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59Fe Uptake Assays in Paracoccidioides Species

副球孢子菌属的59Fe摄取实验

DK Daniel J. Kosman
EB Elisa Flávia Luiz Cardoso Bailão
MS Mirelle Garcia Silva-Bailão
CS Célia Maria de Almeida Soares

发布: 2016年09月20日第6卷第18期 DOI: 10.21769/BioProtoc.1930 浏览次数: 8282

评审: Arsalan DaudiBelen SanzSoazig Le Guyon

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Abstract

Iron is an essential micronutrient required for virtually all organisms. This fact is related to the ability of the transition metal to exist in two oxidation states, the reduced ferrous (Fe2+) and the oxidized ferric (Fe3+). Given the relative availability of aqueous iron (the element which constitutes ~5% of the earth’s crust) one is not surprised that iron is the most common prosthetic element in biology. Usually, fungi can uptake iron through receptor-mediated internalization of a siderophore or heme, and/or reductive iron assimilation (RIA) (Kosman, 2013). In this way, the uptake of iron in the absence or presence of the reducing agent ascorbic acid can be investigated by 59Fe uptake assays, as previously described (Eide et al., 1992). In the presence of ascorbic acid, the reductive-independent 59Fe uptake route is investigated. On the other hand, in the absence of ascorbic acid, the reductive-dependent 59Fe uptake route is stimulated. Using this strategy for the human pathogenic fungus Paracoccidioides species, the results showed that iron uptake by Pb01 in the absence of ascorbic acid was low, unlike what was observed for Pb18. These results suggest that only in Pb18 the iron uptake pathway is coupled to a ferric reductase (Bailão et al., 2015). In this protocol, we describe how to perform 59Fe uptake assays in Paracoccidioides species.

Keywords: Reductive iron assimilation pathway (还原铁同化途径) search Reductive-independent iron uptake route (还原性独立铁吸收途径) search Ascorbic acid (抗坏血酸) search Quench buffer (淬火液) search Gamma counter (γ计数) search

Materials and Reagents

  1. Conical tube
  2. Wide-mouth screw-cap high-density polyethylene (scintillation) vials
  3. 13 x 100 mm test tubes
  4. Petri dish
  5. Filter type A/C 25 mm glass fiber filters from Pall Gelman
  6. 12 x 75 mm glass test tubes
  7. Wooden applicator (a thin wooden stick that is used in this protocol to put the filters containing radioactive cells inside the test tubes that will be read by the G counter)
  8. Disposable vacuum filtration system (TPP, catalog number: 99950 )
  9. Paracoccidioides spp. cells
  10. Culture media
    1. Liquid Brain Heart Infusion Broth (BHI) (Sigma-Aldrich, catalog number: 53286 ) with 4% glucose
    2. Liquid Synthetic Complete (SC medium) (see Recipes)
  11. Deionized water
  12. Yeast Nitrogen Base (YNB) without amino acids (BD, DifcoTM, catalog number: 291940 )
  13. Glucose (VWR, catalog number: 101175P )
  14. Amino acids (Sigma-Aldrich)
    ADE, TRP, HIS, ARG, MET, TYR, ISOLEU, LYS, PHE, GLU. ACID, ASP. ACID, VAL, THR, SER, URA, LEU
  15. Trypan blue solution, 0.4% for microscopy (Sigma-Aldrich, catalog number: 93595 )
  16. Ethylenediamine tetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
  17. Succinic acid (Sigma-Aldrich, catalog number: 398055 )
  18. Tris
  19. NaOH
  20. Analar glucose
  21. MES buffer (Sigma-Aldrich, catalog number: M3671 )
  22. Sodium citrate
  23. 59Fe (Perkin-Elmer, catalog number: NEZ037001MC )
  24. 1 M ascorbic acid (0.176 g in 1 ml sterilized water)
  25. Nitric acid (HNO3)
  26. Bathophenanthrolinedisulfonic acid (BPS) (Sigma-Aldrich, catalog number: B1375 )
  27. Iron (III) chloride (Sigma-Aldrich, catalog number: 157740 )
  28. SC complete media (see Recipes)
  29. Amino acids solution (see Recipes)
  30. Amino acid mix stock (see Recipes)
  31. 59Fe stock solutions (see Recipes)
  32. 10x quench buffer (see Recipes)
  33. Citrate uptake buffer (see Recipes)
  34. Acid-wash glassware treatment (see Recipes)

Equipment

  1. Shaker
  2. Hemocytometer
  3. Microfine forceps
  4. Glassware (erlenmeyers, bottle with blue cap, beakers, cylinders)
  5. Magnetic stirrer
  6. pH meter
  7. Vortex
  8. G counter (LKB Wallac Compu Gamma) with plastic g counting tube carriers
  9. Vacuum filtration manifold (Merck Millipore, model: 1225 )
  10. Source of vacuum, either standard lab pump or water aspirator
  11. Autoclave

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Kosman, D. J., Bailão, E. F. L. C., Silva-Bailão, M. G. and Soares, C. M. D. A. (2016). 59Fe Uptake Assays in Paracoccidioides Species. Bio-protocol 6(18): e1930. DOI: 10.21769/BioProtoc.1930.

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分类

微生物学 > 微生物新陈代谢 > 其它化合物

微生物学 > 微生物生物化学 > 其它化合物

生物化学 > 其它化合物 > 离子

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