Trypanosomatids Methods and Protocols

This volume explores the latest methods used by researchers to study different trypanosomatid parasites. These methods cover numerous disciplines, from organismal biology to molecular mechanism. The chapters in this book cover topics such as high-throughput sequencing; next-generation analysis of trypanosomatid genome stability and instability; DNA repair in cell extracts; ribosome profiling; and the use of CRISPR/Cas9 technology for gene editing. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and practical, Trypanosomatids: Methods and Protocols is a valuable resource for any researcher working with trypanosomatids and trypanosomatid-borne diseases. 

Chapters 14, 15, 16, 23, 24, 30, and 48 are open access under a CC BY 4.0 license.

Preface…
Table of Contents…
Contributing Authors…

Part I Isolation, Culturing, and Genetic Manipulation of Trypanosomatids

1. Field Isolation and Cultivation of Trypanosomatids from Insects 
Julius Lukeš and Jan Votýpka

2. Culturing and Transfection of Pleomorphic Trypanosoma brucei
Sabine Bachmaier, Theresa Thanner, and Michael Boshart

3. In Vitro Culture for Differentiation Simulation of Leishmania spp.
Dan Zilberstein

4. Tsetse Fly Transmission Studies of African Trypanosomes
Lori Peacock and Wendy Gibson

5. Infecting Triatomines with Trypanosomes
Alessandra A. Guarneri

Part II Trypanosomatid Genome-Wide Analyses

6. High-Throughput Sequencing for Trypanosome Transcriptome Characterization
Julius Mulindwa, Kevin Leiss, and Christine Clayton

7. Polysome Profiling and Metabolic Labelling Methods to Measure Translation in Trypanosoma brucei 
Kathrin Bajak and Christine Clayton

8. Immunoprecipitation for the Analysis of Macromolecular Complexes in Trypanosoma cruzi
Bruno A. A. Romagnoli, Samuel Goldenberg, and Lysangela R. Alves

9. Analysis of the In Vivo Translation Process in Trypanosoma cruzi using Ribosome Profiling 
Saloe B. Poubel, Fabiola B. Holetz, Bruno Accioly Alves Romagnoli, Samuel Goldenberg, and Lysangela R. Alves

10. Proteome-Wide Quantitative Phosphoproteomic Analysis of Trypanosoma brucei Insect and Mammalian Lifecycle Stages
Corinna Benz and Michael D. Urbaniak 

11. Genome-Wide Proteomics and Phosphoproteomics Analysis of Trypanosoma cruzi during Differentiation
Michel Batista, Juliana Carolina Amorim, Aline Castro Rodrigues Lucena, Fernanda Grande Kugeratski, Carla Vanessa de Paula Lima, and Fabricio Klerynton Marchini

12. Genome-Wide Proteomics and Phosphoproteomics Analysis of Leishmania spp. during Differentiation
Harsh Pawar, Gajanan Sathe, and Milind S. Patole

13. CRISPR/Cas9 Technology Applied to the Study of Proteins Involved in Calcium Signaling in Trypanosoma cruzi
Noelia Lander, Miguel A. Chiurillo, and Roberto Docampo

14. Application of CRISPR/Cas9 Mediated Genome Editing in Leishmania
Wen-Wei Zhang, Patrick Lypaczewski, and Greg Matlashewski

15. Next Generation Analysis of Trypanosomatid Genome Stability and Instability
Emma M. Briggs, Catarina A. Marques, Joao Reis-Cunha, Jennifer Black, Samantha Campbell, Jeziel Damasceno, Daniella Bartholomeu, Kathryn Crouch, and Richard McCulloch

Part III Molecular Biology and Genetics

16. Transcription Factor Analysis in Trypanosomatids
Arthur Günzl, Ankita Srivastava, and Ujwala Gosavi

17. Identifying Trypanosome Protein-RNA Interactions using RIP-Seq
Elisha Mugo and Esteban D. Erben

18. The Tethering Assay: A Simple Method for the Characterization of mRNA-Fate Regulators
Elisha Mugo and Esteban D. Erben

19. RNA-Binding Proteins and their Targets in Trypanosoma brucei: Single Nucleotide Resolution using iCLIP and iCLAP
Sameer Dixit, Juan D. Alfonzo, and Julius Lukeš

20. In Vivo Tethering System to Isolate RNA-Binding Proteins Regulating mRNA Decay in Leishmania
Hiva Azizi and Barbara Papadopoulou

21. Forward Genetics in African Trypanosomes
Sebastian Hutchinson and Lucy Glover

22. Analysis of Base Excision and Single Strand Break Repair Activities in Trypanosomatid Extracts
Daria M. Kania, Michael L. Ginger, and Sarah L. Allinson

Part IV Cell Biology

23. Light Microscopy in Trypanosomes: Use of Fluorescent Proteins and Tags 
Samuel Dean and Jack Sunter

24. ImageJ for Partially and Fully Automated Analysis of Trypanosome Micrographs
Richard J. Wheeler

25. Motility Analysis of Trypanosomatids
Timothy Krüger and Markus Engstler

26. Electron Microscopy Techniques Applied to Symbiont-Harboring Trypanosomatids: The Association of the Bacterium with Host Organelles 
Maria Cristina M. Motta and Carolina M. C. Catta-Preta

27. AiryScan Super-Resolution Microscopy to Study Trypanosomatid Cell Biology
Jane Harmer, Asma Belbelazi, Martin Carr, and Michael L. Ginger

28. All You Ever Wanted to Know About APOL1 and TLFs and Didn’t Dare Ask
Joseph Verdi, Charles Schaub, Russell Thomson, and Jayne Raper

29. Isolation of Leishmania Promastigote Flagella
Tom Beneke, François Demay, Richard J. Wheeler, and Eva Gluenz

30. Gel Based Methods for the Investigation of Signal Transduction Pathways in Trypanosoma brucei
Balázs Szöor and Mathieu Cayla

31. Methods to Investigate Signal Transduction Pathways in Trypanosoma cruzi: Cyclic Nucleotide Phosphodiesterases Assay Protocols
Alejandra C. Schoijet, Tamara Sternlieb, and Guillermo D. Alonso

32. Methods for the Investigation of Trypanosoma cruzi Amastigote Proliferation in Mammalian Host Cells
Peter C. Dumoulin and Barbara A. Burleigh

33. Isolation of Extracellular Vesicles from Leishmania spp.
Andrea Vucetic, Alonso Da Silva Lira Filho, George Dong, and Martin Olivier

Part V Biochemistry

34. 2D Gel Electrophoresis Analysis of Leishmania Proteomes
Chistina Naula and Richard Burchmore

35. Analysis of the Physiological and Metabolic State of Leishmania using Heavy Water Labelling
Joachim Kloehn and Malcolm J. McConville

36. A Scalable Purification Method for Mitochondria from Trypanosoma brucei
Moritz Niemann and André Schneider

37. Isolation of Glycosomes from Trypanosoma cruzi
Héctor Acosta and Wilfredo Quiñones

38. Sorting the Muck from the Brass: Analysis of Protein Complexes and Cell Lysates
Martin Zoltner, Ricardo Canavante del Pino, and Mark C. Field

39. Measurement of Energy States of the Trypanosomatid Mitochondrion
Mayke Bezerra Alencar, Richard Bruno Marcel Moreira Girard, and Ariel Mariano Silber

40. Isolation and Characterization of Acidocalcisomes from Trypanosomatids
Guozhong Huang, Silvia N.J. Moreno, and Roberto Docampo

41. Metabolic Control Analysis for Drug Target Prioritization in Trypanosomatids 
Zabdi González-Chávez, Citlali Vázquez, Rafael Moreno-Sánchez, and Emma Saavedra

Part VI Biomarkers and Diagnosis

42. Establishment of a Standardized Vaccine Protocol for the Analysis of Protective Immune Responses during Experimental Trypanosome Infections in Mice
Magdalena Radwanska, Hang Nguyen, Sangphil Moon, Emmanuel Obishakin, and Stefan Magez

43. Isolation of Trypanosoma brucei brucei Infection-Derived Splenic Marginal Zone B Cells Based on CD1dHigh/B220High Surface Expression in a Two-Step MACS-FACS Approach
Magdalena Radwanska, Nick Vereecke, and Stefan Magez

44. Cellular Markers for the Identification of Chemo-Resistant Isolates in Leishmania
Maritza Padrón-Nieves and Alicia Ponte-Sucre

Part VII Drug Discovery for Trypanosomatid-Borne Diseases

45. In Vitro Image-Based Assay for Trypanosoma cruzi Intracellular Forms
Amanda G. Eufrásio and Artur T. Cordeiro

46. In Vitro Drug Efficacy Testing against Trypanosoma brucei
Marcel Kaiser and Pascal Mäser

47. In Vitro Growth Inhibition Assays of Leishmania spp.
Sarah Hendrickx, Guy Caljon, and Louis Maes

48. In Vivo Bioluminescence Imaging to Assess Compound Efficacy against Trypanosoma brucei
Ryan Ritchie, Michael P. Barrett, Jeremy C. Mottram, and Elmarie Myburgh