Mass Spectrometry Analysis Reveals Lipids Induced by Oxidative Stress in Candida albicans Extracellular Vesicles

Abstract

Candida albicans is a commensal fungus in healthy humans that causes infection in immunocompromised individuals through the secretion of several virulence factors. The successful establishment of infection is owing to elaborate strategies to cope with defensive molecules secreted by the host, including responses toward oxidative stress. Extracellular vesicle (EV) release is considered an alternative to the biomolecule secretory mechanism that favors fungal interactions with the host cells. During candidiasis establishment, the host environment becomes oxidative, and it impacts EV release and cargo. To simulate the host oxidative environment, we added menadione (an oxidative stress inducer) to the culture medium, and we explored C. albicans EV metabolites by metabolomics analysis. This study characterized lipidic molecules transported to an extracellular milieu by C. albicans after menadione exposure. Through Liquid Chromatography coupled with Mass Spectrometry (LC-MS) analyses, we identified biomolecules transported by EVs and supernatant. The identified molecules are related to several biological processes, such as glycerophospholipid and sphingolipid pathways, which may act at different levels by tuning compound production in accordance with cell requirements that favor a myriad of adaptive responses. Taken together, our results provide new insights into the role of EVs in fungal biology and host–pathogen interactions.

Stress Conditions Affect the Immunomodulatory Potential of Candida albicans Extracellular Vesicles and Their Impact on Cytokine Release by THP-1 Human Macrophages

“…It has been demonstrated previously that EVs released by bacteria and fungi under stress conditions triggered by the presence of antimicrobial drugs differ in terms of size, amount, and immunogenic properties from those produced under controlled conditions, as reported for Enterococcus faecium and Candida auris [19,52]. Furthermore, previous work by Trentin et al [42] demonstrated that the presence of menadione as an oxidative-stress inducer could affect the lipid composition of C. albicans' vesicles, and, additionally, the number of EVs produced under these conditions was higher [42]. Therefore, it might be assumed that such stress-related structures may also differ in functionality from those produced under the conditions of undisrupted redox balance.…”

“…Oxidative stress was induced by the presence of the vitamin K-derived quinone-menadione-leading to the production of intracellular ROS at various cellular locations. Fungal cells were gently scratched from agar plates and suspended in PBS buffer; then, the differential centrifugation technique with the final ultracentrifugation step was applied to eliminate cells and cell debris and subsequently obtain EVs from the supernatant; this method has already been used successfully for the isolation of fungal EVs [42][43][44]. To confirm the presence of EVs in obtained samples, transmission electron microscopy (TEM) imaging was performed, and spherical structures differing in size and surrounded by a lipid bilayer were present in samples from each growth condition (Figure 1A-C).…”

“…In the case of Candida fungi, the production of EVs is currently well documented for C. albicans [32][33][34][35][36][37] and non-albicans Candida species-C. glabrata, C. tropicalis, C. parapsilosis [38,39], C. auris [36], and C. haemulonii [40]. It has been shown recently that the amount of fungal EVs produced and their molecular composition may differ depending on the growth conditions and the presence of stressful milieu [41,42]. Therefore, we hypothesize that the conditions of oxidative stress or host concentration of CO 2 , under which C. albicans EVs are produced, may affect the EVs' release yield, the properties of the vesicles' components, and the effect of EVs on the host cells.…”

Extracellular Vesicle Protein Expression in Doped Bioactive Glasses: Further Insights Applying Anomaly Detection

“…As the Section 1.5 mentions, traditional proteomic techniques may not be optimized for EVs’ unique characteristics [ 69 ], and enriched analysis could support deeper biological understanding. Also, newer technologies and methodologies, such as improved mass spectrometry approaches to recognize molecular networking from MS data [ 70 ], which can reveal interactions or regulatory processes, and single-vesicle analysis techniques, are continually being developed to address these challenges [ 71 ]. Ongoing research efforts are focused on refining protocols and producing innovative techniques to improve the accuracy and reproducibility of proteomic analysis of extracellular vesicles, such as performing extensive quantitative analysis to compare populations of proteins with different features [ 72 ].…”

Extracellular vesicles produced during fungal infection in humans are immunologically active

“…Sphingosine and phytosphingosine are compounds present in the cell membranes of mammals and yeast with the inclusion of specific mammalian tissues such as epidermis, small intestine, and myelin, respectively (38). Furthermore, these compounds have been identified in pathogenic strains of C. albicans (26) and C. neoformans (39), and in the EVs of C. albicans (40). Previous studies revealed the importance of cholesterol and sphingomyelin in controlling the host immune response in phagocytic cells (41,42).…”