Photochromic Fluorophores Enable Imaging of Lowly Expressed Proteins in the Autofluorescent Fungus Candida albicans

Abstract

Fluorescence microscopy is a standard research tool in many fields, although collecting reliable images can be difficult in systems characterized by low expression levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly or endogenously expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungal agents in the fungal pathogen Candida albicans. We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by high background fluorescence. IMPORTANCE Understanding the spatial and temporal organization of proteins of interest is key to unraveling cellular processes and identifying novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans, and resistance mechanisms against these therapeutic agents are rapidly acquired. Fluorescence microscopy is a valuable tool to investigate molecular processes and assess the localization of possible antifungal targets. Unfortunately, fluorescence microscopy of C. albicans suffers from extensive autofluorescence. In this work, we present the use of a photochromic fluorescent protein and stochastic optical fluctuation imaging to enable the imaging of lowly expressed proteins in C. albicans through the suppression of autofluorescence. This method can be applied in C. albicans research or adapted for other fungal systems, allowing the visualization of intricate processes.

A multi-colour fluorogenic tag and its application in Candida albicans

“…As expected, in the Erg11-iFAST strain we found localization of fluorescence at the ER and cortical ER (Fig. 3a) [23,24]. A significant difference is observed in mean fluorescence acquired from the Erg11-iFAST strain compared to the parent SC5314 treated with Coral (P<0.0001).…”

“…However, the strain with an overexpression construct of Erg11-iFAST showed a reduction in growth rate in combination with a delay in the exponential phase compared to the parent strain (respectively P =0.0215, P <0.0001). From this we can conclude that there may be an adverse effect of tagging a protein-of-interest but that this effect is dependent on the manner in which it is attached, and the specific protein-of-interest and its transport throughout the cell [18, 23, 24]. However, some background signal of Coral and Lime may be observed, this background is most obvious in () where endogenous imaging of Ftr1 resulted in a significant background signal in the SC5314 parental strain treated with Lime.…”