Genome-wide Analysis for Variants in Philippine Trypanosoma evansi Isolates with Varying Drug Resistance Profiles
Jose Enrico H. Lazaro1§, Neil Andrew D. Bascos1,3§, Francis A. Tablizo3§,
Nancy S. Abes2, Renlyn Ivy DG. Paynaganan1, Michelle A. Miguel2,
Hector M. Espiritu2, Mary Rose D. Uy2, Claro N. Mingala2, and Cynthia P. Saloma1,3*
1National Institute of Molecular Biology and Biotechnology, College of Science,
University of the Philippines Diliman, Quezon City 1101 Philippines
2Philippine Carabao Center, Department of Agriculture,
Science City of Munoz, Nueva Ecija 3120 Philippines
3Philippine Genome Center, University of the Philippines, Diliman,
Quezon City 1101 Philippines
§These authors contributed equally to this work
*Corresponding Author: This email address is being protected from spambots. You need JavaScript enabled to view it.
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ABSTRACT
Surra, a parasitic disease transmitted by hematophagous flies and caused by Trypanosoma evansi, affects many domesticated animals – including water buffaloes, camels, horses, pigs, dogs, and other carnivores – throughout the world. When left untreated, this disease can cause anemia, significant loss of weight, abortion, and death in affected animals. Among Philippine isolates of T. evansi, variability has been reported in terms of virulence as well as response to drug treatment. In this study, trypanosoma-positive blood was obtained from 15 Philippine water buffalo samples from different sites in the country. The collected T. evansi strains were propagated in mice then subjected to in vivo virulence, in vitro drug sensitivity testing, and whole genome sequencing. One strain (O14) was found to be highly virulent in vivo, and was found to be resistant to three commonly used drugs [i.e., isometamidium chloride (IC), diminazene diaceturate (DD), and melarsamine hydrochloride (CY for Cymelarsan®)] in vitro. This highly resistant sample was compared with two less-virulent strains using genome-wide analysis of single nucleotide polymorphisms (SNPs) and short insertions and deletions (indels) relative to the reference strain STIB 805. Variant analysis between O14 and the less virulent strains (M4 and C117) identified a number of distinctive SNPs, many of which corroborate previous data. Genes with relatively high copy numbers were observed in mutation hotspots. These included genes that code for variant surface glycoproteins (VSGs), expression site-associated genes (ESAGs), retrotransposon hot spot (RHS) proteins, and leucine rich repeat proteins. Notable mutations were also predicted from genes coding for membrane transporters and cysteine peptidases, as well as those involved in RNA degradation. The whole genome sequences acquired from the Philippine isolates (O14, M4, and C117) vary greatly from the reference strain (STIB 805). These WGS data serve as a good resource for the discovery of genetic and phenotypic features that may be translated to effective treatment strategies, relevant to the Philippine setting.
INTRODUCTION
Blood parasitic diseases caused by vector-borne protozoa gravely affect livestock production in many developing countries. Surra, a parasitic disease caused by Trypanosoma evansi, affects many domesticated animal species including buffaloes, camels, and horses in Asia, Africa, and Central and South America. Surra is transmitted by hematophagous flies (e.g., Tabanids and Stomoxys) that act as mechanical vectors of the disease. Infected individuals exhibit highly variable clinical effects, depending on the host and their geographical area (Desquesnes et al. 2013). . . . read more
REFERENCES
ALTSCHUL SF, GISH W, MILLER W, MYERS EW, LIPMAN DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215(3): 403–410.
BALTZ T, BALTZ D, GIROUD C, CROCKETT J. 1985. Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense, and T. gambiense. The EMBO Journal 4(5): 1273–1277.
BERRIMAN M, GHEDIN E, HERTZ-FOWLER C, BLANDIN G, RENAULD H, BARTHOLOMEU DC, LENNARD NJ, CALER E, HAMLIN NE, HAAS B, BÖHME U, HANNICK L, ASLETT MA, SHALLOM J, MARCELLO L, HOU L, WICKSTEAD B, ALSMARK UC, ARROWSMITH C, ATKIN RJ, BARRON AJ, BRINGAUD F, BROOKS K, CARRINGTON M, CHEREVACH I, CHILLINGWORTH TJ, CHURCHER C, CLARK LN, CORTON CH, CRONIN A, DAVIES RM, DOGGETT J, DJIKENG A, FELDBLYUM T, FIELD MC, FRASER A, GOODHEAD I, HANCE Z, HARPER D, HARRIS BR, HAUSER H, HOSTETLER J, IVENS A, JAGELS K, JOHNSON D, JOHNSON J, JONES K, KERHORNOU AX, KOO H, LARKE N, LANDFEAR S, LARKIN C, LEECH V, LINE A, LORD A, MACLEOD A, MOONEY PJ, MOULE S, MARTIN DM, MORGAN GW, MUNGALL K, NORBERTCZAK H, ORMOND D, PAI G, PEACOCK CS, PETERSON J, QUAIL MA, RABBINOWITSCH E, RAJANDREAM MA, REITTER C, SALZBERG SL, SANDERS M, SCHOBEL S, SHARP S, SIMMONDS M, SIMPSON AJ, TALLON L, TURNER CM, TAIT A, TIVEY AR, VAN AKEN S, WALKER D, WANLESS D, WANG S, WHITE B, WHITE O, WHITEHEAD S, WOODWARD J, WORTMAN J, ADAMS MD, EMBLEY TM, GULL K, ULLU E, BARRY JD, FAIRLAMB AH, OPPERDOES F, BARRELL BG, DONELSON JE, HALL N, FRASER CM, MELVILLE SE, EL-SAYED NM. 2005. The Genome of the African Trypanosome Trypanosoma brucei. Supplementary Information. Science 309(5733): 416.
BIRHANU H, GEBREHIWOT T, GODDEERIS BM, BÜSCHER P, VAN REET N. 2016. New Trypanosoma evansi Type B Isolates from Ethiopian Dromedary Camels. PLoS Neglected Tropical Diseases10(4):e0004556.
BRANQUINHA MH, MARINHO FA, SANGENITO LS, OLIVEIRA SS, GONCALVES KC, ENNES-VIDAL V, D'AVILA-LEVY CM, SANTOS AL. 2013. Calpains: Potential Targets for Alternative Chemotherapeutic Intervention Against Human Pathogenic Trypanosomatids. Current Medicinal Chemistry 20 (25): 3174–3185.
BUCHFINK B, XIE C, HUSON DH. 2015. Fast and Sensitive Protein Alignment Using DIAMOND. Nature Methods 12: 59–60.
BULUS T, AHMED AB, ABOI TY, DANBAKI DA. 2016. Determination of IC50 and IC90 values of ethanolic extracts of some medicinal plants against Trypanosoma brucei brucei. Archives of Clinical Microbiology 7: 3.
CAMPOS MC, CASTRO-PINTO DB, RIBEIRO GA, BERREDO-PINHO MM, GOMES LH, DA SILVA BELLIENY MS, GOULART CM, ECHEVARRIA A, LEON LL. 2013. P-Glycoprotein Efflux Pump Plays an Important Role in Trypanosoma cruzi Drug Resistance. Parasitology Research 112(6): 2341–2351.
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, KOŘENÝ L, LAI DH, MACLEOD A, MCDERMOTT SM, MERRITT C, MONNERAT S, MOON W, MYLER P, PHAN I, RAMASAMY G, SIVAM D, LUN ZR, LUKEŠ J, STUART K, SCHNAUFER A. 2015.Genome and Phylogenetic Analyses of Trypanosoma Evansi Reveal Extensive Similarity to T. brucei and Multiple Independent Origins for Dyskinetoplasty. PLoS Neglected Tropical Diseases 9(1): e3404.
CINGOLANI P, PLATTS A, WANG LE L, COON M, NGUYEN T, WANG L, LAND SJ, LU X, RUDEN DM. 2012. 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 6(2): 80–92.
CONESA A, GÖTZ S. 2008. Blast2GO: A Comprehensive Suite for Functional Analysis in Plant Genomics.International Journal of Plant Genomics 2008: 1–12.
DANIELS JP, GULL K, WICKSTEAD B. 2010. Cell Biology of the Trypanosome Genome.Microbiology and Molecular Biology Reviews 74(4): 552–69.
DARGANTES AP, MERCADO RT, DOBSON RJ, REID SA.2009. Estimating the impact of Trypanosoma evansi infection (surra) in buffalo population dynamics in southern Philippines using data from cross-sectional surveys. International Journal of Parasitology 39: 1109–1114.
DESQUESNES M, DARGANTES A, LAI DH, LUN ZR, HOLZMULLER P, JITTAPALAPONG S. 2013. Trypanosoma evansi and surra: A review and perspectives on transmission, epidemiology and control, impact, and zoonotic aspects. Biomed Res Int. 2013: 321237.
DOBSON RJ1, DARGANTES AP, MERCADO RT, REID SA. 2009. Models for Trypanosoma evansi (surra), its control and economic impact on smallhold livestock owners in the Philippines. International Journal of Parasitology 39: 1115–1123.
FIGIEL M, CHON H, CERRITELLI SM, CYBULSKA M, CROUCH RJ, NOWOTNY M. 2011. J Biol Chem 286:10540–10550.
FLORINI F, NAGULESWARAN A, GHARIB WH, BRINGAUD F, RODITI I. 2018. Unexpected Diversity in Eukaryotic Transcription Revealed by the Retrotransposon Hotspot Family of Trypanosoma brucei. Nucleic Acids Research 47(4): 1725–1739.
GRAF FE, LUDIN P, ARQUINT C, SCHMIDT RS, SCHAUB N, KUNZ RENGGLI C, MUNDAY JC, KREZDORN J, BAKER N, HORN D, BALMER O, CACCONE A, DE KONING HP, MÄSER P. 2016. Comparative Genomics of Drug Resistance in Trypanosoma brucei Rhodesiense. Cellular and Molecular Life Sciences 73 (17): 3387–3400.
GREWAL JS, MCLUSKEY K, DAS D, MYBURGH E, WILKES J, BROWN E, LEMGRUBER L, GOULD MK, BURCHMORE RJ, COOMBS GH, SCHNAUFER A, MOTTRAM JC. 2016. PNT1 Is a C11 Cysteine Peptidase Essential for Replication of the Trypanosome Kinetoplast. Journal of Biological Chemistry 291(18): 9492–9500.
HABILA N, INUWA MH, AIMOLA IA, UDEH MU, HARUNA E. 2012. Pathogenic Mechanisms of Trypanosoma evansi Infections. Research in Veterinary Science 93(1): 13–17.
HELMS MJ. 2006. Bloodstream Form Trypanosoma brucei Depend upon Multiple Metacaspases Associated with RAB11-Positive Endosomes. Journal of Cell Science 119(6): 1105–1117.
KUMAR R, SINGH J, SINGH R, KUMAR S, YADAV SC. 2015. Comparative efficacy of different in vitro cultivation media for Trypanosoma evansi isolated from different mammalian hosts inhabiting different geographical areas of India. Journal of Parasitic Diseases 39(2): 174–178.
KÜNDIG C, HAIMEUR A, LÉGARÉ D, PAPADOPOULOU B, OUELLETTE M. 1999. Increased Transport of Pteridines Compensates for Mutations in the High Affinity Folate Transporter and Contributes to Methotrexate Resistance in the Protozoan Parasite Leishmania Tarentolae. The EMBO Journal 18(9): 2342–2351.
KURIAKOSE S, MULEME HM, ONYILAGHA C, SINGH R, JIA P, UZONNA JE. 2012. Diminazene Aceturate (Berenil) Modulates the Host Cellular and Inflammatory Responses to Trypanosoma congolense Infection. PLoS ONE 7(11): e48696.
LI H, DURBIN R. 2010. Fast and Accurate Long-Read Alignment with Burrows-Wheeler Transform. Bioinformatics 26(5): 589–595.
LUCKINS AG. 1988. Trypanosoma evansi in Asia. Trends in Parasitology 4: 137–142.
MANUEL MF. 1998. Sporadic outbreaks of surra in the Philippines and its economic impact. Journal of Protozoology Research 8: 131–138.
MARTIN T, PARKER SE, HEDSTROM R, LE T, HOFFMAN S, NORMAN J, HOBART P, LEW D. 2004. Human Gene Therapy 10(5): 759–768.
MUNGUBE EO, VITOULEY HS, ALLEGYE-CUDJOE E, DIALL O, BOUCOUM Z, DIARRA B, SANOGO Y, RANDOLPH T, BAUER B, ZESSIN KH, CLAUSEN PH. 2012. Detection of multiple drug-resistant Trypanosoma congolense populations in village cattle of south-east Mali. Parasit Vectors. 5: 155.
MCKENNA A, HANNA M, BANKS E, SIVACHENKO A, CIBULSKIS K, KERNYTSKY A, GARIMELLA K, ALTSHULER D, GABRIEL S, DALY M, DEPRISTO MA. 2010. The Genome Analysis Toolkit: A MapReduce Framework for Analyzing Next-Generation DNA Sequencing Data. Genome Research 20: 1297–1303.
NIKOLSKAIA OV, DE A LIMA AP, KIM YV, LONSDALE-ECCLES JD, FUKUMA T, SCHARFSTEIN J, GRAB DJ. 2008. Blood-Brain Barrier Traversal by African Trypanosomes Requires Calcium Signaling Induced by Parasite Cysteine Protease. Journal of Clinical Investigation 118(5): 1974–1974.
PROTO WR, CASTANYS-MUNOZ E, BLACK A, TETLEY L, MOSS C, JULIANO L, COOMBS G, MOTTRAM J. 2011. Trypanosoma brucei Metacaspase 4 is a pseudopeptidase and Virulence Factor. Journal of Biological Chemistry 286(46): 39914–39925.
REID SA. 2002. Trypanosoma evansi control and containment in Australasia. Trends in Parasitology 18: 219–224.
ROY N, NAGESHAN RK, PALLAVI R, CHAKRAVARTHY H, CHANDRAN S, KUMAR R, GUPTA AK, SINGH RK, YADAV SC, TATU U. 2010. Proteomics of Trypanosoma evansi Infection in Rodents. PLoS ONE 5(3): e9796.
SANTOS CC, COOMBS GH, LIMA AP, MOTTRAM JC. 2007. Role of the Trypanosoma brucei Natural Cysteine Peptidase Inhibitor ICP in Differentiation and Virulence. Molecular Microbiology 66(4): 991–1002.
STOKES MJ, GÜTHER ML, TURNOCK DC, PRESCOTT AR, MARTIN KL, ALPHEY MS, FERGUSON MA. 2008. The Synthesis of UDP- N -Acetylglucosamine Is Essential for Bloodstream Form Trypanosoma brucei In Vitro and In Vivo and UDP- N -Acetylglucosamine Starvation Reveals a Hierarchy in Parasite Protein Glycosylation. Journal of Biological Chemistry 283(23): 16147–16161.
TAVARES KC, DA SILVA AS, WOLKMER P, MONTEIRO SG, MILETTI LC. 2011. Cryopreservation of Trypanosoma evansi after DEAE-cellulose purification: Evaluation of infective parameters. Research in Veterinary Science 90(2): 257–259.
TIHON E, IMAMURA H, VAN DEN BROECK F, VERMEIREN L, DUJARDIN JC, VAN DEN ABBEELE J. 2017. Genomic Analysis of Isometamidium Chloride Resistance in Trypanosoma congolense. International Journal for Parasitology: Drugs and Drug Resistance 7(3): 350–361.
TROEBERG L, MORTY RE, PIKE RN, LONSDALE-ECCLES JD, PALMER JT, MCKERROW JH, COETZER TH. 1999. Cysteine Proteinase Inhibitors Kill Cultured Bloodstream Forms of Trypanosoma brucei brucei. Experimental Parasitology 91(4): 349–355.
URBANIAK MD, COLLIE IT, FANG W, ARISTOTELOUS T, ESKILSSON S, RAIMI OG, HARRISON J, NAVRATILOVA IH, FREARSON JA, VAN AALTEN DM, FERGUSON MA. 2013. A Novel Allosteric Inhibitor of the Uridine Diphosphate N-Acetylglucosamine Pyrophosphorylase from Trypanosoma brucei. ACS Chemical Biology 8(9): 1981–1987.
WITTUNG-STAFSHEDE P. 2002. Role of Cofactors in Protein Folding. Accounts of Chemical Research 35. 201–208.
ZHOU J, LE V, KALIA D, NAKAYAMA S, MIKEK C, LEWIS EA, SINTIM HO. 2014. Diminazene or Berenil, a Classic Duplex Minor Groove Binder, Binds to G-Quadruplexes with Low Nanomolar Dissociation Constants and the Amidine Groups Are Also Critical for G-Quadruplex Binding. Molecular BioSystems 10(10): 2724–2734.
ZOLTNER M, HORN D, DE KONING HP, FIELD MC. 2016. Exploiting the Achilles’ Heel of Membrane Trafficking in Trypanosomes. Current Opinion in Microbiology 34: 97–103.
