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

Comparison of VSG structure with modeled structures for ESAG4 and ISG65.

<p>The line diagram at the top shows the location in the extracellular part of the proteins of the real and putative structured domains. The structures below are coloured from blue at the N-terminus to red at the C-terminus. VSG structures are from PDB: 1VSG and 1XU6. The ESAG4 models were made using Phyre2 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005259#ppat.1005259.ref022" target="_blank">22</a>] and default parameters; the programme gave a 100% confidence model for both domains. The ISG65 model was made using an initial structural alignment using Fugue Profile Library Search [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005259#ppat.1005259.ref032" target="_blank">32</a>], and small adjustments were made to align cysteines for disulphide bridge formation. Subsequently, Modeller was used to generate 100 models using standard Modeller scripts [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005259#ppat.1005259.ref031" target="_blank">31</a>], and the model with the lowest Discrete Optimized Protein Energy (DOPE) assessment score [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005259#ppat.1005259.ref033" target="_blank">33</a>] was selected to be shown here.</p

How Does the VSG Coat of Bloodstream Form African Trypanosomes Interact with External Proteins?

<div><p>Abstract</p><p>Variations on the statement “the variant surface glycoprotein (VSG) coat that covers the external face of the mammalian bloodstream form of <i>Trypanosoma brucei</i> acts a physical barrier” appear regularly in research articles and reviews. The concept of the impenetrable VSG coat is an attractive one, as it provides a clear model for understanding how a trypanosome population persists; each successive VSG protects the plasma membrane and is immunologically distinct from previous VSGs. What is the evidence that the VSG coat is an impenetrable barrier, and how do antibodies and other extracellular proteins interact with it? In this review, the nature of the extracellular surface of the bloodstream form trypanosome is described, and past experiments that investigated binding of antibodies and lectins to trypanosomes are analysed using knowledge of VSG sequence and structure that was unavailable when the experiments were performed. Epitopes for some VSG monoclonal antibodies are mapped as far as possible from previous experimental data, onto models of VSG structures. The binding of lectins to some, but not to other, VSGs is revisited with more recent knowledge of the location and nature of N-linked oligosaccharides. The conclusions are: (i) Much of the variation observed in earlier experiments can be explained by the identity of the individual VSGs. (ii) Much of an individual VSG is accessible to antibodies, and the barrier that prevents access to the cell surface is probably at the base of the VSG N-terminal domain, approximately 5 nm from the plasma membrane. This second conclusion highlights a gap in our understanding of how the VSG coat works, as several plasma membrane proteins with large extracellular domains are very unlikely to be hidden from host antibodies by VSG.</p></div

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

VSG models.

<p>(A) A model of VSG121 showing the location of the cyanogen bromide fragment p19 (blue) that contains the epitopes for MoAbs that bound live trypanosomes. From the left, one monomer orientated so the dimerization interface runs vertically up the page; second, rotated approximately 90° so that the dimerization interface has turned away from the observer; third, same view with the surface added. There are potential surface-exposed epitopes along the entire length of the domain. (B) A model of VSG117 showing in blue the location that contained the epitope recognised by a MoAb that bound live cells. (C) Model of VSG WATat1.1 showing the location of differences with the related VSG WATat1.12. A monoclonal antibody that recognises an epitope in WATat1.1 does not bind WATat1.12, so the epitope probably contains one of these differences. An envelope for one possible position of the C-terminal domain is shown in purple.</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

Structure of VSG221 (MITat1.2).

<p>(A) Illustrated model of VSG221 dimer showing the structures of the N-terminal domain, one monomer in blue and one in grey, and the two C-terminal domains in purple (PDB: 1VSG and 1XU6) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005259#ppat.1005259.ref001" target="_blank">1</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005259#ppat.1005259.ref009" target="_blank">9</a>]. The N-linked oligosaccharide in the N-terminal domain is shown in red. Three residues are shown that form the core; there are between one and three further residues not shown. The relative positions of the N- and C-terminal domains are not known, and this illustration is a model [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005259#ppat.1005259.ref001" target="_blank">1</a>]. (B) Space-filling model of VSG221 viewed from the <i>x</i>-, <i>y</i>-, and <i>z</i>-axes. The maximum width dimensions of the VSG are shown below, and the fit of an ellipse of approximately 28 Ų is shown around the structure of a VSG viewed from outside the cell.</p

Stumpy forms without kDNA die rapidly when α-KG is the main carbon source.

<p>(A) Stumpy forms of the genotypes indicated were harvested, purified from blood and placed in Creek’s minimal medium (CMM) with 10% (v/v) FCS, supplemented with either glucose (blue bars) or α-KG (grey bars). N-acetyl glucosamine (GlcNAc, 50 mM) was added to one set of experiments to reduce uptake of residual glucose from FCS (red bars). Cells were stained with PI and the % of live cells was assessed by flow cytometry before (t₀; black) and 24 h after the start of the experiment; n = 3 for each cell line; all three kDNA⁰ cell lines were assessed and data averaged (total n = 9). The gating strategy is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007195#ppat.1007195.s005" target="_blank">S5B Fig</a>. (B) Quantification of dead cells within WT/WTγ cell populations after 24 h in CMM supplemented with either glucose (25 mM) or α-KG (25 mM), with or without azide (0.5 mM). Cells were stained with PI and analysed by flow cytometry, the gating strategy is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007195#ppat.1007195.s005" target="_blank">S5B Fig</a>. Shown are average values ±SD; n = 3.</p

The mathematical model for <i>T</i>. <i>brucei</i> infection dynamics.

<p>(A) Schematic of the mathematical model. Slender cells can become committed to differentiation via a SIF dependent route, proportional to SIF concentration, and a SIF independent route. SIF is produced by both committed and non-committed slender forms, and is cleared over time. The concentration of each cell type depends on the replication rate (applicable to slender forms only), the immune clearance rate, the lifespan of that cell type and the differentiation rate (applicable to slender forms only). (B) Standardised residuals (blue circles) of parasite density and slender fraction, by time (dpi, days post infection), of the model fits with SIF-dependent and -independent differentiation to all mice. Under a true model standardised residuals have an approximately standard normal distribution (i.e., zero mean and unit standard deviation (SD)). Inadequate fit of a model is indicated by its residuals deviating from a standard normal distribution (such as residuals further than ~3 SD from zero, represented by the lightest grey shading, or a set of residuals consistently above or below zero. The red line shows the average, across all mice, of the residuals at a particular time point.</p

Proposed mitochondrial energy metabolism of stumpy form <i>T</i>. <i>brucei</i> cells in the bloodstream.

<p>Schematic representation of key functions we propose to be involved in energy metabolism of stumpy form <i>T</i>. <i>brucei</i>, based on data presented in this work and in earlier studies, as cited in the text. Note that energy metabolism in other compartments such as adipose tissue or skin will very likely be different. Transporters in the inner mitochondrial membrane are shown as coloured squares (MPC, mitochondrial pyruvate carrier; KGC, α-KG carrier; AAC, ATP/ADP carrier). A two-subunit mitochondrial pyruvate carrier, MPC1/2, presumably driven by proton symport, has been identified in <i>T</i>. <i>brucei</i>, but functional studies concluded that at least one additional mitochondrial pyruvate transporter must be present [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007195#ppat.1007195.ref074" target="_blank">74</a>], indicated here by a yellow square with a question mark. Enzymes or enzyme complexes associated with the inner membrane are shown as coloured circles (cI, NADH:ubiquinone oxidoreductase; cV, F₁F_O-ATPase, or respiratory complex V; G3P-DH, glycerol-3-phosphate dehydrogenase; AOX, alternative oxidase; NDH2, type 2 NADH dehydrogenase). Functions that directly depend on kDNA-encoded proteins are indicated by red letters and arrows. Key metabolic reactions in the mitochondrial matrix are indicated by numbers in circles: 1, pyruvate dehydrogenase; 2, acetyl-CoA thioesterase; 3, ASCT; 4, SCoAS; 5, α-KG dehydrogenase complex; 6, L-alanine aminotransferase (co-substrate glutamate and co-product alanine omitted for simplicity). Other abbreviations: UQ, ubiquinone; G3P, glycerol-3-phosphate; DHAP, dihydroxyacetone phosphate; ACoA, acetyl-CoA; SucCoA, succinyl-CoA).</p