Tb927.10.12940 has opposing effects on apoL1 and NHS sensitivity, and localises to the lysosome.

<p>Representative EC₅₀ assays carried out in quadruplicate showing the impact of Tb927.10.12940 RNAi knockdown on <i>T</i>. <i>b</i>. <i>brucei</i> sensitivity to (A) apoL1 and (B) NHS. Inset charts show pooled EC₅₀ data for three independent cell lines. Error bars, standard deviation; <i>P</i>-values derived from paired students t-test. (C) Western blot showing tetracycline (tet)-inducible expression of Tb927.10.12940^GFP. (D) Immunofluorescence localisation of Tb927.10.12940^GFP and the lysosomal membrane protein, p67; counter-staining with the DNA intercalating dye, DAPI, reveals the kinetoplast (k) and nucleus (n). A processed merged image of the region of interest shows the co-localisation of Tb927.10.12940^GFP and p67. Scale bar, 5 μm.</p

Evidence for interdependence between Tb927.9.8000 and Tb927.10.12940.

<p>Representative EC₅₀ assays carried out in quadruplicate showing the impact of Tb927.9.8000 RNAi knockdown in wild type (left hand panels) and <i>12940</i> null (middle panels) <i>T</i>. <i>b</i>. <i>brucei</i> on (A) apoL1 and (B) NHS sensitivity. Right hand panels, pooled EC₅₀ data for at least three independent cell lines. Error bars, standard deviation.</p

TbKIFC1 depletion complements NHS sensitivity of <i>12940</i> null <i>T</i>. <i>b</i>. <i>brucei</i>, but has no additive effect on apoL1 sensitivity or suramin efficacy.

<p>Representative EC₅₀ assays carried out in quadruplicate showing the impact of TbKIFC1 RNAi knockdown in wild type (left hand panels) and <i>12940</i> null (middle panels) <i>T</i>. <i>b</i>. <i>brucei</i> on (A) apoL1, (B) NHS and (C) suramin sensitivity. Right hand panels, pooled EC₅₀ data for at least three independent cell lines. Error bars, standard deviation.</p

Distinct sets of <i>T</i>. <i>b</i>. <i>brucei</i> proteins determine parasite sensitivity to apoL1 and NHS.

<p>(A) Reads containing the 14-base RNAi construct-specific barcode identified following RNAi library selection in human apoL1 (this study) or NHS [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006855#ppat.1006855.ref032" target="_blank">32</a>] plotted as RPKM (plus 0.1) to correct for variations in read depth between the respective RNAi library screens (and to enable plotting of zero read outputs on log₁₀ scales); r² = 0.016. Dashed lines represent the 100-read stringency criterion converted to RPKM for each RNAi library screen (human apoL1, 158; NHS, 404); apoL1 (purple) and NHS (orange)-specific RNAi targets are highlighted and the top hits listed (gene ID and functional annotation; see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006855#ppat.1006855.s008" target="_blank">S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006855#ppat.1006855.s009" target="_blank">S3</a> Tables for further details). (B-D) Representative EC₅₀ assays carried out in quadruplicate confirming that (B) p67 knockdown, (C) <i>ICP</i> deletion and (D) TbCATL depletion does not affect <i>T</i>. <i>b</i>. <i>brucei</i> sensitivity to apoL1. Insets, representative EC₅₀ assays carried out in quadruplicate showing the known impact of the corresponding manipulations on sensitivity to NHS. Error bars, standard deviation.</p

RNAi library screens reveal the distinct paths that human serum TLFs follow to instigate trypanolysis.

<p>(A) The anti-trypanosomal action of TLF1 is particularly vulnerable to changes in the surface receptor, TbHpHbR, and lysosomal function as defined by the V-ATPase, p67, ICP and TbCATL. (B) ApoL1 and TLF2 enter <i>T</i>. <i>brucei</i> via fluid phase endocytosis and an unknown mechanism (represented by the black box), respectively, and are reliant on the support of a network of sensitivity determinants, including the V-ATPase and vesicle and membrane trafficking proteins, such as TbKIFC1 and VAMP7B, which may themselves be regulated by dynamic ubiquitination. The orange bars represent membrane-integrated apoL1. The dashed line highlights the recent finding that apoL1 may also form pores in the parasite’s plasma membrane (omitted from panel (B) for clarity) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006855#ppat.1006855.ref017" target="_blank">17</a>]. Ellipses highlight possible regions of influence of the membrane trafficking (VAMP) and ubiquitination (Ub) proteins described herein.</p

RIT-seq profiles of candidate novel apoL1-sensitivity determinants.

<p>ApoL1 sensitivity determinants include (A) putative ubiquitin modifiers and (B) putative membrane trafficking proteins. RIT-seq profiles show predicted transcripts (open reading frames and untranslated regions of interest in black) and the RNAi targeting fragment reads mapped; total reads (red) and tagged reads (blue; containing the 14-base RNAi construct barcode sequence) mapped to each predicted transcript are shown in the top right corner of each panel. Known or putative annotations based on domain organisation are included beneath each accession number and derived from GeneDB (<a href="http://www.genedb.org/Homepage/Tbruceibrucei927" target="_blank">http://www.genedb.org/Homepage/Tbruceibrucei927</a>). Ub, ubiquitin; DUB, deubiquitinase; VAMP, vesicle-associated membrane protein; VAP, VAMP-associated protein.</p

Selecting a genome-scale <i>T</i>. <i>b</i>. <i>brucei</i> RNAi library identifies parasite determinants of apoL1 sensitivity.

<p>(A) Genome-wide human apoL1 RIT-seq profile representing 7,398 non-redundant predicted transcripts, showing those targeted by 100 or more reads per kilobase containing the 14-base RNAi construct barcode sequence. ‘Hits’ targeting V-ATPase subunits and the kinesin, TbKIFC1, are highlighted in green and red, respectively; novel high confidence hits corresponding to six putative ubiquitin modifiers and four putative membrane transporters are highlighted in black and blue, respectively (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006855#ppat.1006855.g004" target="_blank">Fig 4</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006855#ppat.1006855.s007" target="_blank">S1 Table</a> for further details). (B) RNAi library selection with human or <i>P</i>. <i>papio</i> apoL1 identified similar sets of sensitivity determinants (r² = 0.78); key hits are coloured as in (A), and are similarly abundant in both selected libraries. Data presented as RPKM (plus 0.1; <u>r</u>eads <u>p</u>er <u>k</u>ilobase of transcript per <u>m</u>illion mapped reads) to correct for variations in read depth between the respective RNAi library screens (and to enable plotting of zero read outputs on log₁₀ scales). Dashed lines represent the 100-read stringency criterion converted to RPKM for each RNAi library screen (human apoL1, 158; <i>P</i>. <i>papio</i> apoL1, 132). (C) RIT-seq profiles for three of the V-ATPase subunits and for TbKIFC1; predicted transcripts (open reading frames and untranslated regions) are coloured as in (A); total reads (red) and tagged reads (blue; containing the 14-base RNAi construct barcode sequence) mapped to each predicted transcript are shown in the top right corner of each panel.</p

Generation and complementation of <i>12940</i> null <i>T</i>. <i>b</i>. <i>brucei</i>.

<p>(A) Southern blot confirming <i>Tb927</i>.<i>10</i>.<i>12940</i> deletion; <i>BSD</i>, blasticidin-S-deaminase; <i>NPT</i>, neomycin phosphotransferase. Schematic showing segment deleted from the <i>Tb927</i>.<i>10</i>.<i>12940</i> locus (open triangle); positions of <i>Sal</i>I restriction sites (arrowheads), deletion (12940) and flanking (DS) probes highlighted. (B) Cumulative growth of wild type and three independent <i>12940</i> null <i>T</i>. <i>b</i>. <i>brucei</i> cell lines; error bars showing standard deviation are smaller than the plot symbols. (C, D) Representative EC₅₀ assays carried out in quadruplicate showing the impact of (C) <i>Tb927</i>.<i>10</i>.<i>12940</i> deletion and (D) re-expression on apoL1 sensitivity. Insets, pooled EC₅₀ data for at least two independent cell lines. (E, F) Kinetic analyses of parasite killing in 10 μg.ml^-1 apoL1 following (E) <i>Tb927</i>.<i>10</i>.<i>12940</i> deletion and (F) re-expression; each assay was carried out using three independent cell lines, re-expression was induced in 1 μg.ml^-1 tetracycline for 24 hours prior to exposure to apoL1. Error bars, standard deviation; <i>P</i>-values derived from paired students t-test.</p

An RNAi library screen to identify <i>T</i>. <i>b</i>. <i>brucei</i> apoL1 sensitivity determinants.

<p>(A) <i>T</i>. <i>b</i>. <i>brucei</i> (MITat1.2; strain 2T1) is similarly sensitive to human (<i>H</i>. <i>sapiens</i>) and baboon (<i>P</i>. <i>hamadryas</i> and <i>P</i>. <i>papio</i>) apoL1. EC₅₀ assays were carried out in quadruplicate; error bars, standard deviation. (B) Schematic showing selection of apoL1-resistant parasites from the RNAi library. (C) Selection of populations with reduced sensitivity to apoL1. RNAi induced in 1 μg.ml^-1 tetracycline (Tet) for 24 hours prior to selection (initiated at day-0); arrows indicate culture dilution, and addition of fresh apoL1 and tetracycline at 2X EC₅₀ and 1 μg.ml^-1, respectively. (D) RNAi target fragment-specific PCR amplification from <i>T</i>. <i>b</i>. <i>brucei</i> genomic DNA extracted after eight days’ selection in apoL1 (Hs, <i>H</i>. <i>sapiens</i>; Ph, <i>P</i>. <i>hamadryas</i>; Pp, <i>P</i>. <i>papio</i>).</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