. Geographical location is the main distinction. TgsGP gene, found only in type 1 T. b. gambiense is also a specific distinction between T. b. gambiense strains. The subspecies lack many of the features commonly considered necessary to constitute monophyly. Etymology The genus name is derived from two Greek words: τρυπανον (trypanon or trupanon), which means "borer" or "auger", referring to the corkscrew-like movement; The specific name is after David Bruce, who discovered the parasites in 1894. The subspecies, the human strains, are named after the regions in Africa where they were first identified: T. brucei gambiense was described from an Englishman in Gambia in 1901; T. brucei rhodesiense was found from another Englishman in Northern Rhodesia in 1909. ==Structure==

false colour micrograph of the procyclic form as found in the tsetse fly midgut. The cell body is shown in orange and the flagellum is in red. 84 pixels/μm.T. brucei is a typical unicellular eukaryotic cell, and measures 8 to 50 μm in length. It has an elongated body having a streamlined and tapered shape. Its cell membrane (called pellicle) encloses the cell organelles, including the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, and ribosomes. In addition, there is an unusual organelle called the kinetoplast, which is a complex of thousands of interlinked circles of mitochondrial DNA known as mini- and maxicircles. The kinetoplast lies near the basal body with which it is indistinguishable under microscope. From the basal body arises a single flagellum that run towards the anterior end. Along the body surface, the flagellum is attached to the cell membrane forming an undulating membrane. Only the tip of the flagellum is free at the anterior end. The cell surface of the bloodstream form features a dense coat of variant surface glycoproteins (VSGs) which is replaced by an equally dense coat of procyclins when the parasite differentiates into the procyclic phase in the tsetse fly midgut. Trypanosomatids show several different classes of cellular organisation of which two are adopted by T. brucei at different stages of the life cycle: a lattice structure of proteins unique to the kinetoplastids, 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 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 oscillations along the attached flagellum and cell body in human blood stream and tsetse fly gut, and attachment to the salivary gland epithelium of the fly during the epimastigote stage. The flagellum propels the body in such a way that the axoneme generates the oscillation and a flagellar wave is created along the undulating membrane. As a result, the body moves in a corkscrew pattern. In flagella of other organisms, the movement starts from the base towards the tip, while in T. brucei and other trypanosomatids, the beat originates from the tip and progresses towards the base, forcing the body to move towards the direction of the tip of the flagellum. ==Life cycle==

T. brucei completes its life cycle between tsetse fly (of the genus Glossina) and mammalian hosts, including humans, cattle, horses, and wild animals. In stressful environments, T. brucei produces exosomes containing the spliced leader RNA and uses the endosomal sorting complexes required for transport (ESCRT) system to secrete them as extracellular vesicles. When absorbed by other trypanosomes these EVs cause repulsive movement away from the area and so away from bad environments. They are protected from the host's immune system by producing antigentic variation called variant surface glycoproteins on their body surface. Sometimes, wild animals can be infected by the tsetse fly and they act as reservoirs. In these animals, they do not produce the disease, but the live parasite can be transmitted back to the normal hosts. The short and stumpy trypomastigotes (SS) are taken up by tsetse flies during a blood meal. The procyclic trypomastigotes cross the peritrophic matrix, undergo slight elongation and migrate to the anterior part of the midgut as non-proliferative long mesocyclic trypomastigotes. As they reach the proventriculus, they became thinner and undergo cytoplasmic rearrangement to give rise to proliferative epimastigotes. The short epimastigote migrate from the proventriculus via the foregut and proboscis to the salivary glands where they get attached to the salivary gland epithelium. In the salivary glands, the survivors undergo phases of reproduction. The first cycle in an equal mitosis by which a mother cell produces two similar daughter epimastigotes. They remain attach to the epithelium. This phase is the main reproduction in first-stage infection to ensure sufficient number of parasites in the salivary gland. The trypomastigote detach from the epithelium and undergo transformation into short and stumpy trypomastigotes. The surface procyclins are replaced with VSGs and become the infective metacyclic trypomastigotes. ==Reproduction==

Binary fission The reproduction 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. The events of reproduction are: But it is not always necessary for a complete life cycle. The existence of meiosis-specific proteins was reported in 2011. The haploid gametes (daughter cells produced after meiosis) were discovered in 2014. The haploid trypomastigote-like gametes can interact with each other via their flagella and undergo cell fusion (the process is called syngamy). Thus, in addition to binary fission, T. brucei can multiply by sexual reproduction. Trypanosomes belong to the supergroup Excavata and are one of the earliest diverging lineages among eukaryotes. The discovery of sexual reproduction in T. brucei supports the hypothesis that meiosis and sexual reproduction are ancestral and ubiquitous features of eukaryotes. ==Infection and pathogenicity==

The insect vectors for T. brucei are different species of tsetse fly (genus Glossina). The major vectors of T. b. gambiense, causing West African sleeping sickness, are G. palpalis, G. tachinoides, and G. fuscipes. While the principal vectors of T. b. rhodesiense, causing East African sleeping sickness, are G. morsitans, G. pallidipes, and G. swynnertoni. Animal trypanosomiasis is transmitted by a dozen species of Glossina. In later stages of a T. brucei infection of a mammalian host the parasite may migrate from the bloodstream to also infect the lymph and cerebrospinal fluids. It is under this tissue invasion that the parasites produce the sleeping sickness. Newborn babies can be infected (vertical or congenital transmission) from infected mothers. ==Chemotherapy==

There are four drugs generally recommended for the first-line treatment of African trypanosomiasis: suramin developed in 1921, pentamidine developed in 1941, melarsoprol developed in 1949 and eflornithine developed in 1990. These drugs are not fully effective and are toxic to humans. In addition, drug resistance has developed in the parasites against all the drugs. The drugs are of limited application since they are effective against specific strains of T. brucei and the life cycle stages of the parasites. Suramin is used only for first-stage infection of T. b. rhodesiense, pentamidine for first-stage infection of T. b. gambiense, and eflornithine for second-stage infection of T. b. gambiense. Melarsopol is the only drug effective against the two types of parasite in both infection stages, but is highly toxic, such that 5% of treated individuals die of brain damage (reactive encephalopathy). Another drug, nifurtimox, recommended for Chagas disease (American trypanosomiasis), is itself a weak drug but in combination with melarsopol, it is used as the first-line medication against second-stage infection of T. b. gambiense. Historically, arsenic and mercuric compounds were introduced in the early 20th century, with success particularly in animal infections. German physician Paul Ehrlich and his Japanese associate Kiyoshi Shiga developed the first specific trypanocidal drug in 1904 from a dye, trypan red, which they named Trypanroth. These chemical preparations were effective only at high and toxic dosages, and were not suitable for clinical use. Animal trypanosomiasis is treated with six drugs: diminazene aceturate, homidium (homidium bromide and homidium chloride), isometamidium chloride, melarsomine, quinapyramine, and suramin. They are all highly toxic to animals, and drug resistance is prevalent. Homidium is the first prescription anti-trypanosomal drug. It was developed as a modified compound of phenantridine, which was found in 1938 to have trypanocidal activity against the bovine parasite, T. congolense. Among its products, dimidium bromide and its derivatives were first used in 1948 in animal cases in Africa, and became known as homidium (or as ethidium bromide in molecular biology). Drug development The major challenge against the human disease has been to find drugs that readily pass the blood-brain barrier. The latest drug that has come into clinical use is fexinidazol, but promising results have also been obtained with the benzoxaborole drug acoziborole (SCYX-7158). This drug is currently under evaluation as a single-dose oral treatment, which is a great advantage compared to currently used drugs. Another research field that has been extensively studied in Trypanosoma brucei is to target its nucleotide metabolism. The nucleotide metabolism studies have both led to the development of adenosine analogues looking promising in animal studies, and to the finding that downregulation of the P2 adenosine transporter is a common way to acquire partial drug resistance against the melaminophenyl arsenical and diamidine drug families (containing melarsoprol and pentamidine, respectively). Phytochemicals. Some phytochemicals have shown research promise against the T. b. brucei strain. Aderbauer et al., 2008 and Umar et al., 2010 find Khaya senegalensis is effective in vitro and Ibrahim et al., 2013 and 2008 in vivo (in rats). Ibrahim et al., 2013 find a lower dose reduces parasitemia by this subspecies and a higher dose is curative and prevents injury. ==Distribution==

T. brucei is found where its tsetse fly vectors are prevalent in continental Africa. That is to say, tropical rainforest (Af), tropical monsoon (Am), and tropical savannah (Aw) areas of continental Africa. Hence, the equatorial region of Africa is called the "sleeping sickness" belt. However, the specific type of the trypanosome differs according to geography. T. b. rhodesiense is found primarily in East Africa, while T. b. gambiense is found in Central and West Africa. ==Impact==

T. brucei is a major cause of livestock disease in sub-Saharan Africa. It is thus of tremendous veterinary concern and one of the greatest limitations on agriculture in Africa and the economic life of sub-Saharan Africa. ==Evolution==

Trypanosoma brucei gambiense evolved from a single progenitor ~10,000 years ago. It is evolving asexually and its genome shows the Meselson effect. ==Genetics==

There are two subpopulations of T. b. gambiense that possesses two distinct groups that differ in genotype and phenotype. Group 2 is more akin to T. b. brucei than group 1 T. b. gambiense. All T. b. gambiense are resistant to killing by a serum component — trypanosome lytic factor (TLF) of which there are two types: TLF-1 and TLF-2. Group 1 T. b. gambiense parasites avoid uptake of the TLF particles while those of group 2 are able to either neutralize or compensate for the effects of TLF. In contrast, resistance in T. b. rhodesiense is dependent upon the expression of a serum resistance associated (SRA) gene. This gene is not found in T. b. gambiense. Genome The genome of T. brucei is made up of: • 11 pairs of large chromosomes of 1 to 6 megabase pairs. • 3–5 intermediate chromosomes of 200 to 500 kilobase pairs. • Around 100 minichromosomes of around 50 to 100 kilobase pairs. These may be present in multiple copies per haploid genome. Most genes are held on the large chromosomes, with the minichromosomes carrying only VSG genes. The genome has been sequenced and is available on GeneDB. The mitochondrial genome is found condensed into the kinetoplast, an unusual feature unique to the kinetoplastid protozoans. The kinetoplast and the basal body of the flagellum are strongly associated via a cytoskeletal structure In 1993, a new base, ß-d-glucopyranosyloxymethyluracil (base J), was identified in the nuclear DNA of T. brucei. == VSG coat ==

The surface of T. brucei and other species of trypanosomes is covered by a dense external coat called variant surface glycoprotein (VSG). VSGs are 60-kDa proteins which are densely packed (~5 million molecules) to form a 12–15 nm surface coat. VSG dimers make up about 90% of all cell surface proteins in trypanosomes. They also make up ~10% of total cell protein. For this reason, these proteins are highly immunogenic and an immune response raised against a specific VSG coat will rapidly kill trypanosomes expressing this variant. However, with each cell division there is a possibility that the progeny will switch expression to change the VSG that is being expressed. This VSG coat enables an infecting T. brucei population to persistently evade the host's immune system, allowing chronic infection. VSG is highly immunogenic, and an immune response raised against a specific VSG coat rapidly kills trypanosomes expressing this variant. Antibody-mediated trypanosome killing 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 change the VSG that is being expressed. The frequency of VSG switching has been measured to be approximately 0.1% per division. As T. brucei populations can peak at a size of 1011 within a host this rapid rate of switching ensures that the parasite population is typically highly diverse. Because host immunity against a specific VSG does not develop immediately, some parasites will have switched to an antigenically distinct VSG variant, and can go on to multiply and continue the infection. The clinical effect of this cycle is successive 'waves' of parasitemia (trypanosomes in the blood). The expressed VSG can be switched either by activating a different expression site (and thus changing to express the VSG in that site), or by changing the VSG gene in the active site to a different variant. The genome contains many hundreds if not thousands of VSG genes, both on minichromosomes and in repeated sections ('arrays') in the interior of the chromosomes. These are transcriptionally 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. It is thought that any of these genes can be moved into the active site by recombination for expression. It remains unproven whether the regulation of VSG switching is purely stochastic or whether environmental stimuli affect switching frequency. Switching is linked to two factors: variation in activation of individual VSG genes; and differentiation to the "short stumpy" stage - triggered by conditions of high population density - which is the nonreproductive, interhost transmission stage. it also remains unexplained how this transition is timed and how the next surface protein gene is chosen. These questions of antigenic variation in T. brucei and other parasites are among the most interesting in the field of infection. ==Killing by human serum and resistance to human serum killing==

Trypanosoma brucei brucei (as well as related species T. equiperdum and T. evansi) is not human infective because it is susceptible to innate immune system 'trypanolytic' factors present in the serum of some primates, including humans. These trypanolytic factors have been identified as two serum complexes designated trypanolytic factors (TLF-1 and −2) both of which contain haptoglobin-related protein (HPR) and apolipoprotein LI (ApoL1). TLF-1 is a member of the high density lipoprotein family of particles while TLF-2 is a related high molecular weight serum protein binding complex. The protein components of TLF-1 are haptoglobin related protein (HPR), apolipoprotein L-1 (apoL-1) and apolipoprotein A-1 (apoA-1). These three proteins are colocalized within spherical particles containing phospholipids and cholesterol. The protein components of TLF-2 include IgM and apolipoprotein A-I. Trypanolytic factors are found only in a few species, including humans, gorillas, mandrills, baboons and sooty mangabeys. This appears to be because haptoglobin-related protein and apolipoprotein L-1 are unique to primates. This suggests these genes originated in the primate genome -. Human infective subspecies T. b. gambiense and T. b. rhodesiense have evolved mechanisms of resisting the trypanolytic factors, described below. ApoL1 ApoL1 is a member of a six gene family, ApoL1-6, that have arisen by tandem duplication. These proteins are normally involved in host apoptosis or autophagic death and possess a Bcl-2 homology domain 3. ApoL1 has been identified as the toxic component involved in trypanolysis. ApoLs have been subject to recent selective evolution possibly related to resistance to pathogens. The gene encoding ApoL1 is found on the long arm of chromosome 22 (22q12.3). Variants of this gene, termed G1 and G2, provide protection against T. b. rhodesiense. This glomerulopathy may help to explain the greater prevalence of hypertension in African populations. The gene encodes a protein of 383 residues, including a typical signal peptide of 12 amino acids. The plasma protein is a single chain polypeptide with an apparent molecular mass of 42 kilodaltons. ApoL1 has a membrane pore forming domain functionally similar to that of bacterial colicins. This domain is flanked by the membrane addressing domain and both these domains are required for parasite killing. Within the kidney, ApoL1 is found in the podocytes in the glomeruli, the proximal tubular epithelium and the arteriolar endothelium. It has a high affinity for phosphatidic acid and cardiolipin and can be induced by interferon gamma and tumor necrosis factor alpha. Hpr Hpr is 91% identical to haptoglobin (Hp), an abundant acute phase serum protein, which possesses a high affinity for hemoglobin (Hb). When Hb is released from erythrocytes undergoing intravascular hemolysis Hp forms a complex with the Hb and these are removed from circulation by the CD163 scavenger receptor. In contrast to Hp–Hb, the Hpr–Hb complex does not bind CD163 and the Hpr serum concentration appears to be unaffected by hemolysis. Killing mechanism The association of HPR with hemoglobin allows TLF-1 binding and uptake via the trypanosome haptoglobin-hemoglobin receptor (TbHpHbR). TLF-2 enters trypanosomes independently of TbHpHbR. The trypanosome haptoglobin-hemoglobin receptor is an elongated three a-helical bundle with a small membrane distal head. This protein extends above the variant surface glycoprotein layer that surrounds the parasite. The first step in the killing mechanism is the binding of TLF to high affinity receptors—the haptoglobin-hemoglobin receptors—that are located in the flagellar pocket of the parasite. The bound TLF is endocytosed via coated vesicles and then trafficked to the parasite lysosomes. ApoL1 is the main lethal factor in the TLFs and kills trypanosomes after insertion into endosomal / lysosomal membranes. Resistance mechanisms: T. b. gambiense Trypanosoma brucei gambiense causes 97% of human cases of sleeping sickness. Resistance to ApoL1 is principally mediated by the hydrophobic β-sheet of the T. b. gambiense specific glycoprotein. This is due to a thymidine to cytosine mutation at the second codon position. These mutations may have evolved due to the coexistence of malaria where this parasite is found. Resistance mechanisms: T. b. rhodesiense Trypanosoma brucei rhodesiense relies on a different mechanism of resistance: the serum resistance associated protein (SRA). The SRA gene is a truncated version of the major and variable surface antigen of the parasite, the variant surface glycoprotein. However, it has little similarity (low sequence homology) with the VSG gene (<25%). SRA is an expression site associated gene in T. b. rhodesiense and is located upstream of the VSGs in the active telomeric expression site. The protein is largely localized to small cytoplasmic vesicles between the flagellar pocket and the nucleus. In T. b. rhodesiense the TLF is directed to SRA containing endosomes while some dispute remains as to its presence in the lysosome. SRA binds to ApoL1 using a coiled–coiled interaction at the ApoL1 SRA interacting domain while within the trypanosome lysosome. Experimental mutations allowing ApoL1 to be protected from neutralization by SRA have been shown capable of conferring trypanolytic activity on T. b. rhodesiense. These mutations resemble those found in baboons, but also resemble natural mutations conferring protection of humans against T. b. rhodesiense which are linked to kidney disease. == See also ==