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

INTRODUCTION: A previous study demonstrated a strong emulsification ability of the culture supernatant obtained by cultivation of Candida albicans in a medium containing a ß-1,3-glucan synthesis inhibitor and proposed a novel screening method using emulsification as an indicator for ß-1,3-glucan synthesis inhibition (Nerome et al., 2021. Evaluating ß-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 ß-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. OBJECTIVE: This 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 ß-1,3-glucan. METHODS: C. 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 ß-1,3-glucan or GPI-anchor synthesis by mass spectrometry, their recombinant proteins were prepared, and their emulsification efficacy was evaluated. RESULTS: In GPI-anchor synthesis inhibition, a weak emulsification phenomenon was observed compared to the ß-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 ß-1,3-glucan synthesis inhibition, and recombinant Fba1 showed a strong emulsification activity. CONCLUSIONS: We concluded that the emulsion phenomenon could be used to screen ß-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.

Concentrative Nucleoside Transporter, CNT, Results in Selective Toxicity of Toyocamycin against Candida albicans.

Toyocamycin (TM) is an adenosine-analog antibiotic isolated from Streptomyces toyocaensis. It inhibits Candida albicans, several plant fungal pathogens, and human cells, but many fungi, including Saccharomyces cerevisiae, are much less susceptible to TM. Aiming to clarify why TM and its analogs tubercidin and 5-iodotubercidin are active against C. albicans but not S. cerevisiae, this study focused on the absence of purine nucleoside transport activity from S. cerevisiae. When the concentrative nucleoside transporter (CNT) of C. albicans was expressed in S. cerevisiae, the recombinant strain became sensitive to TM and its analogs. The expression of C. albicans purine nucleoside permease in S. cerevisiae did not result in sensitivity to TM. Clustered regularly interspaced short palindromic repeat-mediated disruption of CNT was performed in C. albicans. The CNTΔ strain of C. albicans became insensitive to TM and its analogs. These data suggest that the toxicity of TM and its analogs toward C. albicans results from their transport via CNT. Interestingly, S. cerevisiae also became sensitive to TM and its analogs if human CNT3 was introduced into cells. These findings enhance our understanding of the mechanisms of action of adenosine analogs toward Candida pathogens and human cells. IMPORTANCE We investigated the mechanism of toxicity of TM and its analogs to C. albicans. Inspired by the effect of the copresence of TM and purine nucleosides on cell growth of C. albicans, we investigated the involvement of CNT in the toxicity mechanism by expressing CNT of C. albicans (CaCNT) in S. cerevisiae and deleting CaCNT in C. albicans. Our examinations clearly demonstrated that CaCNT is responsible for the toxicity of TM to C. albicans. S. cerevisiae expressing the human ortholog of CaCNT also became sensitive to TM and its analogs, and the order of effects of the TM analogs was a little different between CaCNT- and hCNT3-expressing S. cerevisiae. These findings are beneficial for an understanding of the mechanisms of action of adenosine analogs toward Candida pathogens and human cells and also the development of new antifungal drugs.

Evaluating ß-1,3-glucan synthesis inhibition using emulsion formation as an indicator.

INTRODUCTION: The cell wall ß-1,3-glucan of fungal pathogen Candida albicans is an attractive antifungal target. ß-1,3-Glucan is the skeletal structure in the cell wall and the major scaffold for cell wall proteins. In previous studies using Saccharomyces cerevisiae, strong emulsification was detected by mixing cell wall proteins with oil. To date, there have been no reports of applying an emulsification phenomenon to assessing ß-1,3-glucan synthesis inhibition. OBJECTIVE: The aim of this study was to clarify that emulsification is useful as an indicator for evaluating ß-1,3-glucan synthesis inhibition in C. albicans. METHODS: At first, whether cell wall proteins released from cells by ß-1,3-glucanase treatment worked as an effective emulsifier in C. albicans was examined. Next, whether emulsification occurred even in the culture supernatant brought about by treating with bioactive compounds, including ß-1,3-glucan synthesis inhibitors, under osmotic protection was investigated. In addition, the release of cell wall proteins into the culture medium by treating with those compounds was examined. Finally, a simpler evaluation method using emulsion formation was examined for application to screening of inhibitors. RESULTS: Emulsification occurred by cell wall proteins obtained by treating with ß-1,3-glucanase in C. albicans. In addition, cell wall proteins were released into the culture medium by treating with ß-1,3-glucan synthesis inhibitors, resulting in emulsification. However, such phenomena were not observed in the case of other bioactive compounds. Furthermore, emulsification could be detected in the culture broth obtained by static culture on a small scale. CONCLUSIONS: The obtained results strongly implied that emulsification results from decreased ß-1,3-glucan levels in the cell wall. As emulsification can be simply evaluated by mixing the culture broth with oil, in the future application to the initial assessment and screening of ß-1,3-glucan synthesis inhibitors is expected.

Cell surface changes that advance the application of using yeast as a food emulsifier.

A previous study revealed that Saccharomyces cerevisiae mcd4Δ, a cell wall mutant with a defect in the synthesis of the glycosylphosphatidylinositol anchor, has a strong macrophage activation ability. In this study, remarkable emulsion formation after cell suspensions of mcd4Δ and anp1Δ (which exhibit an extreme reduction of mannan) were mixed with oil was found. Moreover, the relationship between cell wall mutation and emulsion formation was investigated, suggesting that och1Δ with a defect in the formation of N-linked glycans also had a strong emulsification ability and that high molecular weight materials released from the cells were involved in emulsion formation. Furthermore, two strains (asc1Δ and scp160Δ) with a strong emulsification ability without a large decrease in mannan content were also found from the wide screening of strains that exhibit an emulsifying activity using more than 5000 gene-deficient strains. These results provide valuable information for the development of a yeast-derived emulsifier.