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Peer-reviewed

Drosophila Model of Leishmania amazonensis Infection

亚马逊利什曼原虫感染的果蝇模型

KO Kendi Okuda
NS Neal Silverman

发布: 2017年12月05日第7卷第23期 DOI: 10.21769/BioProtoc.2640 浏览次数: 6426

评审: Adler R. DillmanPascale GeirardAnonymous reviewer(s)

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Jun 2016

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Abstract

This protocol describes how to generate and harvest antibody-free L. amazonensis amastigotes, and how to infect adult Drosophila melanogaster with these parasites. This model recapitulates key aspects of the interactions between Leishmania amastigotes and animal phagocytes.

Keywords: Drosophila melanogaster (黑腹果蝇) search Leishmania (利什曼虫) search Phagocytosis (吞噬作用) search Plasmatocytes (浆细胞) search Hemocytes (血细胞) search Axenic amastigote (纯培养无鞭毛体) search Amastigote (无鞭毛体) search RNAi screen (RNAi 筛选) search

Background

Leishmaniasis, caused by Leishmania protozoans, affects the skin, mucosa or internal organs, depending on the parasite species and immunological status of the host. Although the mouse model of infection has provided most of our understanding about this parasitic infection, it is not well suited for large-scale exploratory approaches. In our lab, we exploited the tractable and powerful genetics of the fruit fly Drosophila melanogaster to establish a model of Leishmania infection and perform a small-scale screen to identify host genes involved in the phagocytosis of these parasites (Okuda et al., 2016). In particular, a set of genes possibly linked to phagocytosis was specifically knocked-down in the fly hemocytes, using the UAS-GAL4 expression systems (Brand and Perrimon, 1993) and the survival and parasite burden of mutant-infected flies were monitored to identify factors modulating the infection.

Materials and Reagents

  1. 15 ml conical tube (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339650 )
  2. Cell culture flasks (T-flask) 80 cm2 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 178905 )
  3. T-flask 175 cm2 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 178883 )
  4. Cell scraper (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 179707 )
  5. 10 ml serological pipette (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 170356 )
  6. 1.5 ml microcentrifuge tube (Corning, Costar®, catalog number: 3622 )
  7. 96-well plate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 167008 )
  8. Pipette tips (Fisher Scientific, catalog numbers: 21-402-269 , 21-402-569 , 21-402-589 )
    Manufacturer: Thermo Fisher Scientific, catalog numbers: 3752RI , 3782RI .
  9. Disposable counting chamber (Bio-Rad Laboratories, catalog number: 1450003 )
  10. Pellet pestles (Fisher Scientific, catalog number: 12-141-364 )
  11. Fly vials (Genesee Scientific, Flystuff, catalog number: 32-116SB )
  12. Leishmania amazonensis (strain RAT/BA/LV78, Dutta et al., 2005)
  13. BALB/c bone marrow derived macrophages (BMDM)
    Note: Material and reagents are described in the Bio-protocol paper of Chen, 2013. Balb/c mice were obtained from The Jackson Laboratories. The femurs and tibias of one mouse generate approximately 3 x 108 macrophages.
  14. Phosphate-buffered saline (PBS) (Corning, catalog number: 21-031-CV )
  15. RPMI 1640 (Corning, catalog number: 10-040-CV )
  16. Percoll (GE Healthcare, catalog number: 17-0891-01 )
  17. Bromophenol blue (Fisher Scientific, catalog number: BP115-25 )
  18. Fly food (FlyStuff Nutri-Fly Molasses Formulation or Nutri-FlyTM Bloomington Formulation, Genesee Scientific, Flystuff, catalog numbers: 66-116 )
  19. Yellow Cornmeal Fly food (Genesee Scientific, Flystuff, catalog number: 62-101 )
  20. Schneider’s insect medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21720024 )
  21. Penicillin/streptomycin (Corning, catalog number: 30-002-Cl )
  22. Tetracyclin (Sigma-Aldrich, catalog number: T7660 )
  23. Tunicamycin (Cayman Chemical, catalog number: 11445 )
  24. 70% ethanol
  25. 199 media (Corning, catalog number: 90-050-PB )
  26. Heat inactivated fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S12450 )
  27. HEPES (Sigma-Aldrich, catalog number: H3784-100G )
  28. Sodium succinate (Sigma-Aldrich, catalog number: S9637 )
  29. Hemin (Sigma-Aldrich, catalog number: H9039 )
  30. Tryptic soy broth (BD, catalog number: 211825 )
  31. 199 media to grow promastigotes (see Recipes)
  32. 199 media for promastigote to axenic amastigote differentiation (see Recipes)

Equipment

  1. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: SorvallTM LegendTM RT ), equipped with swinging buckets for 15 ml conical tubes
  2. Incubator (VWR® Water Jacketed CO2 Incubators)
  3. Glass dounce with grinding chamber and Teflon rod, clearance of 0.004” to 0.006” (DWK Life Sciences, Wheaton, catalog number: 358011 )
  4. Hemocytometer Hausser Scientific Bright-LineTM Counting Chamber (Hausser Scientific, catalog number: 3110 )
  5. Capillary puller (NARISHIGE, model: PC-100 )
  6. Nanoliter injector (Drummond Scientific, model: Nanoject II )
  7. Drummond Scientific Capillaries for Nanojet II (Pipette.com, catalog number: 3-000-203-G/X)
    Manufacturer: Drummond Scientific, catalog number: 3-000-203-G/X .
  8. Electronic caliper (Mitutoyo, catalog number: 500-197-30 )
  9. CO2 pad (Genesee Scientific, FlyStuff, catalog number: 59-114 )
  10. Stereomicroscope (Nikon Instruments, model: SMZ645 )
  11. Multichannel pipette (Thermo Fisher Scientific, Thermo ScientificTM, model: FinnpipetteTM F2 )
  12. Pellet grinder (Fisher Scientific, catalog number: 12-141-361 )
  13. Fine tweezers (Fisher Scientific, catalog number: 12-000-122 )
  14. Fluorescence microscope (Nikon Instruments, model: Eclipse E400 ) equipped with an imaging system (camera: Nikon Instruments, model: Ds-Qi1 , imaging software: NIS-Elements)

Software

  1. Imaging software: NIS-Elements Basic Reseach, Microscope Imaging Software (Nikon Instruments)
  2. GraphPad Prism7 (GraphPad Software)
  3. CellProfiler cell image analysis software (www.cellprofiler.org)

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Okuda, K. and Silverman, N. (2017). Drosophila Model of Leishmania amazonensis Infection. Bio-protocol 7(23): e2640. DOI: 10.21769/BioProtoc.2640.

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分类

免疫学 > 动物模型 > 果蝇

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