Ergosterol-Dependent Membrane Stress and Calcineurin-HSP90 Signaling Govern Fluorinated Sulfone-Induced Cell Death in Candida albicans.

Ergosterol-Dependent Membrane Stress and Calcineurin-HSP90 Signaling Govern Fluorinated Sulfone-Induced Cell Death in Candida albicans - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Search Log in Primary site navigation Search Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Mycopathologia . 2026 May 29;191(3):57. doi: 10.1007/s11046-026-01081-6 Ergosterol-Dependent Membrane Stress and Calcineurin- HSP90 Signaling Govern Fluorinated Sulfone-Induced Cell Death in Candida albicans Anna Staniszewska 1, 2 , Łukasz Kuryk 3, 5 , Deepak Kala 4 , Izabela Ziuzia-Graczyk 5 , Zbigniew Ochal 6 , Monika Staniszewska 5, ✉ Author information Article notes Copyright and License information 1 Medical University of Lublin, Lublin, Poland 2 Central Clinical Hospital, Medical University of Warsaw, Banacha 1a, 02-097 Warsaw, Poland 3 National Institute of Public Health NIH – National Research Institute, Warsaw, Poland 4 CENTERA Laboratories, Institute of High Pressure Physics, Polish Academy of Sciences, Warsaw, Poland 5 Centre for Advanced Materials and Technologies, Warsaw University of Technology, Poleczki 19, 02-822 Warsaw, Poland 6 Chair of Drug and Cosmetics Biotechnology, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland Handling Editor: Sadri Znaidi. ✉ Corresponding author. Received 2026 Jan 13; Accepted 2026 May 18; Issue date 2026. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13221380  PMID: 42213245 Abstract The antifungal activity of halogenated methyl sulfones depends on both the type and number of halogen atoms in the halogenomethylsulfonyl moiety. In this study, 28 halogenomethylphenyl sulfones were evaluated for minimal inhibitory (MIC) and fungicidal concentrations (MFC) against Candida albicans . Active compounds were further tested for cytotoxicity in vitro using the Vero E6 cell line. Notably, 4-(4-chloro-3,4-dichlorophenoxy)-2-nitrophenyl difluoromethyl sulfone (compound 15 ) showed fungicidal activity while remaining non-toxic at effective doses. Additionally, no adverse effects were observed in Galleria mellonella larvae treated with 15 in vivo. Mechanistic studies with 15 at a sub-inhibitory concentration (2 μg/mL) showed predominant induction of necrosis in C. albicans cells (88.43%) and protoplasts (96.25%), along with increased reactive oxygen species (ROS) production (31.17%). Cell cycle analysis revealed arrest in the G0 phase (99%), consistent with a pro-necrotic mechanism. RT-qPCR indicated that HSP90 gene expression contributes to C. albicans resistance to 15 . Confocal microscopy confirmed the compound’s fungicidal activity against planktonic and sessile C. albicans cells in vitro. Modulation experiments with exogenous ergosterol showed that reducing sterol concentrations decreased MIC values, suggesting that the fungal cell membrane is the primary target of 15 and that its interaction with ergosterol is critical for activity. Overall, 15 demonstrated antifungal activity comparable to that of amphotericin B and is a promising lead candidate for the development of new antifungal agents for local and systemic candidiasis. Supplementary Information The online version contains supplementary material available at 10.1007/s11046-026-01081-6. Keywords: Halogenomethylphenyl sulfone, C. albicans , Necrosis, Ergosterol, HSP90 , G. mellonella Introduction The emergence of antifungal resistance in pathogenic fungi poses a significant challenge to the effective treatment of fungal infections [ 1 – 4 ]. Resistance to first-line antifungal agents, including fluconazole and amphotericin B, is increasingly limiting therapeutic options and worsening clinical outcomes [ 4 – 6 ]. Candida albicans is typically a component of the human microbiome, but can become dangerous in immunocompromised patients [ 6 ]. Given the declining effectiveness and toxicity of commonly used antifungal drugs [ 7 , 8 ], our research suggests that sulfones may be promising alternatives due to their potent fungicidal activity and low toxicity [ 7 – 12 ]. In a previous study, we demonstrated that sulfones represent a promising class of future antifungal agents due to their high fungicidal activity and low cytotoxicity [ 9 – 12 ]. The necroptotic pathway in mammalian cells is a regulated form of necrotic cell death characterized by membrane permeabilization and disruption of ionic homeostasis, ultimately leading to cell lysis via pore formation [ 13 ]. Although canonical components of the mammalian necroptotic machinery have not been identified in fungi, several features of sulfone-induced cell death in C. albicans show striking functional parallels to necroptosis [ 9 ]. Calcineurin signaling is crucial for determining C. albicans susceptibility to sulfones [ 9 ]. Mutations in the CNB1 gene increase sensitivity to sulfones and reduce ergosterol production, further enhancing susceptibility [ 9 ]. This underscores calcineurin's role in membrane homeostasis and sterol biosynthesis [ 9 ]. Therefore, CNB1 mutants were included to evaluate the involvement of calcineurin-dependent pathways in the cellular response to sulfone treatment. We evaluated a series of sulfone-containing compounds as potential novel antifungals against C. albicans . After initial screening, we determined the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the tested compounds. A sulfone found to be non-cytotoxic in vitro with mammalian cell lines was subsequently assessed in vivo using the Galleria mellonella infection model. This selected sulfone was further examined for its effects on the fungal cell cycle and to elucidate its mechanisms of action. The study investigated cell death in C. albicans within the context of fungal pathogenesis by analyzing cellular responses to sulfone treatment and HSP90 expression. Specifically, it explored the relationship between sulfone-induced cell death and HSP90 regulation to clarify the stress-response mechanisms activated during antifungal exposure. The use of CNB1 -deficient strains allowed us to assess whether calcineurin-dependent defense mechanisms modulate the antifungal activity of sulfone. To further characterize the biological activity of the selected sulfone, we investigated its effects on C. albicans -macrophage interactions and biofilm development. Ultimately, based on the collected data, we identified the sulfone as a promising candidate for further development as a novel antifungal. Results Biological Study Anti- C. albicans Activity Tested compounds at concentrations ranging from 0.25 to 16 μg/mL showed fungistatic activity against C. albicans 90,028; growth inhibition exceeded 97% in each case (Table S1 in the Supplementary material). Some compounds showed MIC values, which were visually assessed and are presented in Table 1 and Table S1 (Supplementary Material). Compound 15 displayed fungicidal activity at 8 μg/mL; compounds 24 , 26 , and 32 showed activity at the highest tested concentration (lgR > 3) (Table 1 ). The sulfone solvent did not affect the results (DMSO at a final concentration of 0.96% in Figure S1 ). Table 1. Killing activity against C. albicans (MFC) Sulfon derivative Conc (µg/mL) Colony number mean of treated wells colony number mean of control wells LgR 4* 16 57 65 0.06 5 31 44 0.15 6 12 61 0.91 7 52 61 0.07 8 13 27 0.32 9 4 61 1.18 10 96 65 − 0.17 13 104 65 − 0.20 14 11 44 0.60 15* 8 0 61 6.79 16 16 19 44 0.36 18 184 65 − 0.45 19 93 36 − 0.41 24 0 61 6.79 26* 0 44 6.64 28 1 44 1.64 30 93 36 − 0.41 31* 1 27 1.43 32 0 27 6.43 33 34 61 0.25 35 36 36 0.00 36 1 44 1.64 40 106 65 − 0.21 41 23 44 0.28 48 58 61 0.02 55 41 61 0.17 62 32 27 − 0.07 65 19 44 0,36 Open in a new tab Lg R means decimal logarithm of the reduction in C. albicans viability (R) calculated following the scheme: lg R = lg CFU/ mL GCW—lg CFU/ mL CTW, where GCW means growth control wells and CTW means compound tested well. Colony fungal units CFU/ mL = (mean number of colonies grown on three plates) x (inverse of the dilution = 10 5 ) × 10 (counted per 1 mL). MFC (minimum fungicidal concentration) is the lowest concentration of a compound that kills ≥ 99.9% of viable cells compared to the growth control after 48 h of incubation, and is claimed for lg R ≥ 3 (marked in bold). The MIC values for compounds 4, 15, 26, and 31 were 8, 4, 4, and 2, respectively, as assessed visually after 48 h. Data were presented as means for free independent experiments In Table 1 it was explained in the legend in the brakets The Toxic Effects of Sulfones: An Examination of their Impact in vitro and in vivo In vitro, for the selected sulfone at the tested concentration, where inhibition and/or fungicidal action was observed, there was no toxic effect on the primary Vero cell line (Fig. 1 ). The index CC 50 for 15 was above 8 μg/mL (fungicidal concentration). Fig. 1. Open in a new tab Cytotoxicity of halogenomethylphenyl sulfones on Vero Cell Line ATCC CCL-81 in vitro Legend: Cytotoxicity of selected sulfones; red line points out 50% reduction of cell viability compared to growth control (50% cytotoxic concentration, CC 50 ). After 18 h of incubation, measurements were made using a spectrophotometer at 475 nm and 660 nm. The cytotoxicity was calculated using the formula: %cytotoxicity = (SA test sample–SA positive control)/ (SA negative control–SA positive control) × 100%; SA (specific absorbance) = A 475 (test)–A 475 (blank)–A 660 (blank). Data were presented as mean ± SD for free independent experiments Compound 15 does not exhibit pronounced acute toxicity (Fig. 2 A–D), as indicated by a relatively high survival rate (Fig. 2 A) and a stable health index (Fig. 2 D). However, it induces significant sublethal effects, including reduced melanization (Fig. 2 B) and impaired cocoon formation (Fig. 2 C), suggesting an impact on physiological and developmental processes. These results indicate that 15 induces a time-dependent decrease in survival at both tested concentrations, as evidenced by statistically significant negative slopes ( p = 0.0287 and 0.0443), with a stronger effect at higher concentrations. Fig. 2. Open in a new tab Effects of 15 on G. mellonella viability and physiological parameters after injections. A larvae survival, B melanization, C cocoon formation, and D health score index of larvae treated with compound 15 at 16 or 8 µg/mL. The following conditions were prepared: negative control (untreated larvae); RMPI (control larvae injected with medium); DMSO (control larvae injected with solvent at 0.96%). The larvae were monitored daily for 5 days, focusing on survival, melanization, and cocoon formation to calculate the health score (as described previously by Staniszewska et al . [ 12 ]). Each larval group contained 10 model organisms. Data were presented as mean ± SEM for three independent experiments. All data (A-D) were analyzed using linear regression. with F-test (GraphPad Prism version 11.0.0 (84)). Differences were considered statistically significant at p < 0.05 Histopathological Assessment of Sulfone-Mediated Inhibition of C. albicans Infection in Larvae Treatment with 15 modulated host response to C. albicans infection in a time-dependent manner (Fig. 3 ). In terms of survival (Fig. 3 A), both pre-treatment (− 1 h) and post-treatment (+ 1 h) with 15 resulted in higher survival rates than in the C. albicans group, with a more pronounced protective effect observed with pre-treatment (− 1 h, p = 0.0206). Linear regression analysis indicated a less steep decline in survival over time in the treated groups than in the infected controls. Melanization response (Fig. 3 B) was reduced in the C. albicans group, whereas treatment with 15 partially preserved melanization levels, particularly at pre-treatment (− 1 h), suggested modulation of the host immune response. Cocoon formation (Fig. 3 C) was markedly impaired upon C. albicans infection. Treatment with 15 mitigated this effect, with higher cocoon formation rates observed in treated groups, particularly in pre-treated samples (− 1 h, p = 0.0199). Similarly, the health score index (Fig. 3 D) decreased over time in infected larvae, whereas treatment with 15 stabilized this parameter, indicating an overall protective effect on host physiological condition. Overall, these results suggest that 15 exerts a protective effect against C. albicans -induced damage, particularly when administered prior to infection (− 1 h, p = 0.0199). Fig. 3. Open in a new tab Inhibition of candidiasis in G. mellonella larvae treated with 15 . Treatment of G. mellonella with 15 at 8 µg/mL before and after C. albicans infection: A. larvae survival after C. albicans infection and treatment with 15 : 1 h before (− 1 h) or 1 h after infection (+ 1 h); B. melanization after infection with C. albicans and 15 treatment (− 1 h) and (+ 1 h); C. coccon formation after infection with C. albicans and 15 treatment (− 1 h) and (+ 1 h); D . health score of larvae after infection with C. albicans and 15 treatment (-1 h) and (+ 1 h). Condition names: Negative control means untreated larvae; C. albicans means larvae infected with C. albicans ; infected, 15-1 h means larvae injected with the compound 1 h before infection with C. albicans ; 15 + 1 h means larvae injected with the compound 1 h after infection with C. albicans. Visual monitoring of viability, cocoon formation, and melanization was performed within 5 days, a health score was calculated (as described previously by Staniszewska et al . [ 21 ] , ). Data represented 3 (n = 10) independent experiments; expressed as mean ± SEM. All data (A–D) were analyzed using linear regression with F-test (GraphPad Prism version 11.0.0 (84)). Differences were considered statistically significant at p < 0.05 Microscope observations of the larvae’s semithin sections confirmed that there were no morphological alterations after treatment with 15 at 8 μg/mL (a few small nodules and hemocytes visible in Fig. 4 E) vs. control (untreated, RPMI, 0.96% DMSO in Fig. 4 A–C). Investigation of 15 treatments displayed impeding the development of candidiasis both for (+ 1 h) post- and (− 1 h) pre-infection (fewer nodules in Fig. FG) compared to infected larvae with huge nodules (aggregations of fungi cells and hemocytes in Fig. 4 D). Model treatment 15 (− 1 h) pre-infection (Fig. 4 F) was slightly more effective than (+ 1 h) post-infection (smaller nodules in Fig. 4 G). Fig. 4. Open in a new tab Infected Galleria mellonella was treated with a dosage of 15 , yielding positive results. G. mellonella larvae infected by C. albicans and treated with 15 in killing conc. 8 µg/mL for 6 days, stained with hematoxylin and eosin. Cross-section of the A untreated larvae, B treated with RPMI medium, C treated with 0.96% DMSO, D infected by C. albicans , E treated with 15 , F treated with 15 and after 1 h infected by fungal cells, and G inversely. BM-basement membrane, CT-cuticule, FB-fat body, G-gut, H-hemocytes, M-muscles, N-nodules. Data represent 3 (n = 10) independent experiments Sulfone Action on the Cell Membrane Exogenous ergosterol was used to determine whether 15 targets affect ergosterol-dependent membrane integrity. Partial growth restoration in the presence of ergosterol would indicate a membrane-associated mechanism of antifungal action [ 9 ]. The effect of 15 on fungal growth in the presence of exogenous ergosterol is presented in Fig. 5 and Table S2 . Cultures were supplemented with increasing concentrations of ergosterol (25, 125, and 250 µg/mL) to assess whether ergosterol can mitigate the growth inhibition caused by 15 . At low concentrations of 15 (0.25–2 µg/mL), absorbance values remain relatively high across all ergosterol conditions, indicating limited growth inhibition. As the concentration of 15 rises (4–16 µg/mL), there is a significant decrease in absorbance, reflecting reduced fungal growth. This inhibitory effect is reduced at higher exogenous ergosterol concentrations, particularly at 125 and 250 µg/mL, where absorbance values are consistently higher than in cultures supplemented with 25 µg/mL ergosterol. At higher concentrations of 15 (≥ 32 µg/mL), absorbance values converge across all ergosterol conditions, suggesting that while excess ergosterol can partially counteract the antifungal effect, growth inhibition predominates at elevated drug concentrations. Overall, these results indicate that exogenous ergosterol can partially restore fungal growth in the presence of 15 . This implies that the antifungal activity of 15 is at least partly linked to interference in ergosterol-dependent pathways or membrane integrity. This finding supports a mechanism of action that involves the fungal cell membrane rather than nonspecific cytotoxicity. Fig. 5. Open in a new tab Sulfone 15 influence on ergosterol C. albicans were treated with 15 at 0.25–512 µg/mL in YEPD medium with ergosterol at a concentration of 250, 125, and 25 µg/mL or without sterol. The measurements were made at 405 and 660 nm to determine absorbance (OD405—OD660) as a function of 15 concentrations at various final ergosterol concentrations in the medium after 48 h of incubation. GCW ( growth control wells ) means untreated cells without ergosterol in the medium. Data were presented as mean ± SD for free independent experiments. The MICs (minimal inhibitory concentration) were determined by visual readings for ergosterol at 250 µg/mL in the medium, where MIC 15 = 32 µg/mL, ergosterol at 125 µg/mL where MIC 15 = 16 µg/mL; ergosterol at 25 µg/mL where MIC 15 = 16 µg/mL; without ergosterol (0 µg/mL) where MIC 15 = 4 µg/mL. For 15 , MFCs (minimal antifungal concentrations) were pointed out (lg R ≥ 3) at 64 µg/mL (with ergosterol at 250 µg/mL in the medium); at 128 µg/mL (125 µg/mL); at 128 µg/mL (25 µg/mL); at 8 µg/mL (0 µg/mL) Flow Cytometry Analysis Treatment of C. albicans cells with 15 at 2 μg/mL (fungistatic concentration) caused an increase (~ 40-fold) in generating endogenous reactive oxygen species (ROS) vs. the negative control (Table 2 ), in the same concentration, most of the 15 -treated C. albicans cells were killed mainly through necrosis in both studies: non- and protoplast cells (Table 3 ). Cell death was analyzed by flow cytometry using Annexin V/PI staining (Table 3 ). We distinguished viable cells (Annexin V⁻/PI⁻), early apoptotic cells (Annexin V⁺/PI⁻), late apoptotic cells (Annexin V⁺/PI⁺), and necrotic cells (Annexin V⁻/PI⁺). The results indicate a predominant necrotic mechanism, characterized by loss of cell membrane integrity (Table 3 ). The action of 15 toward protoplast cells induced more rapid cell death than toward non-protoplast cells (7.82% units). Apoptosis was noted (early and late apoptosis of cells = 10.16%; protoplasts = 2.56%). Table 2. Reactive oxygen species (ROS) generated by the C. albicans 90,028 cells after treatment with 15 Sample ROS (%) ± SEM Control 0.10 0.00 Negative control 0.77 0.38 Positive control 84.80 11.59 Test 31.17 5.21 Open in a new tab Control means untreated C. albicans cells; Negative control means cells treated with DMSO; Test means C. albicans cells treated with 15 at MIC = 2 µg/mL for 18 h; Positive control means cells treated with 3% H 2 O 2 . To check the content of ROS (%) by flow cytometry, the dye 2,7-dichlorofluorescein diacetate was added to the samples. Data were presented as mean ± SEM for three independent experiments Table 3. Sulfone 15 influence on cell death (non- and protoplast cells) of C. albicans 90,028 Sample Live (%) Annexin V⁻/PI⁻ Necrosis (%) Annexin V⁻/PI⁺ Late apoptosis (%) Annexin V⁺/PI⁺ Early apoptosis (%) Annexin V⁺/PI⁻ Control 99.67 ± 0,58 0.00 ± 0.00 0.00 ± 0.00 0.33 ± 0.33 Negative control 99.00 ± 1.00 0.00 ± 0.00 0.00 ± 0.00 1.00 ± 1.00 Cells 1.41 ± 0.44 88.43 ± 0.36 8.83 ± 0.48 1.33 ± 0.13 Protoplast cells 1.19 ± 0.44 96.25 ± 0.64 2.03 ± 0.25 0.53 ± 0.02 Open in a new tab Non- and protoplast cells of C. albicans were treated with 15 at 2 µg/mL for 18 h. Control means untreated cells, and a negative control means treated with DMSO. Before taking measurements by flow cytometry, samples (without control) were stained Annexin-V and propidium iodide (PI). The scheme of staining: live cells Annexin-V (−) and PI (−); early apoptosis cells Annexin-V ( +) and PI (-); late apoptosis cells both Annexin-V ( +) and PI ( +); necrosis cells Annexin-V (−) and PI ( +). Data were presented as mean ± SEM for three independent experiments We also investigated changes in the cell cycle. Stress conditions induced by 15 at 2 μg/mL induced cells to enter the resting/metabolic state (G0/G1) phase, whereas untreated control cells were distributed across all phases (G0/G1, S, and G2/M) (Fig. 6 AB). Fig. 6. Open in a new tab Changes in the course of the C. albicans cell cycle induced by 15 Legend: C. albicans cells (test sample) were treated with 15 at 2 µg/mL for 18 h. The test sample and growth control (control) were stained using propidium iodide (PI) and then measured by flow cytometry. Based on DNA content in microorganisms (and their staining), cells were classified into a particular phase of the cell cycle: G0/1, S, or G 2 /M. Data were presented as mean ± SEM for three independent experiments. A percentage content of cells in a particular phase of the cell cycle B histogram of a control b test sample HSP90 Expression Analysis We hypothesized that the antifungal activity of 15 is mediated by disruption of the calcineurin– HSP90 regulatory network. Therefore, HSP90 expression was evaluated in calcineurin-deficient mutants to determine whether stress responses induced by 15 require an intact calcineurin pathway. RT-qPCR analysis of the HSP90 co-chaperone in stress responses induced by 15 at sub-MIC = 2 μg/mL was performed using single- and double- CNB1 gene deletion mutants grown either in calcium-supplemented medium or in the absence of calcium (Fig. 7 A–B and Table S3 ). In both conditions, 15 -treated cnb1Δ / cnb1Δ cells showed higher HSP90 mRNA expression compared to the untreated growth control (without Ca2 + : fourfold; with Ca2 + : ~ 1.96-fold) and to the treated cnb1Δ/CNB1 mutant (without Ca2 + : ~ 1.75-fold; with Ca2 + : ~ 2.26-fold) (Table S 3) . Calcium supplementation increased HSP90 mRNA expression in both 15 -treated mutants, particularly in cnb1Δ/cnb1Δ (Fig. 7 AB). In the single-gene deletion mutant, HSP90 expression decreased after incubation with 15 compared to the control (Fig. 7 A). Conversely, the opposite effect was observed when Ca2 + was supplemented in the medium (Fig. 7 B). Fig. 7. Open in a new tab Level of HSP90 expression in C. albicans under 15 Legend: C . albicans cells were treated with 15 at 2 µg/mL for 18 h A. in YEPD medium with uridine and without calcium ions B. in YEPD medium with uridine and calcium ions at 200 mM. The strain BWP17 was used as a calibrator (2 −ΔΔCt = 1). ACT1 gene was used as the reference gene. RT-qPCR obtained Ct measurements. To calculate the relative expression of the HSP90 gene, the scheme was followed: ΔC t = avg. C t HSP90 —avg. C t ACT1 , ΔΔC t = ΔC t —ΔC t parental strain . Data were presented as mean ± SD for three independent experiments 15-Treated Planktonic and Sessile C. albicans Assessed by CLSM To understand 15 ’s action mode, the phenotype alternations of planktonic C. albicans were investigated ( Fig. 8 A–H ). Treatment with 15 results in an increased number of PI-positive cells, indicating compromised membrane integrity and induction of cell death (Fig. 8 B). AO/PI staining showed limited dye uptake in control cells (Fig. 8 E–G), consistent with intact fungal cell wall (CFW-stained in Fig. 8 E, H) and membrane barriers. Treatment with 15 increased both PI and AO signals, indicating compromised cell envelope integrity (Fig. 8 A–C). Due to restricted dye permeability in C. albicans , AO staining reflects increased permeability rather than viability per se, and the assay primarily indicates loss of membrane integrity rather than specific cell death pathways (Fig. 8 A, C). Fig. 8. Open in a new tab Fluorescence-based assessment of planktonic C. albicans cell death induced by 15 . Representative CLSM images of C. albicans cells treated with 15 (8 µg/mL) (A–D) and untreated control cells (E–H). Cells were stained with propidium iodide (PI; red, indicating loss of membrane integrity/necrosis), acridine orange (AO; green, early apoptosis), and Calcofluor White (CFW; blue, cell wall). Panels show merged fluorescence (A, E), PI channel (B, F), AO channel (C, G), and CFW channel (D, H). Scale bar: 20 µm The study (Fig. 9 IAA’ – EE’) examines the interaction between C. albicans and macrophages and adhesion to macrophages (first step of biofilm formation in Fig. 9IIA-D) when exposed to 15 . Compound 15 induced the macrophages (Fig. 9 IAA’–CC’) to phagocytose blastoconidia (Fig. 9 IDD’ – EE’). Derivative 15 promoted macrophage-mediated phagocytosis of C. albicans blastoconidia (Fig. 9 IDD’–EE’). Fig. 9. Open in a new tab Interaction between C. albicans and macrophages following treatment with 15 .I. Representative fluorescence (A–E) and corresponding merged brightfield (differential interference contrast, DIC; A′–E′) images of unfixed sessile C. albicans cells and macrophages, double-stained with acridine orange (AO) and ethidium bromide (EB). (AA’) Viable macrophages after 18 h incubation, showing green nuclear fluorescence (AO). (BB’) Macrophages treated with compound 15 (16 μg/mL, 18 h) displayed a limited cytotoxic effect, as indicated by weak EB (red) fluorescence. (CC’) Co-culture after 2 h incubation showing C. albicans cells (green fluorescence) and macrophages (red fluorescence). (DD’) treatment with compound 15 (16 μg/mL, 2 h), showing polymorphic nuclei of macrophages (open arrowheads) and phagocytosed C. albicans blastoconidia (closed arrowheads). (EE’) After 18 h incubation, C. albicans cells (open arrowheads) and macrophages (closed arrowheads) are observed. Magnification ranges from 318 × to 438 × . Data represent two independent experiments (n = 3). II. Representative fluorescence (A, C) and merged DIC images (B, D) of unfixed C. albicans sessile cells and macrophages after 18 h treatment with compound 15 (16 μg/mL), stained with calcofluor white (CFW) and propidium iodide (PI). (AB) Overlay of CFW and PI signals showing C. albicans cells (CFW—blue chitin-rich cell wall) and necrotic macrophages (PI, red). (CD) CFW fluorescence highlighting fungal cell walls. Magnification: 432 × . The data represent two independent experiments (n = 3) In contrast, untreated fungal cells (Fig. 9IIA–B) formed a biofilm, predominantly composed of blastoconidia, on the surface of the RAW 264.7 (ATCC) macrophage monolayer (Fig. 9IIC–D). Comparison, untreated fungal cells formed biofilm (mostly of blastoconidia) onto the monolayer surface of the RAW 264.7 ATCC cell line (Fig. 9IIA–C), which caused the C. albicans adhesion process to induce necrotic action on macrophages (2 h-incubation) vs macrophages growth control (18 h-incubation) (Fig. 9 AA’). Discussion Sulfone 15 demonstrated significant potential during preliminary screening, exhibiting an lgR of 6.79 and achieving complete inhibition at 4 μg/mL after 48 h. As shown in Fig. 1 , its cytotoxicity remained below 50%, even at fungicidal concentrations, indicating a strong safety profile that merits further investigation. To evaluate the safety of 15 in vivo, tests were conducted using G. mellonella larvae. The results, presented in Fig. 2 , showed that the compound did not adversely affect larval survival compared with either the negative or solvent controls. No significant decrease in activity was observed, resulting in comparable health scores between the test and control groups. Further analysis, shown in Fig. 3 , indicated that, regardless of treatment regimen, 15 did not significantly affect the larvae's survival rate. Notably, both treatment groups exhibited a marked improvement in health scores after 120 h compared with the candidiasis control. Additionally, the level of melanization remained lower than that of the control group, and the ability to form cocoons was noticeably higher. These findings suggest that compound 15 is a promising candidate for inhibiting candidiasis in larvae and highlight its potential for future research and development. Semi-thin cross-sections of the larvae provided valuable insights into the efficacy of 15 in suppressing candidiasis (Fig. 4 ). The results demonstrated that nodules in both experimental groups were smaller than those in the control group, indicating effective reduction of C. albicans proliferation in the larvae. Consequently, fewer hemocytes surrounded the fungal cells, leading to reduced nodule formation. These results suggest that 15 may hold promise for future applications in candidiasis management. In our study, sulfone 15 induced predominantly necrotic cell death (Table 3 ), accompanied by membrane damage (Fig. 8 ), and a substantial increase in reactive oxygen species (ROS) (Table 2 ). Significantly, the antifungal activity of sulfone 15 was modulated by exogenous ergosterol (Fig. 5 ), with lower sterol concentrations resulting in lower MIC values. This finding suggests that the primary target of 15 is the fungal cell membrane and that its interaction with ergosterol plays a key role in determining cell fate. A similar sterol-dependent antagonism has been well documented for membrane-active antifungals, including polyenes, where exogenous ergosterol sequesters the drug and limits membrane disruption [ 14 ]. Given the central role of ergosterol in maintaining membrane permeability, fluidity, and integrity in C. albicans cells [ 15 ], interference with sterol-dependent membrane architecture represents a plausible trigger for catastrophic membrane failure and necrotic cell death. Notably, necroptosis in mammalian systems is often associated with elevated ROS production and rapid loss of membrane integrity [ 13 ], features that are also evident in 15 -treated C. albicans cells (Table 2 and Fig. 8 ). Although fungal regulated cell death (RCD) pathways differ fundamentally from those of higher eukaryotes, recent studies have proposed the existence of necrosis-like and programmed necrotic processes in fungi that are activated under severe stress conditions, particularly those involving membrane damage and oxidative stress [ 13 ]. In this context, sulfone 15 -induced cell death may represent a fungal analog of regulated necrosis rather than classical apoptosis, consistent with our observation that necrosis predominates (Table 3 ) despite elevated ROS levels (Table 2 ). Furthermore, reports indicating that sulfones can interfere with enzymes of the ergosterol biosynthetic pathway [ 16 ] support a dual mechanism in which sulfone 15 may disrupt membrane integrity directly and, indirectly, compromise sterol homeostasis. Such combined effects would exacerbate membrane instability and promote necrotic cell death, especially when compensatory stress-response pathways are overwhelmed. Taken together, our findings suggest that 15 induces a membrane-centered, ROS-associated necrotic cell death program in C. albicans , functionally reminiscent of necroptosis in mammalian cells, albeit mediated by distinct molecular machinery. Flow cytometry analysis demonstrated that 15 induces ROS generation in fungal cells at inhibitory concentrations, approximately 300-fold higher than in controls (Table 2 ). Elevated ROS levels are typically associated with apoptosis [ 17 , 18 ], as these species play a key role in apoptosis induction. Well-known antifungals like itraconazole also trigger apoptosis and cellular stress when ROS increases. Our observation of apoptotic cells suggests that 15 may kill C. albicans planktonic cells through multiple mechanisms, not only membrane disruption. While various modes of action have been reported for sulfones, these were previously linked to cell cycle modulation rather than ROS formation [ 10 ]. Protoplasts were killed more rapidly than intact cells, and a higher proportion of necrotic death was observed in protoplasts, indicating that the fungal cell wall is permeable to 15 but slows its diffusion to the membrane. This supports the idea that 15 acts by disrupting the cell wall, as reported for other sulfones [ 10 , 11 ]. As shown in Fig. 6 , some control cells enter the G2/M phase, preparing for mitosis, while those treated with 15 at inhibitory concentrations remain in G0 or S phase, indicating stress induction by the sulfone—consistent with previous studies [ 19 , 20 ]. Under oxidative stress, the proportion of C. albicans cells in S phase decreases, as DNA is most vulnerable during replication. Potential anti-candidial compounds may induce such stress [ 19 , 20 ]. The most notable findings emerged from the analysis of HSP90 expression. As shown in Fig. 7 , the double calcineurin gene deletion mutant displayed higher HSP90 levels than the 15 -treatment, regardless of calcium ion supplementation. In contrast, the single deletion mutant showed protein expression levels that varied with calcium ion presence. Without Ca 2+ in the medium, control cells produced more HSP90 than those treated with compound 15 . However, in the presence of Ca 2+ , treated cells exhibited higher HSP90 expression. These results indicate that HSP90 contributes to C. albicans resistance to 15 [ 21 , 22 ]. In our previous study [ 9 ], sulfone significantly increased intracellular rhodamine 123 (RH-123) fluorescence in wild-type C. albicans, but not in the CNB1/cnb1∆ mutant. This indicates that calcineurin is essential for sulfone-induced changes in mitochondrial function and membrane potential. While sulfone and RH-123 uptake occur independently, competition at membrane transporters suggests they share similar transport pathways [ 9 ]. These findings highlight that calcineurin modulates membrane stress responses and transporter availability, influencing the accumulation and efficacy of sulfone derivatives. Our study underscores calcineurin's role in sulfone susceptibility in C. albicans , linking sterol homeostasis, membrane transport, and mitochondrial function. Targeting calcineurin pathways may enhance the effectiveness of antifungal treatments. HSP90 is a key factor in C. albicans virulence and morphogenesis [ 23 ]. Our findings suggest this gene is involved in resistance mechanisms, so we recommend further research on 15 , especially in combination with HSP90 inhibitors. While previous studies with HSP90 inhibitors focused on fluconazole, which acts differently than sulfones, our results show that 15 at concentrations above the MFC increases phagocytosis and limits filamentation in biofilms-important because filamentous growth drives C. albicans virulence. Although 15 -treated blastoconidia show elevated HSP90 expression, more research is needed, as biofilm metabolism differs from that of cell suspensions. Future studies comparing HSP90 expression in different C. albicans morphological forms under sulfone treatment will clarify how sulfones target the fungu s and its virulence factors. Our findings also reveal mechanistic similarities between sulfone 15 and fluorinated tetrazoles [ 21 ]: both cause membrane damage (Fig. 8 ), disrupt fungal physiology, and lead to non-apoptotic, lytic cell death associated with oxidative stress (Tables 2 and 3 ). This suggests that antifungal agents containing fluorine-including tetrazole-benzodiazepine derivatives and sulfones-may engage similar stress-response and cell death pathways. Targeting these pathways could yield effective antifungal treatments with reduced host toxicity. Conclusions 4-(4-chloro-3,4-dichlorophenoxy)-2-nitrophenyl difluoromethyl sulfone ( 15 ) is a highly promising antifungal candidate, showing strong activity against C. albicans and low cytotoxicity. In the G. mellonella infection model, sulfone 15 delayed disease progression without affecting host viability. Mechanistic studies show that sulfone 15 disrupts fungal membrane integrity, perturbs sterol homeostasis, induces oxidative stress, activates apoptotic pathways, and modulates HSP90 expression in a calcineurin-dependent manner. Importantly, sulfone 15 also enhances fungal clearance by host phagocytes and suppresses filamentation in biofilms, a key factor in C. albicans virulence. These findings highlight sulfone 15 as a strong lead for further preclinical development and support its potential use in combination therapies to improve antifungal efficacy and reduce host toxicity. Materials and Methods Synthesis of Sulfones Compounds 15 , 24, and 32 were synthesized by the aromatic nucleophilic substitution reaction of halogen in sulfone derivatives by appropriate phenols, according to the scheme shown in Scheme 1 . Scheme 1. Open in a new tab The aromatic nucleophilic substitution reaction of halogen in sulfone derivatives by appropriate phenols 4-Fluoromethyl-1-chloro-2-nitrophenyl sulfone 1 was obtained according to the procedure described in our previous paper [ 24 ]. Synthesis of 4-bromodichloromethyl-1-chloro-2-nitrophenyl sulfone 2 was presented in our publication [ 12 ], while 4-difluoromethyl-1-chloro-2-nitrophenyl sulfone 3 was prepared according to the protocol presented in the work [ 25 ]. Compounds 26 and 28 were synthesized according to the scheme shown in Scheme 2 . The starting compounds, sulfones 4a and 4b , were prepared as described in our previous work [ 11 ]. These sulfones were converted to hydroxyethyl derivatives 5a and 5b by reaction with formaldehyde in the presence of potassium carbonate. Sulfone 5a was reacted with 3-trifluoromethylisocyanate to give the carbamate derivative 26 , while 5b was converted to the ester 28 by reaction with propionic acid chloride. Scheme 2. Open in a new tab Compounds 26 and 28 synthesis Compound 31 was synthesized starting from benzimidazole 6 , the synthesis of which was presented in publication [ 24 ], by hydroxymethylation of the dichloromethylsulfonyl group using formaldehyde and potassium carbonate in toluene Scheme 3 . Scheme 3. Open in a new tab Compound 31 synthesis The Claisen condensation of ethyl 2 obtained the 1,3-pentanedione derivative 36-(2,4-dichlorophenoxy)propanoate 7 with commercial acetophenone 8 (Scheme 4 ). Ester 7 was prepared according to the procedure described in our previous publication [ 26 ]. The condensation reaction was carried out in toluene and cyclohexane in the presence of sodium hydride as a base. The product was isolated by vacuum distillation. Scheme 4. Open in a new tab Compound 36 synthesis Activity Against C. Albicans The microdilution method M27-A3 [ 27 ] was used to check antifungal activity against C. albicans 90,028 (ATCC, Washington, DC). The cell suspension density was 2.5 × 10^3 CFU/mL. Compounds at final concentrations of 0.25–16 µg/mL in the dimples of microtiter plates were incubated for 48 h at 35 °C with C. albicans in YEPD medium (Gibco). Growth control was C. albicans cells treated with 0.96% DMSO or EtOH. Photometric measurements at 625 nm were performed at 18 and 48 h using a microtiter plate reader, Spark Control M10 (Tecan Group Ltd, Germany). Visual observation determined the MIC (the lowest concentration of the compound that prevents cell growth). The OD measurements after 18 h and 48 h were used to calculate the degree of inhibition (samples were named as: CTW- compound test wells, suspension of C. albicans , sulfone derivatives, YEPD; STW- sterility control wells, sulfone derivatives, YEPD; GCW- growth control wells, suspension of C. albicans , 0.96% DMSO or EtOH, YEPD). The minimal fungicidal concentration (MFC) was determined from selected wells and spread onto YEPD agar plates, which were incubated for 48 h at 30 °C. Three independent experiments were performed. Cytotoxicity in Vitro and in Vivo The toxicity of selected compounds was checked using the protocol ATCC XTT Cell Proliferation Assay Kit. The VeroE6 monolayer (1.2 × 10 5 CFU/ mL, EMEM medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Gibco) and antibiotics (Sigma-Aldrich)) was treated with sulfone derivatives at final concentrations from 0.25 to 512 µg/mL. The control contained EMEM medium and 0.96% DMSO. After 24 h of incubation (at 37 °C, 5% CO 2 ), the solution was removed and replaced with 50 µL XTT per well, and the mixture was incubated for two h in the dark (at 37 °C, 5% CO 2 ). The measurements were taken at 475 and 660 nm (reference) using a microtiter plate reader, Spark Control M10 (Tecan Group Ltd, Germany), to determine the CC 50 (concentration that reduces cell viability by 50% compared to the control) [ 12 ]. 15 (at eight µg/mL) cytotoxicity in vivo against the Galleria mellonella animal model was determined as described previously [ 12 ]. The control larvae were treated with 0.96% DMSO, RPMI, or untreated (negative control). Ten larvae were used for each independent experiment. All of them were incubated at 20 °C in the dark for 5 days. The survival, mobility, melanization, and cocoon formation were monitored each day, and based on these observations, the health index [ 12 ] was calculated. Histopathological Diagnosis Activity anti- C. albicans (strain 90,028 ATCC Washington DC, at 1 × 10 8 CFU/ mL) in vivo was checked for 15 (at eight µg/mL) using the animal model organism G. mellonella as described previously in [ 21 ]. The samples: 15 (+ 1 h) denotes larvae infected with C. albicans , then after one h with a compound; 15 (− 1 h) denotes the first compound after suspension of C. albicans ; C. albicans denotes larvae infected only by a pathogen, incubated for 5 days in the dark. The health index was calculated based on daily observations of survival, mobility, melanization, and cocoon formation. There were three independent experiments (n = 10). On the 6th day, selected larvae were frozen (− 20 °C, three h), cut along the body, and fixed with Bouin’s fluid. Then, they were dehydrated using ethanol (Sigma-Aldrich) (at 30, 50, 70, 70 80, 90, and 96%) for 30 min at RT, twice with chloroform (Sigma-Aldrich) for 30 min, and paraffin (Sigma-Aldrich) overnight. The fragments were cut into the smaller section, incubated twice with xylene (Pol-Aura) for 5 min, ethanol (at 100, 100, 96, 90, 80, 70, 50, and 30%), and distilled water. They were stained with hematoxylin and eosin (Carl Roth) and analyzed under a light microscope (PHG, Stollberg, Germany) [ 21 ]. Sterol Assay Action against C. albicans (strain 90,028 ATCC Washington DC, at 2.5 × 10 3 CFU/ mL) of 15 (0,25–512 µg/mL) with supplemented RPMI medium with ergosterol at 250, 125, and 25 µg/mL or without sterol (Sigma-Aldrich, USA) were checked by spectrophotometric measurements at 405 and 625 nm after 48 h of incubation at 35 °C. According to the M27-A3 method as described above (Activity against C. albicans ) and previously reported in [ 12 ], the MIC and MFC were determined in the presence or absence of exogenous ergosterol. All experiments were performed in triplicate. Flow Cytometric Analysis The cell death of C. albicans ’ non- and protoplast cells (strain 90,028 ATCC Washington DC, at 1–1.2 × 10 6 CFU/ mL) was prepared as previously [ 24 ]. The samples were incubated with 15 at 2 µg/mL for 18 h at 35 °C, and a control with 0.96% DMSO. Then, they were washed twice with PBS, suspended in PBS, and stained with Annexin V and propidium iodide (PI) using the Annexin V FLOUS Staining Kit (Roche Diagnostics GmbH, Mannheim, Germany). The cell cycle of C. albicans ’ cells (strain 90,028 ATCC, Washington, DC, at 1 × 10 3 CFU/ mL). After 18 h of incubation at 35 °C, the treated cells with 15 (2 µg/mL) were washed twice with PBS, the pellet was suspended in ethanol (70%), and the suspension was shaken linearly at 810 rpm for 30 min. Then, cells (1 × 10^6 CFU/mL) were prepared in Tris–HCl buffer (10 mM RNase, 50 mM Tris–HCl), and the pellet was suspended in fresh Tris–HCl and linearly shaken; washed with Trypsin-HCl and linearly shaken. After centrifuging (1000 rpm, five min.), cells were incubated with 1 mL cell cycle buffer (10 mM sodium citrate, 180 mM sodium chloride, 70 mM magnesium (VI) sulfate, 180 mM Tris–HCl) and 2.5 µL iodine propidine in the darkness for 30 min, and then glass beads were added and then vortexed. Experiments were performed on the stained control (untreated cells with PI) and the unstained control (untreated cells without PI) [ 12 ]. Using fluorometric assay with 2,7 2,7-dichlorofluorescein diacetate at 5 µM (DCFH-DA), the ROS generation was determined for samples C. albicans (strain 90,028 ATCC Washington DC, at 1 × 10 3 CFU/ mL) treated with 15 at 8 µg/mL for 18 h at 35 °C. Flow Cytometry BD FACSCanto II made the measurements with BD FACSDiva software (BD Biosciences, San Jose, CA, USA) [ 12 ]. RT-qPCR Assay C. albicans BWP17, cnb1Δ/cnb1Δ , CNB1 / cnb1Δ (1–1.2 × 10 6 CFU/mL) were treated with 15 (2 µg/mL) for 18 h at 35 °C in YEPD supplemented with or without CaCl 2 (200 mM). RNA isolation (as described previously [ 12 ]) and cDNA synthesis (Thermo Scientific Revert Aid First Strand cDNA Synthesis Kit protocol) were carried out. The RT-PCR process was done using primers for ACT1 (reference gene): 5’-GACAATTTCTCTTTCAGCACTAGTAGTGA-3’ (forward), 5’-GCTGGTAGAGACTTGACCAACCA-3’ (reverse), and for HSP90 (tested gene): 5 5'-TTCACTGCTGAGATCTCTCAGTT-3’ (forward), 5’-CAATACCAGAATCTCTAATTTCC-3’. The reaction was carried out on a LightCycler 96 Real-Time PCR Instrument. Confocal Laser Scanning Microscope (CLSM) Assay To investigate the interaction between C. albicans and macrophages, and the effect of the tested compound on fungal biofilm formation, co-culture experiments were performed using the murine macrophage cell line RAW 264.7 (ATCC). Macrophages were cultured in appropriate growth medium and seeded onto polycarbonate filter inserts (Corning®, USA) in 24-well plates. C. albicans cells were prepared at a density of 1.2 × 10 6 cells/mL in PBS and added to the macrophage monolayer. The cells were then treated with the compound at 16 μg/mL. To promote fungal germination, hyphal growth, and biofilm formation in the presence of macrophages, co-cultures were maintained in HAMS F-12 medium supplemented with 2 mM glutamine and 10% fetal bovine serum. Cells were incubated for 18 h at 37 °C in a humidified atmosphere containing 5% CO₂. Following incubation, cell viability and membrane integrity were assessed using fluorescence staining. Propidium iodide (PI; 100 μg/mL, Thermo Fisher Scientific, USA) was used to identify cells with compromised membrane integrity. In parallel, dual staining with acridine orange (AO; 100 μg/mL) and PI (Roche Diagnostics GmbH, Mannheim, Germany) was performed to evaluate changes in cellular permeability. Given the presence of the fungal cell wall (calcofluor white CFW-stained), increased uptake of these dyes was interpreted as indicative of altered cell envelope integrity rather than as a direct marker of specific cell death pathways. The tested samples contained 15 at 16 μg/mL, C. albicans’ cells (1–1.2 × 10 3 CFU/ mL) and dyes; control contained comp. and dyes; negative control contained only dyes; positive control contained cell suspension and dyes. The dye mixtures: 1st contained 100 μg/mL AO and 100 μg/mL PI; 2nd contained 10 mg/mL CFW and 100 μg/mL PI. On the RAW 264.7 ATCC monolayer, the samples were incubated for 2 or 18 h at 37 °C with 5% CO 2 . Then, they were washed with PBS buffer and assessed using a confocal laser scanning microscope (CLSM, Olympus, Center Valley, PA, USA). Using Adobe Photoshop CS3 Extended (France), images were assembled [ 12 ]. All experiments were performed in triplicate. Fluorescence imaging was performed on unfixed samples using an Olympus FLUOROVIEW FV1000 confocal laser scanning microscope (CLSM, Olympus, USA). Two fluorescence channels were recorded for each condition. Images were processed using Adobe Photoshop CS3 Extended (France). Statistics Each experiment was performed with at least three independent repetitions, and the measurements were presented as mean ± SD. Using the t-test, statistically significant differences ( P values ≤ 0.05) were detected between the control and test groups. Linear regression analysis was performed using GraphPad Prism (version 11.0.0 (84)) to evaluate changes in survival over time. The goodness of fit was assessed using the coefficient of determination (R 2 ). The statistical significance of the slope was determined using an F-test. A p -value < 0.05 was considered statistically significant. Supplementary Information Below is the link to the electronic supplementary material. Supplementary file1 (DOCX 1251 kb) (1.2MB, docx) Acknowledgements We thank the National Science Center, Poland [grant no. 2021/43/B/NZ6/0073], for funding this research. Author Contributions AS: Assembly of data, data analysis and interpretation, writing the article, Critical revision of the article, Final approval of the article; DK: Writing the article, critical revision of the article. ŁK: Critical revision of the article, writing the article, final approval of the article. IZG: collection and assembly of data. ZO: Concept and design of chemical studies, collection and/or assembly of chemical synthesis data. Data analysis and interpretation, writing the article, Critical revision of the article. MS: Concept and design, collection and/or assembly of data. Data analysis and interpretation, writing the article, Critical revision of the article, and final approval of the article. Data Availability No datasets were generated or analysed during the current study. 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