Coregulation of extracellular vesicle production and fluconazole susceptibility in Cryptococcus neoformans

Abstract

Resistance to fluconazole (FLC), the most widely used antifungal drug, is typically achieved by altering the azole drug target and/or drug efflux pumps. Recent reports have suggested a link between vesicular trafficking and antifungal resistance. Here, we identified novel Cryptococcus neoformans regulators of extracellular vesicle (EV) biogenesis that impact FLC resistance. In particular, the transcription factor Hap2 does not affect the expression of the drug target or efflux pumps, yet it impacts the cellular sterol profile. Subinhibitory FLC concentrations also downregulate EV production. Moreover, in vitro spontaneous FLC-resistant colonies showed altered EV production, and the acquisition of FLC resistance was associated with decreased EV production in clinical isolates. Finally, the reversion of FLC resistance was associated with increased EV production. These data suggest a model in which fungal cells can regulate EV production in place of regulating the drug target gene expression as a first line of defense against antifungal assault in this fungal pathogen. IMPORTANCE Extracellular vesicles (EVs) are membrane-enveloped particles that are released by cells into the extracellular space. Fungal EVs can mediate community interactions and biofilm formation, but their functions remain poorly understood. Here, we report the identification of the first regulators of EV production in the major fungal pathogen Cryptococcus neoformans . Surprisingly, we uncover a novel role of EVs in modulating antifungal drug resistance. Disruption of EV production was associated with altered lipid composition and changes in fluconazole susceptibility. Spontaneous azole-resistant mutants were deficient in EV production, while loss of resistance restored initial EV production levels. These findings were recapitulated in C. neoformans clinical isolates, indicating that azole resistance and EV production are coregulated in diverse strains. Our study reveals a new mechanism of drug resistance in which cells adapt to azole stress by modulating EV production.

TheCryptococcusextracellular vesicle transcriptome

“…Besides, the H/CA ribonucleoprotein complex subunit 3, Nop10 (CNAG_01049) was found in our EV proteomic analysis (Rizzo et al 2023) which is in agreement with an active targeting of some snoRNAs to EVs. Our research also confirmed the very poor conservation of snoRNA sequences in fungi.…”

“…Reads were then aligned to the C. neoformans H99 reference genome (Janbon et al 2014) and the number of reads aligned to each annotated feature was counted (Wallace et al 2020). We used this counting to compare the mRNA and lncRNA abundances in EVs and in cells producing these EVs (Rizzo et al 2023). Our analysis did not reveal spectacular enrichment of any mRNA in EVs (Figure 6).…”

“…Nevertheless, the methods of growth, EV purification, and RNA sequencing employed in these studies vary significantly. We also know that the growth conditions can have a strong influence on EV production and EV composition in fungi (Reis et al 2021;Amatuzzi et al 2022;Rizzo et al 2023). This suggested that further analyses would be beneficial for gaining a deeper understanding of the fungal EV transcriptome structure.…”

“…After 18h of ultracentrifugation at 100 000 g, we observed a single whitish ring at a centered position within the otherwise transparent gradient (Figure 1B). Examination of twelve gradient fractions using both flow cytometry with a NanoFCM TM apparatus and ergosterol concentration evaluation (Rizzo et al 2021b;Rizzo et al 2023) showed that the majority of EVs are found within the fractions 6, 7 and 8 (Figure 1C). In fact, the 7 th fraction, which corresponds to the whitish ring position, exhibited the highest concentration of EVs, strongly suggesting that this ring corresponds to concentrated EVs.…”

Ergosterol mediates aggregation of natamycin in the yeast plasma membrane

“…The lipidic nature of such structures could be confirmed by staining with Nile Red, a membrane inserting hydrophobic dye, using confocal microscopy (not shown). Formation of such extracellular vesicles could represent a first line of defense to get rid of the drug molecules, as has been shown in C. neoformans upon azole treatment 64 . Distinct aggregates with a size of 350 to 500 nm were occasionally also observed inside the vacuole by SXT (Fig.…”