Structural insights into innate immunity against African trypanosomes

The haptoglobin-haemoglobin receptor (HpHbR) is expressed by the African try- panosome, T. brucei, whilst in the bloodstream of the mammalian host. This allows ac- quisition of haem, but also results in uptake of trypanolytic factor 1, a mediator of in- nate immunity against non-human African trypanosomes. Here, the structure of HpHbR in complex with its ligand, haptoglobin-haemoglobin (HpHb), is presented, revealing an elongated binding site along the membrane-distal half of the receptor. A &Tilde;50&deg; kink allows the simultaneous binding of two receptors to one dimeric HpHb, increasing the efficiency of ligand uptake whilst also increasing binding site exposure within the densely packed cell surface. The possibility of targeting this receptor with antibody-drug conjugates is ex- plored. The characterisation of the unexpected interaction between T. congolense HpHbR and its previously unknown ligand, haemoglobin, is also presented. This receptor is iden- tified as an epimastigote-specific protein expressed whilst the trypanosome occupies the mouthparts of the tsetse fly vector. An evolutionary pathway of the receptor is proposed, describing how the receptor has changed to adapt to a role as a bloodstream form-specific protein in T. brucei. Apolipoprotein L1 (ApoL1) is the pore-forming component of the trypanolytic factors. An expression and purification protocol for ApoL1 is presented here, and the functionality of the protein established. Initial attempts to characterise the pores and structure of ApoL1 are described.</p

The structures of trypanosome surface proteins.

<p><b>A.</b> The structures of the major surface proteins of the <i>T</i>. <i>congolense</i> epimastigote, the glutamic acid rich protein (GARP) and of the <i>T</i>. <i>brucei</i> bloodstream form, the variant surface glycoprotein (VSG), and the structures of the <i>T</i>. <i>brucei and T</i>. <i>congolense</i> haptoglobin–hemoglobin receptors (TbHpHbR and TcHpHbR). Both TbHpHbR and TbVSG are elongated by additional C-terminal domains, which are not represented [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006055#ppat.1006055.ref010" target="_blank">10</a>]. <b>B.</b> The structure of a complex of two TcHpHbR bound to a single hemoglobin tetramer. The receptors are coupled to the cell membrane by a GPI anchor and will tilt in order to simultaneously bind to a single hemoglobin. <b>C.</b> The structure of a complex of two TbHpHbR bound to a haptoglobin–hemoglobin tetramer (silver and gold), showing how the kink in the receptor allows two membrane-linked TbHpHbR to simultaneously bind to a single HpHb.</p

The <i>T. brucei</i> HpHbR is a bloodstream form HpHb receptor.

<p>In the life cycle of <i>T</i>. <i>brucei</i>, the epimastigotes inhabit the salivary glands of the tsetse fly and do not express HpHbR. Instead, TbHpHbR is predominantly expressed in the bloodstream form, in which it acts as an HpHb receptor that exists within the densely packed VSG layer. In the structure figure HpHbR is blue, VSG is blue-white, and HpHb is yellow, orange, and red. The ovals represent the C-terminal domains of the VSG and HpHbR. These lie between the N-terminal domains and the membrane, but their relative locations are uncertain.</p

The <i>T</i>. <i>congolense</i> HpHbR is an epimastigote expressed Hb receptor.

<p>In the life cycle of <i>T</i>. <i>congolense</i>, HpHbR is expressed predominantly in the epimastigotes that inhabit the mouthparts of the tsetse fly, where it binds to Hb present in the blood meal of the fly. Here the receptor functions in the context of the major epimastigote surface protein, GARP. In the structure figure TcHpHbR is green, GARP is light blue, and Hb is orange and red.</p

The structure of serum resistance-associated protein and its implications for human African trypanosomiasis.

Only two trypanosome subspecies are able to cause human African trypanosomiasis. To establish an infection in human blood, they must overcome the innate immune system by resisting the toxic effects of trypanolytic factor 1 and trypanolytic factor 2 (refs. 1,2). These lipoprotein complexes contain an active, pore-forming component, apolipoprotein L1 (ApoL1), that causes trypanosome cell death 3 . One of the two human-infective subspecies, Trypanosoma brucei rhodesiense, differs from non-infective trypanosomes solely by the presence of the serum resistance-associated protein, which binds directly to ApoL1 and blocks its pore-forming capacity3-5. Since this interaction is the single critical event that renders T. b. rhodesiense human- infective, detailed structural information that allows identification of binding determinants is crucial to understand immune escape by the parasite. Here, we present the structure of serum resistance-associated protein and reveal the adaptations that occurred as it diverged from other trypanosome surface molecules to neutralize ApoL1. We also present our mapping of residues important for ApoL1 binding, giving molecular insight into this interaction at the heart of human sleeping sickness