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Functional analysis of selected deletion mutants in Candida glabrata under hypoxia

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Abstract

Increased drug resistance in Candida glabrata (a model non-albicans Candida) calls for the identification of potential molecular targets for the development of effective drugs. Hypoxia (a state of low oxygen) is an important host factor, which affects the virulence of the pathogen and efficacy of drugs. In the present study, in vitro characterization of 13 null mutants of C. glabrata were done under hypoxic condition (1% O2). These mutants have a major role to play in cellular pathways, viability and pathogenesis (cell wall biosynthesis, ergosterol synthesis, calcium–calcineurin, etc.). The in vitro growth, biofilm formation and susceptibility of biofilm to antifungal drugs of these mutants were compared with the control. Hypoxia reduced the susceptibility of planktonic cells to fluconazole. The mutants ecm33Δ, kre1Δ, rox1Δ, and kre2Δ showed maximum reductions in their biofilm activities (>20%). The selected mutants (upc2BΔ, kre2 Δ, ecm7Δ, rox1 Δ, mid1Δ, ecm33Δ, cch1Δ, kre1Δ) showed reduced biofilm activities (>30%) in the presence of 16 μg ml−1 fluconazole under hypoxia. Functional analysis revealed that Kre1, Ecm33, Upc2B, Kre2, Ecm7, Cch1, Mid1 and Rox1 can be explored as a potential drug target for developing novel antifungal drugs.

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Acknowledgements

We thank Prof. Brendan P. Cormack, Professor of Molecular Biology and Genetics, School of Medicine, John Hopkins University, Maryland, Baltimaore, and Dr. Rupinder Kaur, Laboratory of Fungal Pathogenesis, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India, for providing the C. glabrata deletion mutants. PG is supported by the INSPIRE fellowship from the Department of Science and Technology, Government of India. We acknowledge the support of the Director, Defence Institute of Physiology and Allied Sciences (DIPAS), DRDO, Delhi-54, along with Dr. Amitabha Chakrabarti, Sc. ‘F’ and Dr. Anju Bansal, Sc. ‘F’, DIPAS, for allowing us to use the facilities for hypoxia exposure and microbial culture in their laboratory. We are also thankful to Dr. Ashish Thapliyal, HOD, Biotechnology, Graphic Era University, for his constant support during the course of this study. This work was financially supported by Graphic Era University, Dehradun.

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  1. Department of Biotechnology, Graphic Era University, 566/6, Bell Road, Clement Town, Dehradun, Uttarakhand, 248002, India

    Payal Gupta & Navin Kumar

  2. Department of Molecular Biology, Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi, 110054, Delhi, India

    Ramesh Chand Meena

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Correspondence to Navin Kumar.

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13205_2017_821_MOESM1_ESM.eps

Fig. S1 Effect of hypoxia on mutants’ biofilm formation. The biofilm was formed in RPMI media for 42 h at 37 °C under hypoxia and normoxia and the metabolic activity of the biofilm formed was analyzed by XTT reduction assay. Optical density (O.D.) was measured at 492 nm. (EPS 17606 kb)

13205_2017_821_MOESM2_ESM.eps

Fig. S2 Antifungal susceptibility of C. glabrata mutants’ biofilm. Different concentration of fluconazole was added during biofilm formation of C. glabrata mutants. The biofilm formed after 48 h at 37 °C under hypoxic and normoxic conditions. The metabolic activity of biofilm formed was quantified by XTT reduction assay and the per cent reduction in the biofilm activity of each mutant, with reference to the biofilm activity of control strain, is shown as % Relative Metabolic Activity. (EPS 11093 kb)

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Gupta, P., Meena, R.C. & Kumar, N. Functional analysis of selected deletion mutants in Candida glabrata under hypoxia. 3 Biotech 7, 193 (2017). https://doi.org/10.1007/s13205-017-0821-7

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