Suramin is one of the oldest drugs in use today. It is still the treatment of choice for the hemolymphatic stage of African sleeping sickness caused by Trypanosoma brucei rhodesiense, and it is also used for surra in camels caused by Trypanosoma evansi. Yet despite one hundred years of use, suramin's mode of action is not fully understood. Suramin is a polypharmacological molecule that inhibits diverse proteins. Here we demonstrate that a DNA helicase of the pontin/ruvB-like 1 family, termed T. brucei RuvBL1, is involved in suramin resistance in African trypanosomes. Bloodstream-form T. b. rhodesiense under long-term selection for suramin resistance acquired a homozygous point mutation, isoleucine-312 to valine, close to the ATP binding site of T. brucei RuvBL1. The introduction of this missense mutation, by reverse genetics, into drug-sensitive trypanosomes significantly decreased their sensitivity to suramin. Intriguingly, the corresponding residue of T. evansi RuvBL1 was found mutated in a suramin-resistant field isolate, in that case to a leucine. RuvBL1 (Tb927.4.1270) is predicted to build a heterohexameric complex with RuvBL2 (Tb927.4.2000). RNAi-mediated silencing of gene expression of either T. brucei RuvBL1 or RuvBL2 caused cell death within 72 h. At 36 h after induction of RNAi, bloodstream-form trypanosomes exhibited a cytokinesis defect resulting in the accumulation of cells with two nuclei and two or more kinetoplasts. Taken together, these data indicate that RuvBL1 DNA helicase is involved in suramin action in African trypanosomes.

Department of Parasitology Faculty of Science Charles University Prague Biocev Vestec Czech Republic

Swiss Tropical and Public Health Institute Kreuzstrasse 2 4123 Allschwil Switzerland; University of Basel Petersplatz 1 4001 Basel Switzerland

Zobrazit více v PubMed

Abrahao J., Amaro B.T., Peres B.R., Quel N.G., Aragao A.Z.B., Morea E.G.O., Cano M.I.N., Houry W.A., Ramos C.H.I. Leishmania major RUVBL1 has a hexameric conformation in solution and, in the presence of RUVBL2, forms a heterodimer with ATPase activity. Arch. Biochem. Biophys. 2021;703 PubMed

Alsford S., Eckert S., Baker N., Glover L., Sanchez-Flores A., Leung K.F., Turner D.J., Field M.C., Berriman M., Horn D. High-throughput decoding of antitrypanosomal drug efficacy and resistance. Nature. 2012;482:232–236. PubMed PMC

Alsford S., Kawahara T., Glover L., Horn D. Tagging a T. brucei RRNA locus improves stable transfection efficiency and circumvents inducible expression position effects. Mol. Biochem. Parasitol. 2005;144:142–148. PubMed PMC

Alsford S., Turner D.J., Obado S.O., Sanchez-Flores A., Glover L., Berriman M., Hertz-Fowler C., Horn D. High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res. 2011;21:915–924. PubMed PMC

Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. Basic local alignment search tool. J. Mol. Biol. 1990;215:403–410. PubMed

Aslett M., Aurrecoechea C., Berriman M., Brestelli J., Brunk B.P., Carrington M., Depledge D.P., Fischer S., Gajria B., Gao X., Gardner M.J., Gingle A., Grant G., Harb O.S., Heiges M., Hertz-Fowler C., Houston R., Innamorato F., Iodice J., Kissinger J.C., Kraemer E., Li W., Logan F.J., Miller J.A., Mitra S., Myler P.J., Nayak V., Pennington C., Phan I., Pinney D.F., Ramasamy G., Rogers M.B., Roos D.S., Ross C., Sivam D., Smith D.F., Srinivasamoorthy G., Stoeckert C.J., Jr., Subramanian S., Thibodeau R., Tivey A., Treatman C., Velarde G., Wang H. TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res. 2010;38:D457–D462. PubMed PMC

Basavannacharya C., Vasudevan S.G. Suramin inhibits helicase activity of NS3 protein of dengue virus in a fluorescence-based high throughput assay format. Biochem. Biophys. Res. Commun. 2014;453:539–544. PubMed

Carnes J., Anupama A., Balmer O., Jackson A., Lewis M., Brown R., Cestari I., Desquesnes M., Gendrin C., Hertz-Fowler C., Imamura H., Ivens A., Koreny L., Lai D.H., MacLeod A., McDermott S.M., Merritt C., Monnerat S., Moon W., Myler P., Phan I., Ramasamy G., Sivam D., Lun Z.R., Lukes J., Stuart K., Schnaufer A. Genome and phylogenetic analyses of trypanosoma evansi reveal extensive similarity to T. Brucei and multiple independent origins for dyskinetoplasty. PLoS Neglected Trop. Dis. 2015;9 PubMed PMC

Cingolani P., Platts A., Wang le L., Coon M., Nguyen T., Wang L., Land S.J., Lu X., Ruden D.M. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly. 2012;6:80–92. PubMed PMC

Dauden M.I., Lopez-Perrote A., Llorca O. RUVBL1-RUVBL2 AAA-ATPase: a versatile scaffold for multiple complexes and functions. Curr. Opin. Struct. Biol. 2021;67:78–85. PubMed

Desquesnes M., Bossard G., Patrel D., Herder S., Patout O., Lepetitcolin E., Thevenon S., Berthier D., Pavlovic D., Brugidou R., Jacquiet P., Schelcher F., Faye B., Touratier L., Cuny G. First outbreak of Trypanosoma evansi in camels in metropolitan France. Vet. Rec. 2008;162:750–752. PubMed

Desquesnes M., Dargantes A., Lai D.H., Lun Z.R., Holzmuller P., Jittapalapong S. Trypanosoma evansi and surra: a review and perspectives on transmission, epidemiology and control, impact, and zoonotic aspects. BioMed Res. Int. 2013;2013 PubMed PMC

Dickie E.A., Giordani F., Gould M.K., Maser P., Burri C., Mottram J.C., Rao S.P.S., Barrett M.P. New drugs for human african trypanosomiasis: a twenty first century success story. Trav. Med. Infect. Dis. 2020;5 PubMed PMC

Edgar R.C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32:1792–1797. PubMed PMC

El Rayah I.E., Kaminsky R., Schmid C., El Malik K.H. Drug resistance in Sudanese Trypanosoma evansi. Vet. Parasitol. 1999;80:281–287. PubMed

Erzberger J.P., Berger J.M. Evolutionary relationships and structural mechanisms of AAA+ proteins. Annu. Rev. Biophys. Biomol. Struct. 2006;35:93–114. PubMed

Fairlamb A.H., Bowman I.B. Trypanosoma brucei: suramin and other trypanocidal compounds' effects on sn-glycerol-3-phosphate oxidase. Exp. Parasitol. 1977;43:353–361. PubMed

Fairlamb A.H., Bowman I.B. Uptake of the trypanocidal drug suramin by bloodstream forms of Trypanosoma brucei and its effect on respiration and growth rate in vivo. Mol. Biochem. Parasitol. 1980;1:315–333. PubMed

Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985;39:783–791. PubMed

Franco J.R., Cecchi G., Priotto G., Paone M., Diarra A., Grout L., Simarro P.P., Zhao W., Argaw D. Monitoring the elimination of human African trypanosomiasis at continental and country level: update to 2018. PLoS Neglected Trop. Dis. 2020;14 PubMed PMC

Gerhold C.B., Gasser S.M. INO80 and SWR complexes: relating structure to function in chromatin remodeling. Trends Cell Biol. 2014;24:619–631. PubMed

Giordani F., Morrison L.J., Rowan T.G., Hp D.E.K., Barrett M.P. The animal trypanosomiases and their chemotherapy: a review. Parasitology. 2016;143:1862–1889. PubMed PMC

Hanson P.I., Whiteheart S.W. AAA+ proteins: have engine, will work. Nat. Rev. Mol. Cell Biol. 2005;6:519–529. PubMed

Hawking F. Suramin: with special reference to onchocerciasis. Adv. Pharmacol. Chemother. 1978;15:289–322. PubMed

Hertz-Fowler C., Figueiredo L.M., Quail M.A., Becker M., Jackson A., Bason N., Brooks K., Churcher C., Fahkro S., Goodhead I., Heath P., Kartvelishvili M., Mungall K., Harris D., Hauser H., Sanders M., Saunders D., Seeger K., Sharp S., Taylor J.E., Walker D., White B., Young R., Cross G.A., Rudenko G., Barry J.D., Louis E.J., Berriman M. Telomeric expression sites are highly conserved in Trypanosoma brucei. PLoS One. 2008;3 PubMed PMC

Hirumi H., Hirumi K. Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers. J. Parasitol. 1989;75:985–989. PubMed

Iwasaki H., Takahagi M., Nakata A., Shinagawa H. Escherichia coli RuvA and RuvB proteins specifically interact with Holliday junctions and promote branch migration. Genes Dev. 1992;6:2214–2220. PubMed

Jha S., Dutta A. RVB1/RVB2: running rings around molecular biology. Mol Cell. 2009;34:521–533. PubMed PMC

Jones D.T., Taylor W.R., Thornton J.M. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 1992;8:275–282. PubMed

Kelley L.A., Mezulis S., Yates C.M., Wass M.N., Sternberg M.J. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 2015;10:845–858. PubMed PMC

Khurana J., Shrivastava A., Singh A., Gupta A. Exploring potential of Plasmodium RUVBL proteins as anti-malarial drug target. J. Biomol. Struct. Dyn. 2021:1–17. PubMed

Kumar S., Stecher G., Li M., Knyaz C., Tamura K. Mega X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 2018;35:1547–1549. PubMed PMC

Lai D.H., Hashimi H., Lun Z.R., Ayala F.J., Lukes J. Adaptations of Trypanosoma brucei to gradual loss of kinetoplast DNA: Trypanosoma equiperdum and Trypanosoma evansi are petite mutants of T. brucei. Proc. Natl. Acad. Sci. U. S. A. 2008;105:1999–2004. PubMed PMC

Lanham S., Godfrey D. Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Exp. Parasitol. 1970;28:521–534. PubMed

Li H., Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25:1754–1760. PubMed PMC

Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R., Subgroup G.P.D.P. The sequence alignment/map format and SAMtools. Bioinforma Oxf Engl. 2009;25:2078–2079. PubMed PMC

Matias P.M., Gorynia S., Donner P., Carrondo M.A. Crystal structure of the human AAA+ protein RuvBL1. J. Biol. Chem. 2006;281:38918–38929. PubMed

McKenna A., Hanna M., Banks E., Sivachenko A., Cibulskis K., Kernytsky A., Garimella K., Altshuler D., Gabriel S., Daly M., DePristo M.A. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20:1297–1303. PubMed PMC

Morty R.E., Troeberg L., Pike R.N., Jones R., Nickel P., Lonsdale-Eccles J.D., Coetzer T.H. A trypanosome oligopeptidase as a target for the trypanocidal agents pentamidine, diminazene and suramin. FEBS Lett. 1998;433:251–256. PubMed

Mukherjee S., Hanson A.M., Shadrick W.R., Ndjomou J., Sweeney N.L., Hernandez J.J., Bartczak D., Li K., Frankowski K.J., Heck J.A., Arnold L.A., Schoenen F.J., Frick D.N. Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays. Nucleic Acids Res. 2012;40:8607–8621. PubMed PMC

Murakami M.T., Arruda E.Z., Melo P.A., Martinez A.B., Calil-Elias S., Tomaz M.A., Lomonte B., Gutierrez J.M., Arni R.K. Inhibition of myotoxic activity of Bothrops asper myotoxin II by the anti-trypanosomal drug suramin. J. Mol. Biol. 2005;350:416–426. PubMed

Naviaux R.K., Curtis B., Li K., Naviaux J.C., Bright A.T., Reiner G.E., Westerfield M., Goh S., Alaynick W.A., Wang L., Capparelli E.V., Adams C., Sun J., Jain S., He F., Arellano D.A., Mash L.E., Chukoskie L., Lincoln A., Townsend J. Low-dose suramin in autism spectrum disorder: a small, phase I/II, randomized clinical trial. Ann Clin Transl Neurol. 2017;4:491–505. PubMed PMC

Räz B., Iten M., Grether-Bühler Y., Kaminsky R., Brun R. The Alamar Blue assay to determine drug sensitivity of African trypanosomes in vitro. Acta Trop. 1997;68:139–147. PubMed

Redmond S., Vadivelu J., Field M.C. RNAit: an automated web-based tool for the selection of RNAi targets in Trypanosoma brucei. Mol. Biochem. Parasitol. 2003;128:115–118. PubMed

Robinson J.T., Thorvaldsdottir H., Winckler W., Guttman M., Lander E.S., Getz G., Mesirov J.P. Integrative genomics viewer. Nat. Biotechnol. 2011;29:24–26. PubMed PMC

Saitou N., Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987;4:406–425. PubMed

Schrödinger L., DeLano W. 2020. PyMOL.

Schumann Burkard G., Jutzi P., Roditi I. Genome-wide RNAi screens in bloodstream form trypanosomes identify drug transporters. Mol. Biochem. Parasitol. 2011;175:91–94. PubMed

Shinagawa H., Shiba T., Iwasaki H., Makino K., Takahagi M., Nakata A. Properties of the Escherichia coli RuvA and RuvB proteins involved in DNA repair, recombination and mutagenesis. Biochimie. 1991;73:505–507. PubMed

Sievers F., Wilm A., Dineen D., Gibson T.J., Karplus K., Li W., Lopez R., McWilliam H., Remmert M., Soding J., Thompson J.D., Higgins D.G. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 2011;7:539. PubMed PMC

Tamarit A., Gutierrez C., Arroyo R., Jimenez V., Zagala G., Bosch I., Sirvent J., Alberola J., Alonso I., Caballero C. Trypanosoma evansi infection in mainland Spain. Vet. Parasitol. 2010;167:74–76. PubMed

Tammana D., Tammana T.V.S. Human DNA helicase, RuvBL1 and its Chlamydomonas homologue, CrRuvBL1 plays an important role in ciliogenesis. Cytoskeleton (Hoboken) 2017;74:251–259. PubMed

Thomas J.A., Baker N., Hutchinson S., Dominicus C., Trenaman A., Glover L., Alsford S., Horn D. Insights into antitrypanosomal drug mode-of-action from cytology-based profiling. PLoS Neglected Trop. Dis. 2018;12 PubMed PMC

Thorvaldsdottir H., Robinson J.T., Mesirov J.P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Briefings Bioinf. 2013;14:178–192. PubMed PMC

Vansterkenburg E.L.M., Coppens I., Wilting J., Bos O.J.M., Fischer M.J.E., Janssen L.H.M., Opperdoes F.R. The uptake of the trypanocidal drug suramin in combination with low-density lipoproteins by Trypanosoma brucei and its possible mode of action. Acta Trop. 1993;54:237–250. PubMed

Vellmer T., Hartleb L., Fradera Sola A., Kramer S., Meyer-Natus E., Butter F., Janzen C.J. A novel SNF2 ATPase complex in Trypanosoma brucei with a role in H2A.Z-mediated chromatin remodelling. PLoS Pathog. 2022;18 PubMed PMC

Voogd T.E., Vansterkenburg E.L., Wilting J., Janssen L.H. Recent research on the biological activity of suramin. Pharmacol. Rev. 1993;45:177–203. PubMed

Waterhouse A.M., Procter J.B., Martin D.M., Clamp M., Barton G.J. Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25:1189–1191. PubMed PMC

Wendler P., Ciniawsky S., Kock M., Kube S. Structure and function of the AAA+ nucleotide binding pocket. Biochim. Biophys. Acta. 2012;1823:2–14. PubMed

Wiedemar N., Graf F.E., Zwyer M., Ndomba E., Kunz Renggli C., Cal M., Schmidt R.S., Wenzler T., Mäser P. Beyond immune escape: a variant surface glycoprotein causes suramin resistance in Trypanosoma brucei. Mol. Microbiol. 2018;107:57–67. PubMed

Wiedemar N., Hauser D.A., Mäser P. 100 Years of suramin. Antimicrob. Agents Chemother. 2020;64 PubMed PMC

Wiedemar N., Zwyer M., Zoltner M., Cal M., Field M.C., Maser P. Expression of a specific variant surface glycoprotein has a major impact on suramin sensitivity and endocytosis in Trypanosoma brucei. FASEB Bioadv. 2019;1:595–608. PubMed PMC

Willhoft O., Ghoneim M., Lin C.L., Chua E.Y.D., Wilkinson M., Chaban Y., Ayala R., McCormack E.A., Ocloo L., Rueda D.S., Wigley D.B. Structure and dynamics of the yeast SWR1-nucleosome complex. Science. 2018;362 PubMed

Willson M., Callens M., Kuntz D.A., Perie J., Opperdoes F.R. Synthesis and activity of inhibitors highly specific for the glycolytic enzymes from Trypanosoma brucei. Mol. Biochem. Parasitol. 1993;59:201–210. PubMed

Wyatt H.D., West S.C. Holliday junction resolvases. Cold Spring Harbor Perspect. Biol. 2014;6:a023192. PubMed PMC

Yamada K., Kunishima N., Mayanagi K., Ohnishi T., Nishino T., Iwasaki H., Shinagawa H., Morikawa K. Crystal structure of the Holliday junction migration motor protein RuvB from Thermus thermophilus HB8. Proc. Natl. Acad. Sci. U. S. A. 2001;98:1442–1447. PubMed PMC

Zeelen J., van Straaten M., Verdi J., Hempelmann A., Hashemi H., Perez K., Jeffrey P.D., Halg S., Wiedemar N., Mäser P., Papavasiliou F.N., Stebbins C.E. Structure of trypanosome coat protein VSGsur and function in suramin resistance. Nat Microbiol. 2021;6:392–400. PubMed PMC

Zeng J., Weissmann F., Bertolin A.P., Posse V., Canal B., Ulferts R., Wu M., Harvey R., Hussain S., Milligan J.C., Roustan C., Borg A., McCoy L., Drury L.S., Kjaer S., McCauley J., Howell M., Beale R., Diffley J.F.X. Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp13 helicase. Biochem. J. 2021;478:2405–2423. PubMed PMC

Zhou J., Shen J., Liao D., Zhou Y., Lin J. Resistance to drug by different isolates Trypanosoma evansi in China. Acta Trop. 2004;90:271–275. PubMed

Zimmermann S., Hall L., Riley S., Sorensen J., Amaro R.E., Schnaufer A. A novel high-throughput activity assay for the Trypanosoma brucei editosome enzyme REL1 and other RNA ligases. Nucleic Acids Res. 2016;44:e24. PubMed PMC