APOLs with low ph dependence can kill all African trypanosomes
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1 SUPPLEMENTARY INFORMATION Letters DOI: 1.138/s In the format provided by the authors and unedited. APOLs with low ph dependence can kill all African trypanosomes Frédéric Fontaine 1, Laurence Lecordier 1, Gilles Vanwalleghem 1,5, Pierrick Uzureau 1,6, Nick Van Reet 2, Martina Fontaine 3, Patricia Tebabi 1, Benoit Vanhollebeke 1, Philippe Büscher 2, David Pérez-Morga 1,4 and Etienne Pays 1 * 1 Laboratory of Molecular Parasitology, IBMM, Université Libre de Bruxelles, 12, rue des Profs Jeener et Brachet, B-641 Gosselies, Belgium. 2 Unit of Parasite Diagnostics, Institute of Tropical Medicine, 155, Nationalestraat, B-2 Antwerpen, Belgium. 3 Laboratory of Immunobiology, IBMM, Université Libre de Bruxelles, 12, rue des Profs Jeener et Brachet, B-641 Gosselies, Belgium. 4 Center for Microscopy and Molecular Imaging (CMMI), Université Libre de Bruxelles, 12, rue des Profs Jeener et Brachet, B-641 Gosselies, Belgium. Present addresses: 5 School of Biomedical Sciences, The University of Queensland, St Lucia, QLD 472, Australia. 6 Laboratoire de Médecine Expérimentale (ULB222), Hôpital André Vésale, Université Libre de Bruxelles, 76, route de Gozée, B-611 Montigny le Tilleul, Belgium. Frédéric Fontaine and Laurence Lecordier contributed equally to this work. * epays@ulb.ac.be Nature Microbiology Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
2 Parasite density (x1 4 ml -1 ) kda Acetic acid + rapol1 1 + rapol2 + rapol3 + rapol Time (h) Acetic acid + rapol1 + rapol2 + rapol3 + rapol rppapol1 rapol1 rapol2 rapol3 rapol6 Suppl. Fig. 1 Supplementary Figure 1. In vitro trypanolytic potential of various human rapols. The effect of different rapols (1 µg ml -1 rapol1, 3 µg ml -1 rapol3, 5 µg ml -1 rapol2 and rapol6) on T.b. brucei growth kinetics and 24 h survival was evaluated (error bars: s.e.m.; 3 technical replicates; n=1). The panel at the right shows the SDS-PAGE pattern of the rapols, as assessed by Coomassie blue staining.
3 APOL3 APOL1 PpAPOL1 APOL3 APOL1 PpAPOL1 APOL3 APOL1 PpAPOL1 APOL3 APOL1 PpAPOL1 MGLGQGWGWEASCFACLIRSCCQVVTFTFPFGFQGISQSLENVSGYYADARLEVGSTQLRTAGSCSHSFKRS MEGAALLRVSVLCIWMSAL-----FLGVGVRAEE-----AGARVQQNVPSGTDTGDPQSKPLGD MEGAALLRLFVLCIWTSGL-----FLGVGVRAEE-----AAARVQQNVPSGTDTGDPQ *.:.: : *. : * *:. *: * **::... :*.. Helix 2 Helix 3 FL------EKKRFTEEATKYFRERVSPVHLQILLTNNEAWKRFVTAAELPRDEADALYEALKKLRTYAAIED WAAGTMDPESSIFIEDAIKYFKEKVSTQNLLLLLTDNEAWNGFVAAAELPRNEADELRKALDNLARQMIMKD ESNVSLEDYLNYFQKNVSPQEMLLLLSDHKAWERVVATAELPRDEADELYKALNKLIRHMVMKD *.. *: :**.:.**. : :**::::**:.*::*****:*** * :**.:* ::* Helix 4 Helix 5+6 Helix 7+8 EYVQQKDEQFREWFLKEFPQVKRKIQESIEKLRALANGIEEVHRGCTISNVVSSSTGAASGIMSLAGLVLAP KNWHDKGQQYRNWFLKEFPRLKSELEDNIRRLRALADGVQKVHKGTTIANVVSGSLSISSGILTLVGMGLAP KNWLEEVQQHRKRFLEEFPRLERELQDKIRRLCDLAGQVQKVHKGATIANAFSSTLGVASGVLTFLGLGLAP : :: :*.*:.**:***.:: ::::.*.* **. :::**.* **:*..*.:. :**:::: *: *** BH3 Helix 9 FTAGTSLALTAAGVGLGAASAVTGITTSIVEHSYTSSAEAEASRLTATSIDRLKVFKEVMRDITPNLLSLLN FTEGGSLVLLEPGMELGITAALTGITSSTMDYGKKWWTQAQAHDLVIKSLDKLKEVREFLGENISNFLSLAG FTAGSSLVLLEPVTGLGITAALTGITSGSVEYAKKRWAQAEAHELVNKSLDTVEEMNEFLYHNIPNFISLRV ** * **.*. ** ::*:****:. :::.. ::*:* *..*:* ::..*.:.*::** APOL3 APOL1 PpAPOL1 APOL3 APOL1 PpAPOL1 NYYEATQTIGSEIRAIRQARAR ARLPVTTWRISAGSGG------QAERTIAGTTRAVSRGARIL NTYQLTRGIGKDIRALRRARANLQSVPHASASRPRVTEPISAESGE------QVERVNEPSILEMSRGVKLT NLVKFTEDTGKAIRAIRQARANPHSVSHVPASLHRVTEPVSATSVEERARVVEMERVAESRTTEVIRGAKIV * : * *. ***:*.***. *.* :** * : **. : **..: C-terminal helix SATTSGIFLALDVVNLVYESKHLHEGAKSASAEELRRQAQELEENLMELTQIYQRLNPCHTH DVAPVSFFLVLDVVYLVYESKHLHEGAKSETAEELKKVAQELEEKLNILNNNYKILQADQEL DKVFEGALFVLDVVGLVCQLKHLHEGAKSKTAEELKKVAQELEKKLNILNKKYETLRQEP--... ::.**** ** : ********* :****.. *****::* *.: *: *. Supplementary Figure 2. Sequence comparison between APOLs. Asterisks and dots respectively refer to identical and similar amino acids. The helices have been defined in 6. BH3 = Bcl2 homology domain 3.
4 a Tb gambiense b k - FMK + FMK rapol3 rat anti-apol3 TgsGP KO Tb gambiense n rapol1 25 µg ml -1 rapol1 5 µg ml -1 rapol3 25 µg ml -1 Supplementary Figure 3. Uptake of rapol3 in trypanosomes. (a) Immunodetection of rapol3 in T.b. gambiense LiTat 1.3 parasites incubated or not in the presence of the cathepsin inhibitor FMK-24. (scale bar=5 µm; n=nucleus and k=kinetoplast, both stained with DAPI). (b) Effect of temperature on trypanolysis of T.b. gambiense TgsGP KO LiTat 1.3 parasites by rapol1 or rapol3. Results are expressed as % control growth at each temperature (error bars: s.e.m.; 3 technical replicates; n=2 independent experiments).
5 Supplementary Figure 4. Doxycycline-mediated induction of TbKIFC1 RNAi. Western blot of induced (+Dox) and non-induced (-Dox) parasite extracts. See 7 for details.
6 Nucleus Mitochondrion + rapol3 M FP K FP M K M K FP K M M Scale = 5 nm Supplementary Figure 5. Mitochondrial and nuclear phenotype of rapol3-mediated trypanolysis. TEM micrographs of T.b. brucei after 2 h incubation with 3 µg ml -1 rapol3 (FP; flagellar pocket; K=kinetoplast; M=mitochondrion; N=nucleus; arrows: heterochromatin patches).
7 rmut4 probability rmut3 probability rapol3 WT probability TMHMM ph 5.5 ph 7.5 T. b. brucei T. b. gambiense transmembrane inside outside % Glc + 75 M IPTG mm NH 4 Cl mm NH4 Cl RGCTISNVVSSSTGAASGIMSLAGLVLAPFTAGTSLALTAAGVGLGAASAVTGITTSIVEHSYTSSA rapol3 (µg ml -1 ) rapol3 (µg ml -1 ) % Glc + 75 M IPTG mm NH4 Cl + 2 mm NH4 Cl RGCTISNVVSSSTGAASGIMSLAGLVLAPFTEGTSLALTAAGVGLGAASAVTGITTSIVEHSYTSSA rmut3 (µg ml -1 ) rmut3 (µg ml -1 ) % Glc + 75 M IPTG mm NH4 1 Cl + 2 mm NH4 Cl RGCTISNVVSSSTGAASGIMSLAGLVLAPFTAGTSLALTEAGVELGAASAVTGITTSIVEHSYTSSA rmut4 (µg ml -1 ) rmut4 (µg ml -1 ) Supplementary Figure 6. Relative activity of glutamic acid mutants in the helix 9 region of rapol3. In each row, the left panels show the predicted patterns of transmembrane spans (TMHMM program, Expasy) in the APOL sequences defined underneath (numbers refer to key helices in the pore-forming domain 6 ), followed by measurements of the colicin-like activity of the different rapols in E. coli as determined by scoring the plating efficiency after overnight incubation at 37 C, comparing induction of protein expression following IPTG addition with control following glucose (Glc) addition 16, and comparing the effect of ph. The right panels show the trypanolytic potential of the different rapols, as determined on T.b. brucei and T.b. gambiense LiTat 1.3 after 24 h incubation in vitro with or without 2 mm NH 4 Cl (error bars: s.e.m.; 3 technical replicates; n=3 independent experiments).
8 Supplementary Figure 7. Trypanolytic activity of 1 µg ml -1 rapol1/apol3 chimaeras (a) or 1 µg ml -1 rapol1 mutants (b) on T.b. brucei and T.b. gambiense LiTat 1.3 after 24 h incubation in vitro. The restriction endonuclease sites used to generate the constructs are indicated (italics: generated by site-directed mutagenesis without changing the amino acid code; underlined: inserted in the PCR primers used to generate the gene fragments). Panel (c) illustrates the activity measurements reported as positive in table a. C=chimaeras; M=mutants. (error bars: s.e.m.; 3 technical replicates; n=3 independent experiments).
9 Intensity value (A.U) Intensity value (A.U) kda Non injected h 24h 48h rapol1 (1µg) 72h 96h kda Non injected h 2h 6h rapol3 (1µg) 24h R 2 = R 2 = Time (h) Time (h) rapol1 half-life = ~5h rapol3 half-life = ~13h Supplementary Figure 8. Measurement of the rapol1 and rapol3 decay in mouse blood. Western blot detection in the blood of 6 weeks-old female BALB/c mice, of different rapols (1 µg) intravenously injected at t=. We used ImageJ to quantify the signal strength from Fig. 4a western blots images (middle panel). Using these values, we applied a linear regression to evaluate the recombinant proteins half-life in mice blood.
10 rmut-null probability rapol3 WT probability TMHMM ph 5.5 ph 7.5 T. b. brucei T. b. gambiense transmembrane inside outside % Glc + 75 M IPTG mm NH 4 Cl mm NH 4 Cl RGCTISNVVSSSTGAASGIMSLAGLVLAPFTAGTSLALTAAGVGLGAASAVTGITTSIVEHSYTSSA RGCTISNVVSSSTGAASGIMSLAGLVQSSFTASTSLALTAAGVGLGAASAVTGITTSIVEHSYTSSA % Glc + 75 M IPTG mm NH 4 Cl rapol3 (µg ml -1 ) rapol3 (µg ml -1 ) rmut-null (µg ml -1 ) rmut-null (µg ml -1 ) mm NH 4 Cl Supplementary Figure 9. Lack of trypanolytic activity of rapol3 137 QQSFTAS 143 (rmut-null). In each row, the left panels show the predicted patterns of transmembrane spans (TMHMM program, Expasy) in the APOL sequences defined underneath (numbers refer to key helices in the pore-forming domain 6 ), followed by measurements of the colicinlike activity of the different rapols in E. coli as determined by scoring the plating efficiency after overnight incubation at 37 C, comparing induction of protein expression following IPTG addition with control following glucose (Glc) addition 16, and comparing the effect of ph. The right panels show the trypanolytic potential of the different rapols, as determined on T.b. brucei and T.b. gambiense LiTat 1.3 after 24 h incubation in vitro with or without 2 mm NH 4 Cl (error bars: s.e.m.; 3 technical replicates; n=3 independent experiments).
11 Supplementary Figure 1. Trypanolysis differences between different T.b. gambiense populations: relative adaptation to in vitro growth conditions, influence of the type of VSG and TgsGP level. (a) Trypanolysis and growth curves of different cloned T.b. gambiense populations: in vitro adapted LiTat 1.3 clone from the ELIANE strain, LiTat 1.3 parasites freshly isolated from blood and bloodstream parasites from the MBA strain expressing the LiTat 1.3 isotype AnTat 11.8, or bloodstream parasites from the ELIANE strain expressing LiTat 1.2 (error bars: s.e.m.; 3 technical replicates; n=3 independent experiments). (b) Western blots of protein extracts from the different T.b. gambiense populations, incubated with anti-litat 1.3 VSG antibodies. (c) Expression levels of TgsGP in the different T.b. gambiense populations and in T.b. brucei negative control parasites, as measured by qrt- PCR.
12 Supplementary Figure 11. Selective precipitation of rapol3 in the trypanosome incubation medium. rapol1 and rapol3 (1 µg ml -1 each) were incubated for different periods at 37 C in the incubation medium, then the centrifugation supernatant was analyzed by Western blotting with anti-apol antibodies. =no protein.
13 From Fig. 4a From Suppl. Fig. 4 Supplementary Figure 12. Raw version of Western blots.
14 Supplementary Table Effect of intravenous injection of different rapols on mice health (3 mice per group; 1=no effect; 2=mobility reduced; 3=mobility reduced + fur bristling; 4=death).
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DOI:.38/ncb3399 a b c d FSP DAPI 5mm mm 5mm 5mm e Correspond to melanoma in-situ Figure a DCT FSP- f MITF mm mm MlanaA melanoma in-situ DCT 5mm FSP- mm mm mm mm mm g melanoma in-situ MITF MlanaA mm mm
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