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Understanding human sleeping sickness

Related publication: Zoll S., Lane-Serff H., Mehmood S., Schneider J., Robinson C. V, Carrington M. & Higgins M. K. The structure of serum resistance-associated protein and its implications for human African trypanosomiasis. Nat. Microbiol. 3, 295–301 (2018). DOI: 10.1038/s41564-017-0085-3

Publication keywords: African trypanosomes; Innate immunity; Apolipoprotein L1; SRA

The worst forms of human sleeping sickness (African trypanosomiasis) are caused by a species of trypanosome (single-celled parasite) known as T. b. rhodesiense. Most African trypanosomes are unable to infect humans as they are killed by a component of human blood, known as trypanolytic factor, and only two trypanosome subspecies are able to infect humans. T. b. rhodesiense is able to do so due to the presence of a single molecule called SRA. Although it was clear that SRA is the sole molecule responsible for allowing this parasite to develop human resistance, we did not know how it worked, or what it looked like. We also did not know how it binds to the active component of the trypanolytic factor, a molecule called ApoLI.

A team of researchers from the Universities of Oxford and Cambridge investigated how SRA stops the trypanolytic factor from working, using the Macromolecular Crystallography beamlines I03 and I04-1 to collect diffraction data from crystals of SRA bound to an antibody, and to determine the structure of the molecule. They were able to work out what SRA looks like, and to map onto the structure the regions of SRA that bind to the trypanolytic factor. This provides a first view of how SRA gives a trypanosome the ability to infect humans. However, it still leaves many questions, and understanding exactly how ApoLI interacts with SRA remains an exciting future research challenge.

African trypanosomes are single-celled eukaryotic pathogens that are transmitted by tsetse flies, and live within the blood streams and tissue spaces of infected mammals. Infection causes disease, including the debilitating wasting illness of cattle, nagana, which restricts the productivity of livestock in sub-Saharan Africa. While there are numerous species of African trypanosome, only two subspecies, T. b. gambiense and T. b. rhodesiense, can proliferate in humans. This is due to lytic factors, present only in humans and some other primates, which are taken up by trypanosomes and causes their death¹. Human-infective species are able to negate the action of this lytic factor. This confers the ability to grow within infected humans, and to cause the disease Human African trypanosomiasis, also known as sleeping sickness. The aim of our work is to understand how human-infective trypanosomes inactivate the lytic factor, and then to determine whether we can make them susceptible to lytic factor mediated killing.

Our first contribution towards these goals used beamline facilities at Diamond Light Source to understand how lytic factor is taken up by trypanosomes. The lytic factor is a complex of several components, including the pore-forming protein, apolipoprotein LI (ApoLI), which acts as the toxin and a complex of haptoglobin related-protein bound to haemoglobin (HprHb). Trypanosomes have a receptor on their cell surface that binds to haptoglobin-haemoglobin (HpHb), allowing them to scavenge this valuable nutrient from the blood. However, the presence of HprHb in lytic factor allows this toxin to hitch a ride on the receptor, taking it into the trypanosome cell, where it mediates its toxic effect. We used beamlines I03 and I04-1 to solve structures of HpHb receptors from human-infective and non-infective trypanosomes, alone and also bound to HpHb^2-4. This revealed how the lytic factor is recognised by trypanosomes. It also helped us to understand how T. b. gambiense becomes human-infective, as it has a mutation of the HpHb receptor which reduces lytic factor uptake. Our structures revealed how this mutation decreases the affinity of the receptor for lytic factor, and how it helps to protect this trypanosome subspecies from destruction^2,3.

We more recently turned our attention to T. b. rhodesiense, which counteracts the action of lytic factor by expressing an anti-toxin that binds to, and inactivates, ApoL1. The anti-toxin, known as the serum resistance associated protein (SRA), is found primarily within the lysosome of trypanosomes, and is sufficient to allow a trypanosome to become human infective. Our goal was to determine the structure of SRA, and to investigate how it binds to, and inactivates, ApoLI.

Determining the structure of SRA proved challenging. As we later discovered, a large loop at one end of SRA is flexible, and prevented the formation of well-ordered crystals. We were able to solve this problem by producing a panel of monoclonal antibodies that bind to SRA, and discovered that the complex of SRA with one of these antibodies formed crystals of sufficient quality⁵. Armed with data collected from beamline I03 at Diamond, we were then able to determine the structure of SRA. Together with data from hydrogen-deuterium exchange mass spectrometry, and validated by mutagenesis, this allowed us to map onto SRA the residues that interact with ApoLI.

This provided the first detailed structural insight into SRA and its mode of interaction with ApoLI⁵. It also revealed that prevailing models for how SRA prevents ApoLI function were not compatible with our structural findings. However, it still leaves many questions, and understanding exactly how ApoLI forms pores, and how the binding of SRA prevents these pores from forming, remains an exciting future research challenge.

: Figure: The centre of the Figure shows a schematic of a blood stream form T. b. brucei. The left-hand side of the figure shows the T. b. rhodesiense haptoglobin-haemoglobin receptor (HpHbR, blue) bound
to a complex of the SP domain of haptoglobin (Hp, yellow), the a-subunit of haemoglobin (Hba, orange) and the b-subunit of haemoglobin (Hbb, red). This is found on the cell surface, and within the
flagella pocket of trypanosomes. The right-hand panel shows the surface resistance associated protein (SRA, rainbow) of T. b. rhodesiense, which is found primarily in the lysosome of trypanosomes.

References:

Pays E. et al. The molecular arms race between African trypanosomes and humans. Nat. Rev. Microbiol. 12, 575 (2014). DOI: 10.1038/nrmicro3298.
Higgins M. K. et al. Structure of the trypanosome haptoglobin{\ textendash}hemoglobin receptor and implications for nutrient uptake and innate immunity. Proc. Natl. Acad. Sci. 110, 1905–1910 (2013). DOI: 10.1073/pnas.1214943110
Lane-Serff H. et al. Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor. Elife 3, e05553 (2014). DOI: 10.7554/eLife.05553
Lane-Serff H. et al. Evolutionary diversification of the trypanosome haptoglobin-haemoglobin receptor from an ancestral haemoglobin receptor. Elife 5, e13044 (2016). DOI: 10.7554/eLife.13044
Zoll S. et al. The structure of serum resistance-associated protein and its implications for human African trypanosomiasis. Nat. Microbiol. 3, 295–301 (2018). DOI: 10.1038/s41564-017-0085-3

Funding acknowledgement:

MRC (MR/L008246/1 and MR/P001424/1); Wellcome Trust.

Corresponding authors:

Prof Matt Higgins, University of Oxford, matthew.higgins@bioch.ox.ac.uk and Prof Mark Carrington, University of Cambridge, mc115@cam.ac.uk

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Understanding human sleeping sickness