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The haptoglobin-haemoglobin receptor of the African trypanosome species, Trypanosoma brucei, is expressed when the parasite is in the bloodstream of the mammalian host, allowing it to acquire haem through the uptake of haptoglobin-haemoglobin complexes. Here we show that in Trypanosoma congolense this receptor is instead expressed in the epimastigote developmental stage that occurs in the tsetse fly, where it acts as a haemoglobin receptor. We also present the structure of the T. congolense receptor in complex with haemoglobin. This allows us to propose an evolutionary history for this receptor, charting the structural and cellular changes that took place as it adapted from a role in the insect to a new role in the mammalian host. Trypanosomes are single-celled parasites that infect a range of animal hosts. These parasites need a molecule called haem to grow properly and are mostly spread by insects that feed on the blood of mammals. Most haem in mammals is found in red blood cells and is bound to a protein called haemoglobin. When it is released from these cells, haemoglobin forms a complex with another protein called haptoglobin as well. The best-studied trypanosomes from Africa have a receptor protein on their surface that recognizes the haptoglobin-haemoglobin complex and allows the parasites to obtain haem from their hosts. An African trypanosome called T. brucei causes sleeping sickness in humans, and has a receptor that can only recognize haemoglobin when it is in complex with haptoglobin. However, few trypanosome receptors have been studied to date, and so it was not clear if they all work in the same way. Trypanosoma congolense is a trypanosome that has a big impact on livestock farmers in sub-Saharan Africa and infects cattle, pigs and goats. Lane-Serff, MacGregor et al. now report that the receptor protein from T. congolense can bind to haemoglobin on its own. A technique called X-ray crystallography was used to reveal the three-dimensional structure of the T. congolense receptor and haemoglobin in fine detail. Further experiments then confirmed that the receptor actually binds more strongly to haemoglobin than it does to the haptoglobin-haemoglobin complex. Experiments with living parasites showed that T. congolense produces its receptor when it is in the mouthparts of its insect host, the tsetse fly. This is unlike what occurs in T. brucei, which only produces its receptor while it is in the bloodstream of its mammalian host. Lane-Serff, MacGregor et al. suggest that T. congolense’s receptor is more like the receptor found in ancestor of the trypanosomes. This means that, at least once during the evolution of these parasites, this receptor evolved from being a haemoglobin receptor produced in the tsetse fly to a haptoglobin-haemoglobin receptor produced in an infected mammal. The next step is to investigate the details of the role played by the T. congolense receptor when the parasite is in the tsetse fly. It will also be important to understand how this parasite is still able to grow in the mammalian host’s bloodstream even though it does not produce much of the receptor during this stage.

The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50o kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface. African Trypanosomes are a group of single-celled parasites that are a major concern for livestock farmers in sub-Saharan Africa. They are carried by the tsetse fly and can cause disease in domestic livestock that diminishes productivity through reduced growth, and may ultimately lead to death. The parasites are coated in a dense layer of protein that help them evade the host’s immune system by preventing immune cells from identifying them. Humans have evolved immunity to many trypanosome species by exploiting a weakness in their lifestyle. Trypanosomes need to get haem—a molecule found in the protein haemoglobin—from their host to survive. In blood plasma, haemoglobin is found associated with a carrier protein called haptoglobin. To acquire haem, the parasites have a protein called HpHbR that binds to these haptoglobin-haemoglobin ‘complexes’. However, in humans there are two complexes of proteins called TLFs that contain haemoglobin and a protein related to haptoglobin. The TLFs are also able to bind to HpHbR and are taken into the parasite. Once inside, TLFs cause internal compartments called lysosomes to swell, which leads to the death of the trypanosome. Two subspecies of Trypanosoma brucei are the only trypanosomes that infect humans as they can overcome the TLF1 defense. However, the details of how TLFs cause cell death at the molecular level are not clear. Lane-Serff et al. used a technique called x-ray crystallography to generate 3-D images of the HpHbR protein from T. brucei bound to the haptoglobin-haemoglobin complexes. These images show that HpHbR is elongated so that it only binds to haemoglobin and haptoglobin when they are together as a complex. The images also reveal that the shape of HpHbR enables it to hold apart the proteins in the protective layer that coats the trypanosome. This allows the haptoglobin-haemoglobin complex to bind to HpHbR, but in humans also makes HpHbR more likely to bind to TLF1. These findings may help to guide future efforts to protect humans and livestock from the diseases caused by trypanosomes.

Human innate immunity against most African trypanosomes, including Trypanosoma brucei brucei, is mediated by a minor subclass of toxic serum HDL, called trypanosome lytic factor-1 (TLF-1). This HDL contains two primate specific proteins, apolipoprotein L-1 and haptoglobin (Hp)-related protein, as well as apolipoprotein A-1. These assembled proteins provide a powerful defense against trypanosome infection. Trypanosoma brucei rhodesiense causes human African sleeping sickness because it has evolved an inhibitor of TLF-1, serum resistance-associated (SRA) protein. Trypanosoma brucei gambiense lacks the SRA gene, yet it infects humans. As transfection of T. b. gambiense (group 1) is not possible, we initially used in vitro-selected TLF-1-resistant T. b. brucei to examine SRA-independent mechanisms of TLF-1 resistance. Here we show that TLF-1 resistance in T. b. brucel is caused by reduced expression of the Hp/Hb receptor gene (TbbHpHbR). Importantly, T. b. gambiense (group 1) also showed a marked reduction in uptake of TLF-1 and a corresponding decrease in expression of T. b. gambiense Hp/Hb receptor (TbgHpHbR). Ectopic expression of TbbHpHbR in TLF-1-resistant T. b. brucei rescued TLF-1 uptake, demonstrating that decreased TbbHpHbR expression conferred TLF-1 resistance. Ectopic expression of TbgHpHbR in TLF-1-resistant T. b. brucei failed to rescue TLF-1 killing, suggesting that coding sequence changes altered Hp/Hb receptor binding affinity for TLF-1. We propose that the combination of coding sequence mutations and decreased expression of TbgHpHbR directly contribute to parasite evasion of human innate immunity and infectivity of group 1 T. b. gambiense.