TRYRAC-WP2 4th PMC meeting - July 2015 - PowerPoint Presentation

1. TRYRAC-WP2 4th PMC meeting - July 2015Institute of Tropical Medicine AntwerpVeterinary Protozoology Unit

2. 2WP2 Overview

3. Development of molecular tools to detect drug resistanceTransfer molecular tools and protocols to regional laboratories and control qualityIdentify drug quality control laboratories3WP2 Objectives

4. Focus on T.congolenseCurrently - two main drugs in use : Diminazene (DA) and Isometamidium-chloride (ISM)In vivo drug-resistance testing of field trypanosome strains: laborious; no large scale screening/monitoring; lab animal use; standardization. Molecular tool: exploiting genetic polymorphism that is linked to the drug-sensitivity phenotype; goal: ‘easy-to-use’ PCR-based assay  use as an ‘early’ warning systemfor DA: exists and has been validatedfor ISM: non-existing4WP2 Molecular tools to detect drug resistance

5. 5WP2 Molecular tools to detect DA-resistanceBased on the detection of a single nucleotide polymorphism (SNP) of the TcoNT10 gene, a paralogue of the T.brucei TbAT1/P2 transporter gene in T.brucei to be involved in the diamidine and arsenical drug uptake.Vitouleyh et al. (2011) PLoS NTD, 5, e1223PCR-Ade2 fragment (part of TcoNT10 gene) DpnII-PCR-RFLPSSSSSRM

6. However:6WP2 Molecular tools to detect DA-resistanceTcoAT1 = P1-type purine nucleoside transporter BUT does not mediate DA-uptake and is therefore unlikely to play a direct role in DA-resistance; Rename TcoAT1  TcoNT10 TcoNT10 SNP is not directly linked with the DA-resistance phenotype  more research needed on DA-transporters in T.congolense and resistance

7. 7WP2.1 Molecular tools to detect ISM-resistanceMolecular tool for Isometamidium-chloride resistance: does NOT exist  to be developed (possible?) Genetical basis / Molecular mechanism of ISM resistance?Molecular mechanism of ISM uptake (at cell membrane and mitochondrium membrane level) ?ISMISMISM

8. 8WP2.1 T.congolense ISM-resistanceISMISMISMNo mutation of genes coding for type II topoisomerases  No polymorphism related to ISM resistance observedInsertion of a conserved GAA codon in a gene coding for an ATP-binding cassette (ABC) transporter ?Not observed in all T congolense resistant strains? Involvement w/ ISM uptakeDecrease in the mitochondrial membrane potential (generated through the hydrolysis of ATP by the mitochondrial FO/F1 ATPase )  Reduced diffusion through the MM ? Cause of this decrease? Modification of transporter(s) located in the mitochondrial inner membrane? Decrease in drug uptake? Increase in drug extrusionV-ATPase(T.brucei; Baker et al., PNAS 2015)

9. WP2.1 T.congolense ISM-resistanceStill poorly characterizedMolecular basis of transport and accumulation of ISM, and acquisition of resistance are mainly unknownAs ISM has mutagenic properties, the use of the drug to target T. congolense infections induces genomic modifications that enable the parasite to survive and adapt in the presence of ISM, resulting in a drug resistant phenotypeMajor aim of WP 2.1 To identify genetic signatures associated with ISM resistance in T.congolense Development of an (‘easy-to-use’) molecular tool to determine the ISM-sensitivity status of a T.congolense field sample9

10. 10WP2.1: T.congolense ISM-resistanceISMISMISM1. Differential ‘omics approach:Genomic levelTranscript levelProtein levelMetabolomic level2. Functional experimental workLab-induced ISM resistance2. Field isolates from cattle with treatment failure + confirmed in vivo resistanceTRYRAC-VPU PhD student: Eliane Tihon; collaborative work with Molecular Parasitology Unit (ITM)

11. Strains selection Whole genome sequencing(MiSeq)SNPsIndelsPloidyCopy number variationsRecombination WP 2.1 Methodology – Whole Genome Sequencing11

12. WP 2.1 Starting material 56 field-isolated strains of T congolenseSavannah sub-group: 52 strainsForest sub-group: 4 strains* 19 Sensitive (S to 0,1 mg/kg) * 7 Intermediate * 30 Resistant (R to 1 mg/kg)Sampling period: 1971-2014 10 ≠ countries ISM-R induced strainsIL1180 (Tanzania)MSORO-M7 (Zambia)12 2 field-isolates from Togo

13. WP 2.1 T.congolense genetic diversity - Results Summary of the sequencing dataSNPs:Whole data set: 614 859 SNPsSavannah strains only: 411 781 SNPs 251 333 in coding regions and 209 933 were non-synonymous mutations. Small indels : Whole data set: coding: 3308; non-coding: 24 796Savannah only: coding: 1698; non-coding: 8982High genetic diversity among the samplesNo association found between SNPs/small indels and the ISM-S or R phenotype13

14. 14WP 2.1 T.congolense genetic diversity - Results SNP network – T. congolense SavannahWest/Central/East Africa(Northern Hemisphere)ZambiaZambia/Congo/Uganda/KenyaTogo samples

15. WP 2.1 T.congolense genetic diversity - Results SAVANNAHFORESTSNP network, phylogenic tree & structure analysis15The strains expose a clear division by regions High genetic diversity among Zambian strains

16. 16WP 2.1 T.congolense genetic diversity - Results

17. 17WP 2.1 T.congolense genetic diversity - Results Recombination and complex genetic exchanges seems to be frequent in T. congolense, especially when looking at the Zambian strains

18. No changes in PloidyLong deletions and duplications Small indels (deletions & insertions)1300 bpSmall insertionSmall deletionWP 2.1 T.congolense genetic diversity - Results 18No link between structural variations and ISM-S or R phenotype

19. The number of genome-wide SNPs identified in the whole population revealed a high genetic diversity among samplesPhylogenic and structure analysis highlighted a geographically asymmetric genetic diversity in T. congolense, underlying an extremely high diversity among the strains collected in Zambia compared to the rest of the samples.Recombination and complex genetic exchanges seems to be frequent in T. congolense, especially when looking at the Zambian strainsThe strains belonging to a same geographic area tend to share the same structural variationsNo associations found between genetic variations and the ISM-sensitive/resistant phenotype of the T.congolense populations WP 2.1 T.congolense genetic diversity - Results summary19

20. 20WP2.1 T.congolense genetic diversity versus ISM-resistanceHigh diversity in T. congolense natural populationNot possible to determine which mutations (SNPs, indels, CNV) are associated with the acquisition of ISM-resistance Induction of ISM resistance in vivo and sequencing of the genome of the Resistant clones + Intermediate resistant clones versus the Sensitive ‘mother’ strain to better understand the evolution of the acquisition of ISM resistanceThe induced resistant strains should enable us to better identify genetic signatures linked to ISM resistanceComparison between the results obtained for the in vivo induced clones and the field-isolated strains

21. WP 2.1 In vivo induced ISM Resistant clones - Methodology105 Tryp Infected mouseX 10ISM105 TrypIncreasing doses106,6 Tryp / mlISMSurvival & Proliferation of resistant parasites107,8 Tryp / mlDecrease in parasitaemia106,6 Tryp / ml21DNA extraction and genome analysis Two different induction‘tracks’: Without immune-suppression 2. With immune-suppression (+ Endoxan)

22. WP 2.1 In vivo induced ISM Resistant clones – Results 1Cycle 1ISM 1 : 0,001 mg/kgISM 2 : 0,005 mg/kgCycle 2ISM 1 : 0,005 mg/kgISM 2 : 0,01 mg/kg Cycle 3ISM 1 : 0,01 mg/kgISM 2 : 0,02 mg/kgCycle 4ISM 1 : 0,02 mg/kgISM 2 : 0,04 mg/kg Cycle 5ISM 1 : 0,04 mg/kgISM 2 : 0,08 mg/kg MSORO-M7-A1 (7/10/2013)MSORO-M7-A2(7/11/2013)MSORO-M7-A3 (0,01 mg/kg) passage to MSORO-M7-A3b (6/01/2014) MSORO-M7-A3b (0,01 mg/kg) passage to MSORO-M7-A3c (6/03/2014)MSORO-M7-A3c (cured) New stabilate: MSORO-M7 A3d (24/4/2014)MSORO-M7-A3D (30/06/2014)MSORO-M7-A4 (cured)MSORO-M7-B1(26/11/2013)MSORO-M7-B2 (cured)MSORO-M7-C1(26/11/2013)MSORO-M7-C2(15/01/2014)MSORO-M7-C3 (cured) New stabilate: MSORO-M7-C3b (13/3/2014)MSORO-M7-C3b (cured) New stabilate: MSORO-M7-C3c (12/6/2014)MSORO-M7-C3c (30/06/2014)MSORO-M7-C4 (0,02 mg/kg)passage to MSORO-M7-C4bPassage to MSORO-M7-C4c Passage to MSORO-M7-C4dPassage to MSORO-M7-C4ePassage to MSORO-M7-C4fMSORO-M7-D1(26/11/2013)MSORO-M7-D2(5/01/2014)MSORO-M7-D3 (cured) New stabilate: MSORO-M7-D3b (4/03/2014)MSORO-M7-D3b (17/04/2014)MSORO-M7-D4 (0,02 mg/kg) passage to MSORO-M7-D4b (13/06/2014)MSORO-M7-D4b (cured)MSORO-M7-E1(9/12/2013)MSORO-M7-E2(27/12/2013)MSORO-M7-E3(12/02/2014)MSORO-M7-E4 (7/04/2014)MSORO-M7-E5 (cured) New stabilate: MSORO-M7-E5b (13/6/2015)MSORO-M7-E5b (cured)22R: 80x; >18 months

23. WP 2.1 In vivo induced ISM Resistant clones – Results 2 (+ Endoxan)Cycle 1ISM 1 : 0,001 mg/kgISM 2 : 0,005 mg/kgCycle 2ISM 1 : 0,005 mg/kgISM 2 : 0,01 mg/kg Cycle 3ISM 1 : 0,01 mg/kgISM 2 : 0,02 mg/kgCycle 4ISM 1 : 0,02 mg/kgISM 2 : 0,04 mg/kg Cycle 5ISM 1 : 0,04 mg/kgISM 2 : 1 mg/kg MSORO-M7-F1 (12/09/2014)MSORO-M7-F2(30/09/2014)(cured)MSORO-M7-F2b(17/10/2014)(cured)MSORO-M7-G1(12/09/2014)MSORO-M7-G2(22/09/2014)MSORO-M7-G3(13/10/2014)MSORO-M7-G4(24/10/2014)DEADMSORO-M7-G4b(04/11/2014)DEADMSORO-M7-G4cMSORO-M7-G5(21/12/2014)MSORO-M7-H1(12/09/2014)MSORO-M7-H2(22/09/2014)MSORO-M7-H3(13/10/2014)MSORO-M7-H4(24/10/2014)DEADMSORO-M7-H4b(04/11/2014)MSORO-M7-H5(1/12/2014)MSORO-M7-I1(24/10/2014)MSORO-M7-I2(24/11/2014)MSORO-M7-I3(22/01/2015)(cured)MSORO-M7-I3b(28/01/2015)MSORO-M7-I4(14/02/2015)MSORO-M7-I5(11/03/2015)23R: 1000x; 3-6 months

24. ISM-induced clones Track 1 & 2; Sensitive – Intermediate – ResistantWhole genome sequencing(MiSeq)WP 2.1 In vivo induced ISM Resistant clones: WGS ongoing SNPsIndelsCopy number variations24 ? Genomic ‘hotspots’ that mutate under ISM-selection pressure  ISM-R ?

25. UP and CIRDES are already well-equiped and have the hands-on expertise to perform molecular identification through PCR. Tools to monitor DA-resistance are already installed in CIRDES and UP. 25WP2.2: Transfer of molecular tools and protocols to regional laboratories

26. 26WP2.2 Transfer of molecular tools and protocols to regional laboratories

27. 27WP2.2 Transfer of molecular tools and protocols to regional laboratories

28. 28WP 2.3 Drug quality control

29. 29WP2 Time frame

30. 30ITM Veterinary Protozoology Unit: Eliane TIHON (PhD student) Lieve VERMEIREN Vincent DELESPAUXITM Molecular Parasitology Unit Hideo IMAMURA Frederik VAN DEN BROECK Jean-Claude DUJARDINVetTOGO