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A Reliable Assay to Evaluate the Virulence of Aspergillus nidulans Using the Alternative Animal Model Galleria mellonella (Lepidoptera)

以大蜡螟（鳞翅目）为动物替代模型评估构巢曲霉毒力的可靠实验方法

CF Caroline Mota Fernandes
FF Fernanda Lopes Fonseca
GG Gustavo Henrique Goldman
Marcos Dias Pereira Marcos Dias Pereira *
EK Eleonora Kurtenbach *

 (*contributed equally to this work) 发布: 2017年06月05日第7卷第11期 DOI: 10.21769/BioProtoc.2329 浏览次数: 11363

评审: Valentine V TrotterShahin S. AliAnonymous reviewer(s)

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Abstract

The greater wax moth Galleria mellonella has emerged as an effective heterologous host to study fungal pathogenesis and the efficacy of promising antifungal drugs (Mylonakis et al., 2005; Li et al., 2013). Here, a methodology describing the Aspergillus nidulans infection in G. mellonella larvae, along with insect survival analysis, is reported. This protocol allowed the distinction between virulent A. nidulans strains (such as TNO2A3), which induced high larval mortality rates, to those in which gene deletion was accompanied by reduced pathogenicity such as ∆gcsA and ∆sdeA (Fernandes et al., 2016).

Keywords: Aspergillus nidulans (小巢状曲菌) search Galleria mellonella (大蜡螟) search Fungal pathogenicity ( 真菌致病性) search Fungal virulence ( 真菌毒力) search Alternative models (模型替代) search

Background

G. mellonella is an inexpensive model, easy to handle and its innate immune response shares functional similarities with the mammalian immune system. Additionally, larvae and mice infected with fungal mutant strains exhibited similar survival rates (Brennan et al., 2002). Therefore, larvae constitute a convenient animal host to substitute the use of vertebrates in fungal pathogenesis analysis. Despite all the advantages of the insect model, only a few reports have shown the effect of Aspergillus infection in G. mellonella. This protocol describes an efficient methodology to analyze Aspergillus nidulans pathogenesis in G. mellonella larvae.

Materials and Reagents

  1. Sterile toothpick
  2. Inoculation loop (microstreaker) (Thermo Fischer Scientific, Thermo ScientificTM, catalog number: SL1S )
  3. 200-1,000 µl pipette tips (Corning, Axygen®, catalog number: T-1000-B )
  4. 2-20 µl pipette tips (Corning, Axygen®, catalog number: T-200-Y )
  5. 15 ml conical tube (Greiner Bio One International, catalog number: 188271 )
  6. Miracloth filter, pore size 22-25 µm (EMD Millipore, catalog number: 475855 )
  7. 20 ml syringe (BD, catalog number: 302830 )
  8. 1.5 ml microcentrifuge tube (Corning, Axygen®, catalog number: MCT-150-R )
  9. Gloves
  10. Weighing paper
  11. Glass wool (Sigma-Aldrich, catalog number: 18421 )
  12. 280 ml plastic boxes
  13. Galleria mellonella larvae
  14. Aspergillus nidulans strains (TNO2A3 strain is available commercially in Fungal Genetics Stock Center as #A1149; ∆sdeA and ∆gcsA mutants can be provided by us upon request)
  15. Sterile deionized water
  16. Yeast extract (BD, BactoTM, catalog number: 212750 )
  17. Glucose (Sigma-Aldrich, catalog number: G8270 )
  18. Agar (BD, BactoTM, catalog number: 214010 )
  19. Uridine (Sigma-Aldrich, catalog number: U3750 )
  20. Uracil (Sigma-Aldrich, catalog number: U0750 )
  21. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: V000283 )
  22. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: V000263 )
  23. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Sigma-Aldrich, catalog number: 221279 )
  24. Iron(II) sulfate heptahydrate (FeSO4·7H2O) (Sigma-Aldrich, catalog number: V000119 )
  25. Cobalt(II) chloride hexahydrate (CoCl2·6H2O) (Sigma-Aldrich, catalog number: V000213 )
  26. Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Sigma-Aldrich, catalog number: V000118 )
  27. Ammonium molybdate tetrahydrate (Sigma-Aldrich, catalog number: V000122 )
  28. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: V000114 )
  29. 10 N sodium hydroxide (NaOH) solution
  30. Ethanol
  31. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: V000106 )
  32. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  33. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: V000317 )
  34. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: V000225 )
  35. Hydrogen chloride (HCl)
  36. Honey (Local supermarket)
  37. Glycerol (Sigma-Aldrich, catalog number: V000123 )
  38. Milk (Local supermarket)
  39. Wheat germ (Local supermarket)
  40. Wheat flour (Local supermarket)
  41. Wheat bran (Local supermarket)
  42. Complete media (YUU media) for A. nidulans growth (see Recipes)
  43. Trace elements solution for Aspergillus (see Recipes)
  44. 70% ethanol solution (see Recipes)
  45. PBS buffer (see Recipes)
  46. Artificial diet for Galleria mellonella (see Recipes)

Equipment

  1. 100 x 10 mm glass Petri dishes (Corning, PYREX®, catalog number: 3160-100 )
  2. Autoclave (Prismatec, model: CS-18 )
  3. Laminar flow cabinet (TROX Technik, model: TLF-A1 )
  4. 250 ml beaker (Roni Alzi, catalog number: 2-570 )
  5. Blunt tipped tweezer (EMD Millipore, catalog number: XX6200006P )
  6. Bunsen burner/lighter
  7. Hemocytometer (Hausser Scientific, catalog number: 3500 )
  8. Optical microscope with a 100x objective (American Optical Corporation)
  9. 10 µl Hamilton 700 series cemented syringe (Hamilton, catalog number: 80300 )
  10. Analytical balance (Denver Instrument, model: XL-410 , catalog number: 8515.1)
  11. Spatula (Sigma-Aldrich, catalog number: Z282774 )
  12. Graduated cylinder (Roni Alzi, catalog number: 2-950 )
  13. Glass bottle (Corning, PYREX®, catalog number: 1395-1L )
  14. Manual adjustable pipette (200-1,000 µl, Pipetman P1000) (Gilson, catalog number: F123602 )
  15. Manual adjustable pipette (2-20 µl, Pipetman P20) (Gilson, catalog number: F123600 )
  16. Thermostatic incubators (Fanem®, model: 515 )

Software

  1. GraphPad Prism 7.0 program (GraphPad Software; https://www.graphpad.com/)

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

Fernandes, C. M., Fonseca, F. L., Goldman, G. H., Pereira, M. D. and Kurtenbach, E. (2017). A Reliable Assay to Evaluate the Virulence of Aspergillus nidulans Using the Alternative Animal Model Galleria mellonella (Lepidoptera). Bio-protocol 7(11): e2329. DOI: 10.21769/BioProtoc.2329.

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分类

微生物学 > 微生物-宿主相互作用 > 真菌

微生物学 > 微生物-宿主相互作用 > 体内实验模型 > 节肢动物

细胞生物学 > 细胞活力 > 细胞死亡

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