Extracellular vesicles of Candida albicans regulate its own growth through the L-arginine/nitric oxide pathway

Abstract

Candida albicans is the main conditional pathogenic fungus among the human microbiome. Extracellular vesicles (EVs) secreted by C. albicans are important for its pathogenesis. However, the effects and mechanisms of EVs on C. albicans own growth are not clear. Here, we isolated EVs from C. albicans cells grown in four culture media, including RPMI 1640, DMEM, YPD, and YNB, and measured their effects on the own growth of C. albicans in these media. All the C. albicans EVs from the four media could promote the growth of C. albicans in RPMI 1640 and DMEM media, but had no effects in YPD and YNB media, indicating that the effects of EVs on C. albicans growth were dependent on some media contents. By comparing the media contents and transcriptome analysis, arginine was identified as the key factor for the growth promotion of C. albicans EVs. EVs activated the L-arginine/nitric oxide pathway to promote the growth of C. albicans through that EVs increased the NO levels and upregulated the expression of NO dioxygenase gene YHB1 to reduce the intracellular reactive oxygen species (ROS) and cell apoptosis. During the host cell infections, C. albicans EVs synergistically enhanced the destructive effects of C. albicans to host cells, including RAW264.7, HOK, TR146, and HGEC, suggesting that the growth promotion by EVs enhanced the pathogenesis of C. albicans. Our results demonstrated the important roles of EVs on C. albicans own growth for the first time and highlight its synergism with C. albicans to increase the pathogenesis. KEY POINTS: • C. albicans extracellular vesicles (EVs) promoted its own growth. • EVs activated the l-arginine/NO pathway to reduce ROS and apoptosis of C. albicans. • EVs enhanced the damage to the host cell caused by C. albicans.

Keywords: Cell apoptosis; Extracellular vesicles; Fungal infection; Intracellular ROS; Pathogenesis.

Fig. 1

C. albicans EVs promoted its growth. a SEM of C. albicans EVs isolated from YNB medium. Scale bar, 1 μm. b TEM of C. albicans EVs isolated from YNB medium. Scale bar, 100 nm. c Range of size distribution of C. albicans EVs isolated from YNB medium measured by nanoparticle tracking analysis (NTA). d CFUs of C. albicans grown in RPMI 1640 medium treated with different concentrations of EVs and PBS were served as control. e Growth curves of C. albicans treated with 15 μg/mL EVs and PBS were served as control. f C. albicans treated with different EVs grew in different media. All of the experiments were performed in three distinct replicates, and the data are presented as the means ± SD, **p < 0.05, ****p < 0.0001, no significance (ns) p > 0.05

Fig. 2

Transcriptomic analysis of C. albicans treated by EVs. a The enriched KEGG pathways of C. albicans treated with 15 μg/mL EVs compared to that from C. albicans treated with PBS. b Heat map of shifted specific genes from the arginine biosynthesis and arginine degradation pathways

Fig. 3

The growth promotion of EVs was dependent on arginine. a Effects of different arginine concentrations on the growth regulation induced by 15 μg/mL EVs. EVs were isolated from YNB medium and C. albicans grew in YNB medium. PBS was served as control. b Effects of the growth regulation induced by 15 μg/mL EVs in YPD medium with or without arginine. EVs were isolated from YNB medium. PBS was served as control. c Effect of media containing different amino acids on the growth regulation induced by 15 μg/mL EVs. EVs were isolated from YNB medium and C. albicans grew in YNB medium. All of the experiments were performed in three distinct replicates, and the data are presented as the means ± SD, *p < 0.05, ***p < 0.001, ****p < 0.0001, no significance (ns) p > 0.05

Fig. 4

EVs activated the l-arginine/NO pathway. a YHB1 mRNA expression in C. albicans after 15 μg/mL EVs’ treatment. EVs were isolated from YNB medium and C. albicans grew in YNB medium with 0.2% arginine. b Intracellular NO content in C. albicans after treatment with 15 μg/mL EVs in different medium. EVs were isolated from YNB medium. All of the experiments were performed in three distinct replicates, and the data are presented as the means ± SD, **p < 0.01, ***p < 0.001, ****p < 0.0001, no significance (ns) p > 0.05

Fig. 5

EVs decreased ROS accumulation of C. albicans to reduce fungal cell apoptosis. a Intracellular ROS content in C. albicans. The triangle-labeled group was C. albicans grew in YNB medium with 0.2% arginine, and the circle-labeled group was C. albicans grew in YNB medium without arginine. The control group was C. albicans treated with PBS, and the experimental group was C. albicans treated with 15 μg/mL EVs. The percentages shown in the figure are C. albicans treated with EVs vs C. albicans treated with PBS. b Effect of oxidant (H₂O₂) treatment on the growth regulation of C. albicans by 15 μg/mL EVs. c Effect of antioxidant (GSH) treatment on the growth regulation of C. albicans by 15 μg/mL EVs. d Flow cytometry is used to detect the proportion of early apoptosis, late apoptosis, and cell necrosis in C. albicans. The first three figures are PBS-treated groups, and the last three figures are 15 μg/mL EV-treated groups. e Statistical analysis of flow cytometry. All of the experiments above were used EVs isolated from YNB medium and C. albicans grew in YNB medium with 0.2% arginine. All the experiments were performed in three distinct replicates, and the data are presented as the means ± SD, *p < 0.05, ****p < 0.0001, no significance (ns) p > 0.05

Fig. 6

EVs’ synergies with C. albicans to destroy host cells. a Percentage of cytotoxicity of different cells after 15 μg/mL EVs and PBS control treatment. EVs were isolated from RPMI 1640 medium and the cells grew in DMEM medium. b Cytotoxicity of different concentrations of C. albicans and EVs in RAW264.7 cells. All of the experiments were performed in three distinct replicates, and the data are presented as the means ± SD, ***p < 0.001, ****p < 0.0001

Fig. 7

Schematic diagram of growth promotion of EVs’ pathway. EVs activated the l-arginine/nitric oxide pathway to increase the intracellular NO levels, then inhibited the ROS accumulation to reduce the cell apoptosis and increased its pathogenicity