Expression of <i>T</i>. <i>b</i>. <i>brucei HpHbR</i> in <i>T</i>. <i>b</i>. <i>gambiense</i> group 1 increases sensitivity to <i>P</i>. <i>papio</i> serum lysis.

<p>The percentage of viable trypanosomes following a 24-hour exposure to serial dilutions of A) human serum and B) <i>P</i>. <i>papio</i> serum was determined for <i>T</i>. <i>b</i>. <i>gambiense</i> (strain ELIANE) and <i>T</i>. <i>b</i>. <i>gambiense</i> expressing a functional <i>T</i>. <i>b</i>. <i>brucei HpHbR</i> receptor (strain ELIANE <i>TbbHpHbR</i>^<i>-/+</i>) alongside representative <i>T</i>. <i>brucei</i> sub-species strains <i>T</i>. <i>b</i>. <i>brucei</i> (STIB247) and <i>T</i>. <i>b</i>. <i>rhodesiense</i> (EATRO98). Mean percentage cell survival ± SD is expressed, relative to FBS control. Dose–response curves and IC₅₀ values with 95% confidence intervals (CI) were determined using GraphPad Prism software version 7.</p

A Primate APOL1 Variant That Kills <i>Trypanosoma brucei gambiense</i>

<div><p>Humans are protected against infection from most African trypanosomes by lipoprotein complexes present in serum that contain the trypanolytic pore-forming protein, Apolipoprotein L1 (APOL1). The human-infective trypanosomes, <i>Trypanosoma brucei rhodesiense</i> in East Africa and <i>T</i>. <i>b</i>. <i>gambiense</i> in West Africa have separately evolved mechanisms that allow them to resist APOL1-mediated lysis and cause human African trypanosomiasis, or sleeping sickness, in man. Recently, APOL1 variants were identified from a subset of Old World monkeys, that are able to lyse East African <i>T</i>. <i>b</i>. <i>rhodesiense</i>, by virtue of C-terminal polymorphisms in the APOL1 protein that hinder that parasite’s resistance mechanism. Such variants have been proposed as candidates for developing therapeutic alternatives to the unsatisfactory anti-trypanosomal drugs currently in use. Here we demonstrate the <i>in vitro</i> lytic ability of serum and purified recombinant protein of an APOL1 ortholog from the West African Guinea baboon (<i>Papio papio)</i>, which is able to lyse examples of all sub-species of <i>T</i>. <i>brucei</i> including <i>T</i>. <i>b</i>. <i>gambiense</i> group 1 parasites, the most common agent of human African trypanosomiasis. The identification of a variant of APOL1 with trypanolytic ability for both human-infective <i>T</i>. <i>brucei</i> sub-species could be a candidate for universal APOL1-based therapeutic strategies, targeted against all pathogenic African trypanosomes.</p></div

Trypanolytic activity of recombinant APOL1.

<p>The percentage of viable trypanosomes was determined following a 24-hour exposure to media containing serial dilutions of A) recombinant Human APOL1 protein and B) recombinant <i>P</i>. <i>papio</i> APOL1 protein. Representative <i>T</i>. <i>brucei</i> sub-species strains were tested: <i>T</i>. <i>b</i>. <i>brucei</i> (strain STIB247), <i>T</i>. <i>b</i>. <i>rhodesiense</i> (strain EATRO98), <i>T</i>. <i>b</i>. <i>gambiense</i> group 1 (strain ELIANE), and <i>T</i>. <i>b</i>. <i>gambiense</i> group 2 (strain STIB386). Additional assays were performed with different strains of <i>T</i>. <i>b</i>. <i>gambiense</i> group 1 and are provided in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004903#pntd.0004903.s004" target="_blank">S4 Fig</a>. The mean percentage cell survival ± SD, relative to protein-free control, was calculated from at least three independent experiments. Dose–response curves and IC₅₀ values with 95% confidence intervals (CI) were determined using GraphPad Prism software version 7. APOL1-mediated lysis of each isolate was prevented by the inclusion of an acidotropic agent (10 mM NH₄Cl) in the assay.</p

Recombinant APOL1 uptake and localisation.

<p>The localisation of Alexa488 (green) labelled anti-pentaHis antibody (APOL1), AlexaFluor594 (red) labelled anti-p67 (lysosomal membrane protein) and DAPI after a two-hour exposure to recombinant Human and <i>P</i>. <i>papio</i> APOL1 featuring an N-terminal 6xHis-tag. The panels represent human serum sensitive <i>T</i>. <i>b</i>. <i>brucei</i>, strain STIB247, and human serum resistant <i>T</i>. <i>b</i>. <i>gambiense</i> group 1, strain ELIANE.</p

Titration of the trypanolytic activity of Human (<i>H</i>. <i>sapiens</i>) and Guinea baboon (<i>P</i>. <i>papio)</i> sera against representative examples of the <i>T</i>. <i>brucei</i> sub-species.

<p>The percentage of viable trypanosomes was determined following a 24-hour exposure to serial dilutions of (A) Human (<i>H</i>. <i>sapiens</i>) or (B) Guinea baboon (<i>P</i>. <i>papio)</i> sera. Representative <i>T</i>. <i>brucei</i> sub-species strains were tested: <i>T</i>. <i>b</i>. <i>brucei</i> (strain STIB247), <i>T</i>. <i>b</i>. <i>rhodesiense</i> (strain EATRO98), <i>T</i>. <i>b</i>. <i>gambiense</i> group 2 (strain STIB386), and <i>T</i>. <i>b</i>. <i>gambiense</i> group 1 (strains ELIANE, TOBO and S1/1/6 [Côte d'Ivoire], PA [Republic of the Congo], and BIM [Cameroon]). Mean percentage cell survival ± SD is expressed relative to FBS control, calculated from three independent experiments. Dose–response curves and IC₅₀ values with 95% confidence intervals (CI) were determined using GraphPad Prism software version 7.</p

Membrane rigidity changes and physiological consequences of SHP.

<p>The rigidity of the interior (<b>a</b>) or interfacial (<b>b</b>) region of the plasma membrane of BSF <i>T. b. brucei</i> treated with increasing concentrations of SHP-1 (blue •), SHP-2 (grey ⧫), SHP-3 (red ▪), SHP-3ΔR (green ▴), SHP-3ΔK (purple ▴), SHP-3ΔE (orange-open ▵), SHP-1swap (black-open ○) or solvent alone (DMSO, black ×) was determined by measuring the fluorescence depolarization of DPH or TMA-DPH respectively. (<b>c</b>) FRAP analysis of the mobile fraction of BSF <i>T. b. brucei</i> VSG in the presence (red ▪) or absence (black ▪) of 8 µM SHP-1. (<b>d</b>) Live BSF <i>T. b. brucei</i> treated with equivolume DMSO (grey), 40 µM SHP-1 (blue), SHP-3 (red) or SHP-3ΔR (green) were visualized by DIC microscopy and scored for normal, hyperactivated and constricted motility as well as death at the indicated timepoints (see Movies S1, S2, S3 and S4 for examples of the normal, hyperactivated and constricted motilities respectively).</p

Orientation and structure of SHP in lipid bilayers.

<p>(<b>a</b>) The depth of peptide penetration into the hydrocarbon region of model liposomes was determined via parallax analysis. Assuming a hydrocarbon bilayer thickness of 29 Å, the depths of tryptophans spanning SHP-1 (blue •) and SHP-3 (red ▪) are plotted against a background of the outer leaflet of a POPC bilayer. (<b>b</b>) Circular dichroism spectroscopy of SHP-1 in aqueous buffer (green •) and in the presence of egg phosphatidylcholine liposomes (blue •). (<b>c</b>) Molecular dynamic modeling of SHP-1 in a lipid environment. The backbone trace (top) illustrates a predominantly α-helical structure with disordered termini and an internal disordered region. Surface potential representations (N to C terminal, middle; C to N terminal, bottom) indicate positively charged patches (blue) formed by the N-terminal amino acid and the arginine at position 14. Non-polar and negatively charged regions are shown in white and red, respectively.</p

Small hydrophobic peptide mediated killing of African trypanosomes.

<p>(<b>a</b>) The metacyclic developmental form (the developmental stage injected during a tsetse fly bite) of <i>T. b. brucei</i> was assayed for susceptibility to SHP-1 in 2 h <i>in vitro</i> killing assays (green ⧫, metacyclic; orange ▪, BSF). (<b>b</b>) The veterinary pathogenic African trypanosomes, <i>T. vivax</i> (red ▪, SHP-1; red-open □, DMSO) and <i>T. congolense</i> (blue ▴, SHP-1; blue-open ▵, DMSO), were assayed for susceptibility to SHP-1 in 2 h <i>in vitro</i> killing assays. (<b>c</b>) The sequences of trypanolytic and non-trypanolytic SHP are shown from N- to C-terminus and aligned to the C-terminus in order to emphasize the identity of the amino acid at position -5 relative to the putative signal peptidase cleavage site. Positively charged amino acids are in red, negatively charged amino acids are in green and non-polar amino acids are underlined. (<b>d</b>) The SHP listed in (c) were tested against BSF <i>T. b. brucei</i> in 2 h killing assays (blue •, SHP-1; red ▪, SHP-3; green ▴, SHP-3ΔR; purple ▪, SHP-3ΔK; orange ▴, SHP-3ΔE; black •, SHP-1swap). Colors correspond to the peptide names in (c).</p

Polymorphisms and heterozygosity of <i>T. brucei</i> at the Côte d’Ivoire focus (N = 45).

<p>Polymorphisms and heterozygosity of <i>T. brucei</i> at the Côte d’Ivoire focus (N = 45).</p

Genetic analysis of the <i>Trypanosoma brucei</i> population at the Côte d’ivoire focus.

<p><b>a.</b> Dendrogram of multi-locus genotypes (MLG) for the <i>T. brucei</i> isolates collected from several townships in Côte d’Ivoire, over the period of time 1978–1983 in addition to DAL972 and ELIANE. Bootstrap values from 100 iterations are indicated for branch nodes with a bootstrap value above 10. The presence of <i>TgSGP</i> using primers spanning the 5′ and 3′ ends is indicated by *. Isolates that displayed human serum resistance, the classical <i>T. b. gambiense</i> isoenzyme profile and possess <i>TgSGP</i> can be inferred to be group 1 <i>T. b. gambiense</i> (Red). Isolates that display a degree of resistance or were isolated from humans but did not possess the classical isoenzyme profile or <i>TgSGP</i> were determined to be group 2 <i>T. b gambiense</i> (Blue). Strains that exhibited no human serum resistance and were isolated from animals are most likely <i>T. b. brucei</i> (Black). <b>b.</b> Predicted structure of the Côte d'Ivoire <i>T. brucei</i> focus for the most likely population number (K = 2). The proportion of each population that an isolate is a member of is indicated by red and blue in the histogram. <b>c.</b> Principal component analysis (PCA) of the Côte d’Ivoire <i>T. brucei</i> isolates using a pair-wise genetic distance comparison between each isolates MLG. The x-axis explains 56.45% of the variability in the populations and the y-axis 13.98%, for a total of 70.45%. Isolates are colored as outlined in 1a. The circled numbers indicate the centroids of the two clusters identified by <i>k</i>-means analysis. The limits of these clusters are also indicated.</p