Fungal Extracellular Vesicles Are Involved in Intraspecies Intracellular Communication

Abstract

Fungal infections are associated with high mortality rates in humans. The risk of fungal diseases creates the urgent need to broaden the knowledge base regarding their pathophysiology. In this sense, the role of extracellular vesicles (EVs) has been described to convey biological information and participate in the fungus-host interaction process. We hypothesized that fungal EVs work as an additional element in the communication routes regulating fungal responses in intraspecies interaction systems. In this respect, the aim of this study was to address the gene regulation profiles prompted by fungal EVs in intraspecies recipient cells. Our data demonstrated the intraspecies uptake of EVs in pathogenic fungi, such as Candida albicans, Aspergillus fumigatus, and Paracoccidioides brasiliensis, and the effects triggered by EVs in fungal cells. In C. albicans, we evaluated the involvement of EVs in the yeast-to-hypha transition, while in P. brasiliensis and A. fumigatus the function of EVs as stress transducers was investigated. P. brasiliensis and A. fumigatus were exposed to an inhibitor of glycosylation or UV light, respectively. The results demonstrated the role of EVs in regulating the expression of target genes and triggering phenotypic changes. The EVs treatment induced cellular proliferation and boosted the yeast to hyphal transition in C. albicans, while they enhanced stress responsiveness in A. fumigatus and P. brasiliensis, establishing a role for EVs in fungal intraspecies communication. Thus, EVs regulate fungal behavior, acting as potent message effectors, and understanding their effects and mechanism(s) of action could be exploited in antifungal therapies. IMPORTANCE Here, we report a study about extracellular vesicles (EVs) as communication mediators in fungi. Our results demonstrated the role of EVs from Candida albicans, Aspergillus fumigatus, and Paracoccidioides brasiliensis regulating the expression of target genes and phenotypic features. We asked whether fungal EVs play a role as message effectors. We show that fungal EVs are involved in fungal interaction systems as potent message effectors, and understanding their effects and mechanisms of action could be exploited in antifungal therapies.

Keywords: Aspergillus fumigatus; Candida albicans; Paracoccidioides brasiliensis; cellular communication; extracellular vesicles; fungal biology; fungal infections.

FIG 1

Evaluation of extracellular vesicles (EVs) uptake in different fungal species. The absorption of radioactive VEs was evaluated after 0, 1, 6, 12, and 24 h in the following yeast cells: P. brasiliensis (A), A. fumigatus (B), and C. albicans (C).

FIG 2

EVs from P. brasiliensis as cellular communicator during ER stress. (A) Schematic representation of EVs obtained after tunicamycin exposure. The relative expression of UPR genes HACA (B) and IRE1 (C) was determined after EVs uptake (4 × 10⁹/ml EVs in 10⁵/ml recipient cells). Significantly different values are indicated by asterisks as determined using ANOVA followed by Tukey’s post hoc test (P < 0.05). The calibrator was the NO EVs condition, and the positive control was tunicamycin (TM).

FIG 3

EVs from A. fumigatus as stress message effectors. (A) Schematic representation of EVs obtained from regular cultures, without UV exposure (CONTROL EVs), and EVs obtained from UV-exposed cultures (UV EVs). The thunder diagram represents UV light exposure. The EVs uptake was performed with 5 × 10⁸/ml of EVs in 10⁴/ml recipient cells. (B) The colony formation was counted and a percentage value was determined after EVs uptake. (C to E) RT-qPCR for A. fumigatus genes mpkC (C), akuA (D), and nimA (E). Relative expression was assessed using the NO EVs condition as a reference sample and 18S and βtub as a reference for normalization. Significantly different values are indicated by asterisks as determined using ANOVA followed by Tukey’s post hoc test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 4

EVs heighten the cellular proliferation and pseudohypha formation in C. albicans. (A) Schematic representation of EVs obtained from yeast cultures (CONTROL EVs) and yeast-to-hypha cultures (TRANS EVs). (B) Morphological appearance was investigated by microscope images of C. albicans cells grown on YPD, pH 7.4, at 37°C after EVs uptake for 2 h. Green arrows depict clump structures observed in cultures that underwent TRANS EVs uptake. (C) XTT reduction assay to assess cellular proliferation after EVs uptake during 2 h of growth on transition condition. Identification of hypha and pseudohypha morphologies in C. albicans grown on transition condition for 4 h is shown. (D) Black arrows depict pseudohyphae and blue arrows depict hypha structures. (E) Estimation of morphotype abundance after 4 h of C. albicans growth on transition condition.

FIG 5

EVs prompted a boost in yeast-to-hypha transition gene expression response. The EVs uptake was carried out with EVs (5 × 10⁸/ml) and C. albicans in cellular density adjusted to an OD₆₀₀ of 0.100 to 0.130. RT-qPCR evaluated a set of C. albicans genes after EVs uptake during 1 h and 4 h of growth on transition cultures. HWP1 (A), SEC24 (B), CHT2 (C), and SAP5 (D). The relative expression was assessed using NO EVs condition at the 1-h time point as the reference sample after normalization with the RPP2B and TDH3 genes. Significantly different values are indicated by asterisks, determined using ANOVA followed by Tukey’s post hoc test (*, P < 0.05; **, P< 0.01; ***, P < 0.001).