Identification of emulsification proteins released from the cells by inhibiting the synthesis of GPI-anchor or β-1,3-glucan in Candida albicans

Abstract

IntroductionA previous study demonstrated a strong emulsification ability of the culture supernatant obtained by cultivation of Candida albicans in a medium containing a β-1,3-glucan synthesis inhibitor and proposed a novel screening method using emulsification as an indicator for β-1,3-glucan synthesis inhibition (Nerome et al., 2021. Evaluating β-1,3-glucan synthesis inhibition using emulsion formation as an indicator. J Microbiol Methods. 190:106327). The emulsification was presumed to be caused by the proteins released from the cells; however, which proteins have a strong emulsification ability was unclear. Furthermore, as many cell wall proteins are connected to β-1,3-glucan via the carbohydrate moiety of the glycosylphosphatidylinositol (GPI)-anchor, which remains when detached from the cell membrane, emulsification might be detected by inhibiting GPI-anchor synthesis. ObjectiveThis study aimed to confirm whether emulsification could be detected by inhibiting GPI-anchor synthesis and identifying emulsification proteins released by inhibiting the synthesis of GPI-anchor or β-1,3-glucan. MethodsC. albicans was cultured in a medium containing a GPI-anchor synthesis inhibitor, and the emulsification by the culture supernatant was evaluated. We identified cell wall proteins released from the cells upon inhibition of β-1,3-glucan or GPI-anchor synthesis by mass spectrometry, their recombinant proteins were prepared, and their emulsification efficacy was evaluated. ResultsIn GPI-anchor synthesis inhibition, a weak emulsification phenomenon was observed compared to the β-1,3-glucan synthesis inhibition. Phr2 protein was released from the cells upon GPI-anchor synthesis inhibition, and recombinant Phr2 showed a strong emulsification activity. Phr2 and Fba1 proteins were released upon β-1,3-glucan synthesis inhibition, and recombinant Fba1 showed a strong emulsification activity. ConclusionsWe concluded that the emulsion phenomenon could be used to screen β-1,3-glucan and GPI-anchor synthesis inhibitors. Also, the two kinds of inhibitors could be distinguished by differences in the growth recovery by osmotic support and strength of emulsification. In addition, we identified the proteins involved in emulsification.