白念珠菌生物膜细胞内cAMP的测定

Liuliu Jiang; Shengyan Chen; Kairui Sun; Peng Zhou; Xin Wei

doi:10.21769/BioProtoc.3461

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Peer-reviewed

Intracellular cAMP Measurements in Candida albicans Biofilms

白念珠菌生物膜细胞内cAMP的测定

XW Xin Wei email

发布: 2019年12月05日第9卷第23期 DOI: 10.21769/BioProtoc.3461 浏览次数: 3626

评审: Alexandros AlexandratosSadri ZnaidiMichael Tscherner

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Dec 2018

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Abstract

Candida albicans is the most common cause of fungal infections worldwide. Infection by C. albicans is closely associated with its ability to form a biofilm, closely packed communities of cells attached to the surfaces of human tissues and implanted devices, in or on the host. When tested for susceptibility to antifungals, such as polyenes, azoles, and allylamines, C. albicanscells in a biofilm are more resistant to antifungal agents than C. albicans cells in the planktonic form. Cyclic Adenosine monophosphate (cAMP) is one of the key elements for triggering hyphal and biofilm formation in C. albicans. It is hard to detect or extract molecular markers (e.g., cAMP) from C. albicans biofilms because the biofilms have a complex three-dimensional architecture with an extracellular matrix surrounding the cell walls of the cells in the biofilm. Here, we present an improved protocol that can effectively measure the level of intracellular cAMP in C. albicans biofilms.

Keywords: Candida albicans (白色念珠菌)

Biofilm (生物膜)

Second messenger (第二信使)

Cyclic Adenosine monophosphate (环磷酸腺苷)

cAMP enzyme immunoassay (环腺苷酸酶免疫分析)

Enzyme linked immunosorbent assay (酶联免疫吸附测定)

Background

Candida albicans is a member of the fungal community in the human gastrointestinal tract, mouth, and genital tract. C. albicans does not only cause superficial diseases, but also systemic infections in immunocompromised patients (Brown et al., 2012; Kim, 2016; Lohse et al., 2018). In clinical infections of C. albicans, the fungal cells commonly form biofilms; C. albicans biofilms are associated with chronic infection and antifungal resistance (Rajendran et al., 2016). The cyclic adenosine monophosphate (cAMP) signaling pathway has been considered to be one of the most important signal transduction pathways that can cause a switch from budding to hyphal morphogenesis, a switch to hyphal growth promotes biofilm formation and invasive growth in C. albicans (Wang, 2013; Tao et al., 2017). Activation of adenylyl cyclase to produce cAMP is thought to promote hyphal induction, and maximal levels of cAMP coincide with maximal germ tube formation (Parrino et al., 2017). In brief, it is generally accepted that biofilm formation of C. albicans requires elevated levels of cAMP. Therefore, measurement of cAMP levels in C. albicans biofilms is important to understand biofilm formation and antifungal drug resistance and has the potential to be an important reference value for future drug development efforts.

C. albicans biofilms are comprised of complex, surface-associated cell populations embedded in an extracellular matrix (Lohse et al., 2018). The three-dimensional hyphal structure of biofilms and the matrix surrounding the cells serve as physical barriers that make it difficult to detect or extract specific molecular markers such as cAMP from C. albicans biofilms. Yun et al. (1998) presented a method of to measure the intracellular cAMP level of planktonic Saccharomyces cerevisiae; however, no protocol has been reported on how to measure the level of intracellular cAMP in C. albicans biofilms. This article provides an improved protocol to measure the intracellular cAMP level of C. albicans biofilms. In this protocol, the levels of intracellular cAMP of cells within biofilms are examined by using an enzyme-linked immunosorbent assay (ELISA) with modifications; this is a modification of the method originally described in Yun et al. (1998).

Materials and Reagents

Stainless steel tweezer (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: DS0399-0001)
Pipettes (Eppendorf, P1000, catalog number: 3120000062; P200, catalog number: 3120000054; P10, catalog number: 3120000020; P2.5, catalog number: 3120000011, respectively)
Flasks (Duran, 50 ml, catalog numbers: 21-216-17; 100 ml, catalog number: 21-216-24, respectively)
Cell culture dish (Thermo Fisher Scientific, Thermo Scientific^TM, Nunc^TM EasYDish^TM, catalog number: 150466)
1.5 ml microtubes (Axygen^®, catalog number: MCT-150-C)
Tubes (Eppendorf, 15 ml, catalog number: 0030122151; 50 ml, catalog number: 0030122178, respectively)
Cell scraper (Costar, catalog number: 3010)
Pipette tips (Eppendorf, 1,000 µl, catalog number: 0030000919; 200 µl, catalog number: 0030000870; 10 µl, catalog number: 0030000811, respectively)
Aluminum foil
Parafilm
Strain (Candida albicans, SC5314) (ATCC, catalog number: 10231)
Yeast extract (BD Biosciences, catalog number: 212750)
Peptone (BD Biosciences, catalog number: 211677)
Dextrose (BD Biosciences, catalog number: 215530)
Agar (BD Biosciences, catalog number: 214040)
RPMI 1640 medium (Sigma-Aldrich, catalog number: R6504)
Sodium phosphate monobasic monohydrate (Thermo Fisher Scientific, catalog number: S369-500)
Sodium phosphate dibasic anhydrous (Thermo Fisher Scientific, catalog number: BP332-500)
Sodium chloride (Thermo Fisher Scientific, catalog number: S671-3)
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 59223C)
Distilled water
2-(N-Morpholino)ethanesulfonic acid (MES) (Sigma-Aldrich, catalog number: M5287)
Trichloroacetic acid (Sigma-Aldrich, catalog number: T9159)
Ice
Liquid nitrogen
YPD medium
Ether (Sinopharm Chemical Reagent Co., Ltd, catalog number: 10009318)
cAMP Enzyme Immunoassay Kit (Sigma-Aldrich, catalog number: CA200)
1. Goat Anti-Rabbit IgG Coated 96-well
2. cAMP-Alkaline Phosphatase Conjugate
3. cAMP EIA Antibody Rabbit Anti-cAMP
4. 0.1 M Hydrochloric Acid
5. 0.1 M HCI in water
6. Neutralizing Reagent
7. Wash Buffer Concentrate
8. Cyclic AMP Standard
9. p-Nitrophenyl Phosphate Substrate Solution
10. Stop Solution
11. Triethylamine
12. Acetic Anhydride
13. Plate Sealer
MES Buffer (see Recipes)
Sabouraud dextrose agar medium (SDA) (see Recipes)
YPD broth (see Recipes)
YPD agar medium (see Recipes)
1x PBS (see Recipes)

Equipment

Ultrasonic cell crusher (Celton, model number: UH-500B)
30 °C shaking incubator (Infors, model number: Multitron Standard)
CO₂ incubator (ESCO, CelMate^®, model number: CLM-170B-8-NF)
Multiwell plate shaker (Thermo Fisher Scientific, Thermo Scientific^TM, Sorvall^TM ST 40R, catalog number: 75004524)
Multi-mode Microplate Reader (Molecular Devices, SpectraMax M5)
Autoclave
Centrifuge

Procedure

English

中文翻译

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如何引用

Jiang, L., Chen, S., Sun, K., Zhou, P. and Wei, X. (2019). Intracellular cAMP Measurements in Candida albicans Biofilms. Bio-protocol 9(23): e3461. DOI: 10.21769/BioProtoc.3461.