Vacuole and mitochondria patch protein Mcp1 of Saccharomyces boulardii impairs the oxidative stress response of Candida albicans by regulating 2-phenylethanol

Abstract

Background: Vacuole and mitochondria patch (vCLAMP) protein Mcp1 is crucial in eukaryotic cells response to environmental stress, but the mechanism of Mcp1 in Saccharomyces boulardii (S. boulardii) against pathogenic fungi is unclear.

Results: This work first explored the role of Mcp1 in S. boulardii against Candida albicans (C. albicans). The results showed that Mcp1 located on the vacuolar and mitochondrial membrane of S. boulardii. Overexpression of Mcp1 inhibited the adhesion and hyphal formation of C. albicans in vitro. The mice model of intestinal infection revealed that WT-pGK1-MCP1 mutant enhanced the ability of S. boulardii antagonize C. albicans infecting gut. High performance liquid chromatography-mass spectrometry analysis demonstrated that overexpressing Mcp1 promoted the production of 2-phenylethanol. The latter is a secondary metabolite of S. boulardii, and can inhibit the adhesion and biofilm formation of C. albicans. The reverse transcription polymerase chain reaction and western blotting results confirmed Mcp1 promoted the production of 2-phenylethanol by regulating the expression level of Aro10. Notably, RNA-sequencing and Gene Ontology enrichment analyses showed that 2-phenylethanol impaired the oxidative stress response of C. albicans.

Conclusion: This work reveals the critical role of Mcp1 in S. boulardii against C. albicans by regulating 2-phenylethanol metabolism, which provide a theoretical basis for S. boulardii as antifungal biologic therapy to prevent and treat of Candida infection.

Keywords: 2-phenylethanol; Antagonism; Mcp1; Organelle membranes; Oxidative stress response.

Fig. 1

Delection of MCP1 damaged mitochondrial functions of S. boulardii. (A) Mcp1-GFP both located on vacuolar and mitochondrial membrane. After cultured in SC-Uri, the cells were collected and stained by Mito Tracker Red and FM4-64 and observed by fluorescence microscope. Bar = 5 μm. (B) The morphology of mitochondria in S. boulardii (WT, mcp1Δ/Δ, and WT + pGK1-MCP1). The samples were stianed by Mito Tracker Red. Bar = 5 μm. Deletion of MCP1 decreased (C) cellar activity and (D) ATP production of S. boulardii. After the stains were mixed with MTT at 30℃ for 1 h, then the samples were resuspended in DMSO, and measured at OD_570nm by Multi functional enzyme-linked immunosorbent assay reader. The intracellular ATP contents were detected using an ATP kit. This value represents the mean ± SD from three replicates. Statistical significance was defined based on the different p-values: *p < 0.05, **p < 0.01, and ***p < 0.001, ns: The comparison between the two sets of data was not statistically significant

Fig. 2

Overexpression of Mcp1 of S. boulardii inhibited the (A) adhesion and (B) biofilm formation of C. albicans. (C) The heart, liver, lung, and kidney tissues of mice by H&E staining after oral-gavage treatment. Bar = 50 μm. (D) Overexpression of Mcp1 reduced the ability of C. albicans infecting the host gut. Bar = 50 μm. This value represents the mean ± SD from three replicates. Statistical significance was defined based on the different p-values: *p < 0.05, **p < 0.01, and ***p < 0.001, ns: The comparison between the two sets of data was not statistically significant

Fig. 3

The secondary metabolites of S. boulardii inhibited the (A and B) adhesion and (C) biofilm formation of C. albicans. (D) The characteristic peak of 2-phenylethanol is 105.0698 (m/z). (E) Characteristic of 2-phenylethanol content in secondary metabolites of S. boulardii. Cells (WT, mcp1Δ/Δ, and WT-pGK1-MCP1) cultured medium were collected, measured and analyzed by HPLC-MS. (F) 2-phenylethanol inhibited hyphal development of C. albicans. After C. albicans cultured in YPD at 30℃, the cells were collected and resuspended in RPMI-1640 medium with 2-phenylethanol (0.002 mg/mL, 0.02 mg/mL, and 0.2 mg/mL) at 37℃ for 4 h. Then, the samples were stained by CFW, and observed by microscope. Bar = 10 μm. (G) 2-phenylethanol reduced the adhesion ability of C. albicans. (H) Deletion of MCP1 decreased the gene expression level of ARO10. The genes related to the metabolism of 2-phenylethanol (ARO1, PHA2, and ARO10) were analyzed by RT-PCR using ACT1 as the normalization genes. (I) Deletion of MCP1 led to abnormal expression of Aro10 in S. boulardii. The cells were collected, and the total protein was extracted. After the samples were detected by western blotting using anti-GFP and anti-tubulin antibodiers. The results were presented through an exposure device. (J) Gray value analysis of Aro10 by Image J. This value represents the mean ± SD from three replicates. Statistical significance was defined based on the different p-values: *p < 0.05, **p < 0.01, and ***p < 0.001, ns: The comparison between the two sets of data was not statistically significant

Fig. 4

(A) Transcriptomic analysis of differential gene expression of C. albicans. After the C. albicans was cultured with 2-phenylethanol, the genes expression of C. albicans were detected by RNA-seq. (B) GO enrichment analyses of differential gene expression. (C) After cultured with 2-phenylethanol, the expression level of genes related to oxidative stress response in C. albicans, including TRR1, SOD1 and CAT1 were assayed by RT-PCR. (D) The expression level of hypha-associated genes including ALS1, ALS3 and HWP1 in C. albicans after cultured with 2-phenylethanol. (E) ROS level, (F) SOD activity, and (G) CAT activity in C. albicans after cultured with 2-phenylethanol. (H) The impact on intracellular ROS levels by deleting and overexpressing CAT1 in C. albicans. This value represents the mean ± SD from three replicates. Statistical significance was defined based on the different p-values: *p < 0.05, **p < 0.01, and ***p < 0.001, ns: The comparison between the two sets of data was not statistically significant