Abstract
Candida albicans is a leading human fungal pathogen that uses several metabolic adaptations to escape immune cells and causes systemic disease. Here, we describe a protocol for measuring one of these adaptations, the ability to thrive in hypoxic niches. Hypoxia was generated after successful subdermal infection with C. albicans in a murine infection model. Hypoxia was measured using a fluorescent dye for carbonic anhydrase 9, a host enzyme active under hypoxic conditions. Emitted fluorescence was subsequently quantified using an IVIS system. This protocol was optimized for the use in subdermal infection in mice but has the potential to be adapted to other models of fungal infection.
Keywords: Candida albicans, Subdermal infection, Hypoxia, IVIS, Mycology
Background
Fungal colonizers of humans have evolved to sense and adapt to niches available in the host (Grahl and Cramer, 2010). Oxygen is a changing environmental parameter. Levels change in different tissues and during different stages of infection and immune activation (Carreau et al., 2011; Wenger et al., 2015). The ability to sustain growth and to survive in low oxygen environments has been linked to virulence in several fungi (Shepardson et al., 2013; Gresnigt et al., 2016; Pradhan et al., 2018). We have developed a protocol to follow the generation of hypoxia in a mouse infected with C. albicans (Lopes et al., 2018). We chose subdermal infection as a local, non-disseminated model of mycosis with acute onset which allows analysis of hypoxia in a confined space, where C. albicans and host cells interact (Urban et al., 2009; Santus et al., 2018). Thereby, natural variation of oxygen levels in other tissues can be eliminated and secondary effects from distant locations arising in systemic infection can be avoided. During infection, hypoxia is created mainly by neutrophil influx to the site of infection. Neutrophil extravasation and the activation of oxygen-consuming enzymes create environments with low oxygen levels. In turn, C. albicans exploits this environmental shift to avoid immune recognition by changing cell wall composition leading to masking of recognized entities (Lopes et al., 2018).
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Image treatment to quantify average epi-fluorescence:
Notes
Recipes
Acknowledgments
We thank Roland Nordfelth for his assistance with the IVIS equipment. This study was funded by grants to C.F.U. from the Swedish Research Council VR-M 2014-02281 and 2017-01681 and the Kempe Foundation SMK-1453. Additional grants to J.P.L. from J. C. Kempes Minnes Stipendiefond, Arneska Stiftelse, and the Helge ax:son Johnsons Stiftelse are acknowledged. This method is briefly described in Lopes et al. (2018). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.
Competing interests
All authors declare no competing interests.
Ethics
The animal experiments were conducted in accordance with animal ethical guidelines stated in permission A79-14 from the Swedish Board of Agriculture.
References
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