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0INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
APPLICATION OF THE TRADITIONAL SPECTROPHOTOMETRY METHOD FOR MEASURING CELLULAR AND FREE IRON POOLS OF CANDIDA ALBICANS
1Shikha Chandra, 2Sana Akhtar Usmani, 3Khushboo Arya, 4Saumya Chaturvedi, 5Deeksha
Jattan, 6Nitin Bhardwaj, 7Ashutosh Singh
1Research Scholar- Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India.2Research Scholar- Department of Biochemistry, University of Lucknow,
Lucknow, Uttar Pradesh, India
3Research Scholar- Department of Biochemistry, University of Lucknow, Lucknow, Uttar
Pradesh, India
4Research Scholar- Department of Biochemistry, University of Lucknow, Lucknow, Uttar
Pradesh, India
5Research Scholar- Department of Biochemistry, University of Lucknow, Lucknow, Uttar
Pradesh, India
6Department of Zoology and Environmental Science, Gurukula Kangri (Deemed University),
Haridwar, 249404, Uttarakhand, India 7Assistant Professor - Department of Biochemistry, University of Lucknow, Lucknow, Uttar
Pradesh, India https://doi.org/10.5281/zenodo.13847121
Abstract. The availability of iron is crucial for pathogenicity in Candida albicans. Using a traditional visible spectrophotometry based method, this study aims to measure the extra- and intra- cellular iron concentration in wild type culture of C. albicans. Our findings indicate that the method is simple, cost effective and reproducible, and should be useful to determine iron levels under various growth conditions in C. albicans.
Keywords: candida, iron, spectrophotometry, detection.
Introduction
Iron is a crucial component for various metabolic processes, and its importance in the pathogenicity of Candida albicans cannot be overstated. Iron is required for morphogenetic transition of C. albicans from the yeast to the pathogenic hyphal form. This hyphal form facilitates the invasion of body tissues by C. albicans, leading to heightened infection. A sufficient iron level also promotes biofilm formation in the fungus, thereby playing a crucial role in its pathogenicity and virulence [1]. The organism has evolved sophisticated mechanisms to acquire iron from its host despite the host's efforts to sequester it within proteins, making it less accessible. A promising approach to inhibit Candida's growth is using iron chelators alone or in combination with known antifungal agents [2]. By using knockouts of proteins involved in iron acquisition, we could significantly reduce iron availability, leading to decreased survival rates for the organism [3].
Defining the exact role of iron in various physiological processes requires the accurate measurement of iron levels (both ferric (Fe3+) and ferrous (Fe2+) in C. albicans cells [4,5]. There are several advanced methods to measure iron content of the cells such as atomic absorption spectroscopy (ABS) and inductively coupled plasma mass spectrometry (ICP-MS) [6,7]. Both these techniques are routinely used in laboratories. While ABS is used to determine the total iron content, ICP-MS can detect both Fe3+ and Fe2+ ions. However, both the techniques require
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
extensive instrumentation and expertise, which adds to the cost of analysis. Iron estimation kits (MQuant®, Supelco) in market are also not very cost effective, and use of radioactive iron (55Fe) requires extensive laboratory setup [4]. On the other hand, traditional spectrophotometry based iron estimations are cost effective, produce relatively reproducible results and require simple laboratory setup. This is study is aimed to utilize a visible spectrophotometry based estimation of Fe3+ and Fe2+ levels in growth medium and C. albicans cell for practical implications.
Materials and Methodology
Strains and cultural conditions:
About 106 wild type C. albicans cells were cultured in yeast peptone dextrose (YPD) media, agar, or broth at 30°C for 24h in a shaker incubator until full growth [2]. Cells and medium were separated using centrifugation, and media kept separately for estimation of extracellular iron pool. After washing the harvested cells with distilled water, cells were broken in distilled water using glass beads (Sigma, 0.4-0.6 mm), via alternate vortexing and sonication. The cell debris was removed by centrifugation and supernatant was stored to measure the intracellular iron pool.
Preparation of standard solutions:
The standard iron solution was prepared using ferric chloride (for Fe3+) and ferrous sulphate (for Fe2+) at 1 mg/mL, in a volumetric flask and diluted to the mark. 0.3% ortho-phenanthroline and 10% hydroxylamine were prepared separately in 100ml of distilled water.
Iron estimation Protocol:
Here we have used a spectrophotometry based method described earlier [8]. Briefly, for estimation of Fe3+ ions, in 500 |iL of sample, 100 ^L ortho-phenanthroline was added, vortexed and incubated at room temperature for 30 minutes. Absorbance was recorded at 508 nm. For estimation of Fe3+ ions, in 500 |iL of sample, 100 ^L of hydroxylamine was added, vortexed and incubated for 10 minutes. This was followed by addition of 100 ^L ortho-phenanthroline, vortexing and incubation at room temperature for 30 minutes. Absorbance was recorded at 508 nm. Estimation of Fe3+ and Fe3+ ions was performed in reference to the obtained calibration curves (Fig1)
Fig. 1. Calibrations curves obtained for estimation of iron. Fe3+ (A) and Fe2+ (B) ions were measured using ferric chloride and ferrous sulphate as standards.
Results and Discussion
The extra- and intra- cellular Fe3+ ion concentration was found to be in the range of 1745±100 and 3793±248 |ig/mL in C. albicans cells, respectively (Fig. 2A). The extra- and intracellular Fe2+ ion concentration was found to be in the range of 153±6 and 442±180 |ig/mL in C.
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
albicans cells, respectively (Fig. 2B). Based on the data obtained we can safely conclude that the visible spectrophotometry based method is a simple, cost effective and reproducible method to
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Fig. 2. Measurement of Fe3+ and Fe2+ ion levels in C. albicans culture. Values are shown of Fe3+ and Fe2+ ion levels in media (extracellular) and cells (intracellular). Datasets are represented as Mean ± SEM (n=2, represents 2 separate experimental replicates). This method can be optimized for 96 well plate and be used estimate iron levels in large datasets. Considering the importance of iron in C. albicans physiology, this approach should allow us to screen a large number of samples for iron related changes, such as in drug treatment conditions, genetic knockouts and other altered growth parameters.
Acknowledgements: AS thanks support from ICMR (No.52/08/2019-B10/BMS), DST-PURSE program (SR/PURSE Phase 2/29(C)), UP Higher Education (No. 10/2021/281/-4-Sattar-2021-04(2)/2021 and No. 39/2024/242/Sattar-4-2024-001-4(33)/2023), DBT (BT/PR38505/MED/29/1513/2020) and DST (CRG/2022/001047) and the University of Lucknow.
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INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
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