Fluorescently Labelled Aerolysin (FLAER) Labelling of Candida albicans Cells

Background

The GPI (glycosylphosphatidylinositol) anchor is a post translational modification occurring in the endoplasmic reticulum (ER). The preformed GPI anchor is attached in the lumen of the ER to the C-terminus of specific proteins that carry a GPI anchor attachment signal sequence. These proteins are subsequently transported (and extensively further modified) by the secretory pathway to the cell surface where the proteins are present anchored to the extracellular leaflet of the plasma membrane or covalently linked to the cell wall in organisms that have a wall. A variety of proteins get GPI anchored proteins in eukaryotes, for example, hydrolytic enzymes, cell surface adhesion molecules or receptor proteins (Orlean and Menon, 2007). In fungal pathogens, such as Candida albicans, many of the adhesins involved in host recognition and adherence, as well as several pathogenesis and virulence factors are GPI anchored proteins; besides, several GPI-anchored proteins in Candida albicans have proteolytic activity (Richard and Plaine, 2007; Nobile et al., 2008). The pathway is essential in Candida albicans and downregulating it or targeting it could be a useful strategy to combat Candida infections. The protocol used for assessing GPI anchor levels in Candida albicans is elaborated here.

Fluorescently Labelled Aerolysin (FLAER) is a fluorescein labelled inactive derivative of aerolysin, a Gram-negative bacterial toxin, which binds to GPI-anchored proteins in the cell membrane of the eukaryotic host and forms pores (Howard et al., 1987; Parker et al., 1994). In FLAER, proaerolysin is tagged with Alexa Fluor 488 dye which can bind to GPI anchors of eukaryotic cells without any harm to the cell (Sutherland et al., 2007). FLAER is also clinically used to detect GPI anchor levels in Paroxysmal Nocturnal Hemoglobinuria (PNH) cells, a disease caused by somatic mutations in PIGA (a subunit of glycosylphosphatidylinositol-N-acetylglucosamine transferase enzyme complex) in mammals (Brodsky et al., 2000). Once stained with FLAER, the fluorescence of cells can be monitored/quantified under a confocal fluorescence microscope in the GFP channel and the data used as a measure of GPI anchored proteins on the cell surface. In a previous study, we monitored the levels of GPI-anchored proteins on the cell surface of a GPI biosynthetic mutant, Cagpi14, in Candida albicans by using FLAER (Singh et al., 2016). CaGPI14 encodes for the catalytic subunit of the first mannosyltransferase of the GPI-anchor biosynthesis pathway. Deficiency in CaGpi14 severely affects cell growth and wall integrity in the organism although low levels of expression appear to be sufficient to make the cells viable. Its homolog in S. cerevisiae is essential (Kim et al., 2007). The protocol for FLAER labelling used to assess GPI anchor levels in this mutant of Candida albicans is described here.