Deciphering cargo contents in extracellular vesicles of Candida haemulonii var. vulnera

Abstract

Candida haemulonii comprises a group of pathogenic fungi with notable resistance to antifungal treatments and diagnostic challenges. The Candida haemulonii variety vulnera is of particular clinical importance due to its multidrug resistance and association with invasive infections, particularly in immunocompromised patients such as diabetics and neonates. Moreover, it has been implicated in hospital outbreaks, posing a significant challenge for infection control and antifungal treatment. Extracellular vesicles (EVs) released by these fungi play critical roles in pathogen-host interactions, potentially influencing antifungal resistance, immune evasion, and virulence. Previous studies conducted by our group have demonstrated that EVs carry immunogenic cargo that can influence the host's immune response. A comprehensive understanding of the molecular composition of these EVs is crucial for unraveling the mechanisms that govern resistance and virulence in C. haemulonii var. vulnera. This study characterizes the proteomic and miRNA-like cargo of EVs from C. haemulonii var. vulnera, revealing components that contribute to its adaptation and survival mechanisms. Proteomic analysis identified 124 EV-specific proteins, including BMH1, TEF1, CDC19, and PDC11, which are linked to processes such as metabolic adaptation, cell wall remodeling, and biofilm formation. miRNA-like molecules associated with mitochondrial function, such as the electron transport chain and regulation of the citric acid cycle, were also detected. These findings provide insights into EV-mediated molecular mechanisms driving fungal pathogenesis and resistance. By characterizing the EV cargo, this study highlights potential targets for antifungal therapies and offers a framework for understanding EV roles in fungal adaptation and pathogenicity.

Importance: The Candida haemulonii complex poses significant clinical challenges due to its intrinsic resistance to conventional antifungal therapies and diagnostic complexities. This research explores the cargo of EVs released by C. haemulonii var. vulnera, revealing a selective mechanism for exporting proteins and RNA molecules critical to the fungus' adaptation and survival in diverse environments. Proteomic and RNA analyses identified molecules involved in metabolic reprogramming, immune modulation, and stress response. These findings highlight the role of EVs in mediating host-pathogen interactions, facilitating immune evasion, and contributing to fungal virulence. Understanding the EV cargo expands our knowledge of fungal biology and underscores the therapeutic potential of targeting EV-associated molecules in antifungal strategies.

Keywords: Candida haemulonii species complex; Candidiasis; Emergent Yeast; Extracellular vesicles.

Graphical abstract

Fig. 1

Characterization of vesicles secreted by Candida haemulonii var. vulnera cells. Representative graph depicting the size distribution and concentration of EVs of four different replicates (A), which were isolated from Candida haemulonii var. vulnera cells after cultivation in SDA for 48 h. (B) Representative TEM images of Candida haemulonii var. vulnera EVs. The scale bars correspond to 200 nm. (C) Representative SEM images show the association of fungal EVs (indicated in blue) with the yeast’s cell wall, were manually colored using GIMP2® software Magnification of 20,000x.

Fig. 2

Proteomic profile of proteins found in Candida haemulonii var. vulnera 1112/2016 yeast and exclusive proteins transported by extracellular vesicles (EVs) from Candida haemulonii var. vulnera 1112/2016. A) The Venn diagram illustrates the number of proteins identified as either common or exclusive to EVs from Candida haemulonii yeast. B) Percentage of proteins common to Candida haemulonii cells involved in biological processes, molecular functions, and cellular processes. C) Percentage of proteins exclusive to EVs from Candida haemulonii involved in biological processes, molecular functions, and cellular processes.

Fig. 3

Interaction networks of proteins from yeast and extracellular vesicles (EVs) from Candida haemulonii var. vulnera. A) Protein-protein interaction network derived from commonly expressed proteins with increased expression in both yeast cells and EVs of Candida haemulonii var. vulnera. (B) Network of protein-protein interactions involving ribosomal proteins with common expression in both yeast cells and EVs of Candida haemulonii var. vulnera. C) Protein-protein interaction network based on proteins showing increased expression, specifically in EVs of Candida haemulonii var. vulnera. All protein interactions depicted are statistically significant (p < 0.05). Highlighted terms represent relevant biological functions. For the comparison, the STRING database (v.12.0) protein-protein association networks and functional enrichment analyses were used [REF PMID: 36370105].

Fig. 4

Functions of key proteins in diverse biological processes and transported by the extracellular vesicles (EVs) of Candida haemulonii var. vulnera. A) Identified proteins: 14–3–3 protein (BMH1); Pyruvate kinase (CDC19); Elongation factor 1-alpha 1 (TEF 1). B) Proteins enriched in the EVs of Candida haemulonii var. vulnera, significantly contributing to a molecular pathway. The numbers indicate p-values adjusted for list size, where at least 95% of matches are statistically significant.

Fig. 5

Expression of miRNA-like in yeast and the extracellular vesicles (EVs) of Candida haemulonii var vulnera. (A-C) Heatmaps illustrate genes with the strongest associations to pathways related to biological processes (A), cellular components (B), and molecular functions (C) based on Gene Ontology (GO) analysis. (D) Heatmap showing the top 25 miRNA-like molecules with the highest expression levels in Candida haemulonii yeast and extracellular vesicles (EVs).

Fig. 6

Gene ontology function profile of proteins corresponding to high-coverage RNA sequences identified in miRNA-enriched fractions from extracellular vesicles (EVs) and yeast cells of Candida haemulonii var. vulnera. The x-axis indicates the number of hits identified for each term in the gene ontology function profile of proteins associated with high-coverage RNA sequences isolated from miRNA-enriched fractions of both extracellular vesicles and yeast cells of Candida haemulonii var. vulnera.

Fig. 7

Key genes and miRNA-mediated interactions in Candida haemulonii var. vulnera. A) Molecular interaction networks of target genes associated with GO functional pathways enriched by miRNA-like from Candida haemulonii var. vulnera. Networks were constructed using STRING and visualized in Cytoscape based on co-expression analysis, with Candida albicans chosen as the reference organism (NCBI taxonomy Id: 237,561). B) Hub genes within the molecular interaction networks observed in Candida haemulonii var. vulnera yeast cells and EVs.