Iturin A disrupts Cryptococcus neoformans membrane integrity, attenuates virulence, and synergizes with amphotericin B

Abstract

Cryptococcus neoformans is a major fungal pathogen with severely limited therapeutic options. We evaluated the antifungal activity of the lipopeptide iturin A against eight clinical isolates of C. neoformans. Iturin A exhibited robust anti-cryptococcal activity, with minimum inhibitory concentrations ranging from 12.5 to 25 μg/mL, and demonstrated minimal toxicity in A549 lung epithelial cells and HepG2/C3A liver cells. Combination treatment with amphotericin B was synergistic (fractional inhibitory concentration index, 0.37). Mechanistic analyses showed that iturin A disrupts cryptococcal membrane integrity and induces intracellular reactive oxygen species accumulation, leading to fungal cell death. Iturin A also reduced biofilm formation and impaired capsule production. In the Caenorhabditis elegans infection model, treatment with iturin A alone or in combination with amphotericin B prolonged host survival and reduced fungal burden. Although iturin A decreased cryptococcal phagocytosis by J774 macrophage- and NR-9460 microglia-like cells, it enhanced intracellular killing by these phagocytes. Collectively, these findings demonstrate that iturin A exhibits multifaceted activity against C. neoformans through membrane disruption, oxidative stress induction, and interference with virulence determinants, supporting its potential as a novel anti-cryptococcal therapeutic agent.

Introduction

Fungi represent a serious and underrecognized threat to global health¹. Fungal infections globally affect ≥6.5 million people and cause ~2.5 million deaths each year, exceeding the annual mortality caused by either malaria or tuberculosis^2,3. Due to their severity, the World Health Organization (WHO) has designated fungal infections as a global health priority and categorized Cryptococcus neoformans as one of the most critical fungi of medical importance^1,4. C. neoformans is an encapsulated yeast-like fungus that infects humans when desiccated or poorly encapsulated yeasts/basidiospores are inhaled⁵. The fungus causes cryptococcal meningoencephalitis (CME) predominantly in immunocompromised individuals such as those with HIV/AIDS^6,7. CME causes high mortality even with antifungal therapy⁸, killing ~20% of individuals with AIDS⁹, particularly in sub-Saharan Africa¹⁰. Therefore, CME remains a major global public health challenge.

The pathogenesis of C. neoformans is driven primarily by its polysaccharide capsule synthesis¹¹. The capsule provides protection against host immune recognition¹², with its main polysaccharide, glucuronoxylomannan (GXM), playing a central role in biofilm formation¹³ as well as disease progression¹⁴. Cryptococci secrete GXM to generate an extracellular matrix¹³ that confers resistance to antifungal therapy¹⁵ and immunity-derived molecules¹⁶. In the central nervous system (CNS), C. neoformans can form cryptococcomas or biofilm-like masses¹⁷ that make the fungus impervious to eradication¹⁸. C. neoformans biofilm formation also occurs in medical prosthetics such as ventriculoatrial shunt catheters¹⁹. In addition, the application of ventriculoperitoneal shunt catheters for treating elevated intracranial pressure in CME patients underscores the critical need to develop novel anti-cryptococcal therapies.

Current treatment for cryptococcosis and CME follows a three-phase approach: induction, consolidation, and maintenance therapy²⁰. The preferred induction regimen combines amphotericin B (AmpB) with flucytosine for at least two weeks, which has demonstrated superior fungicidal activity and improved survival rates²¹. In resource-limited settings where flucytosine is unavailable, AmpB monotherapy or combined with fluconazole serves as an alternative²². Following induction, consolidation therapy with high-dose fluconazole is administered for eight weeks to further reduce fungal burden²¹. Maintenance therapy, employing lower-dose fluconazole, continues for at least one year to prevent relapse, particularly in immunocompromised patients²³. Despite these established protocols, treatment faces significant challenges, including AmpB-associated nephrotoxicity, limited access to flucytosine globally, prolonged therapy duration, and high mortality rates ranging from 20% to 70% depending on geographic location and patient population²⁴. These limitations underscore the urgent need for novel therapeutic strategies.

Iturin A is a cyclic lipopeptide with antifungal properties synthesized by various Bacillus species, such as B. subtilis and B. amyloliquefaciens. Structurally, this compound consists of a heptapeptide ring attached to a β-amino fatty acid chain²⁵. Iturin A demonstrates broad-spectrum efficacy against both yeast, including Saccharomyces cerevisiae and Candida albicans²⁶ and filamentous fungi, such as Fusarium, Penicillium, and Aspergillus²⁷. It disrupts fungal cell membrane integrity by forming pores and increasing permeability, leading to cellular leakage and death^28,29. Moreover, iturin A triggers oxidative damage by elevating intracellular reactive oxygen species (ROS) levels³⁰. Notably, iturin A has preferential toxicity toward fungal cells while exhibiting minimal mammalian cytotoxicity at therapeutically relevant doses, underscoring its promise as a therapeutic or adjunct antifungal option^31,32. In this study, we investigated the anti-cryptococcal activity of iturin A alone or combined with conventional antifungal drugs (e.g., AmpB) in vitro and in vivo, using the soil nematode Caenorhabditis elegans as a model of infection. We also evaluated the activity of this lipopeptide on C. neoformans viability, capsule production, biofilm inhibition, and phagocyte function. Our findings suggest that iturin A is a promising candidate for further development as an alternative therapy to combat cryptococcocosis.

Results

Iturin A kills C. neoformans cells and exhibits minimal toxicity in A549 human pulmonary epithelial cells and HepG2/C3A liver cells

We first determined the minimum inhibitory concentration (MIC; μg/mL) of iturin A by testing it against eight C. neoformans clinical isolates (55, 57, 59, 68, 72, 83, and 88), including standard reference strain H99 (Table 1). We used amphotericin B (AmpB) and fluconazole, two commonly used antifungal drugs in the clinical setting, as positive controls. Iturin A showed anti-cryptococcal activity against all the strains with MIC ranging between 12.5–25 μg/mL compared to 0.06–0.5 μg/mL for AmpB and 1–8 μg/mL for fluconazole.

Table 1.

Minimum inhibitory concentration (MIC) of iturin A, amphotericin B, and fluconazole against C. neoformans isolates

C. neoformans isolates	MIC (μg/mL)
	Iturin A	Amphotericin B	Fluconazole
H99	12.5	0.5	2
55	25	0.125	1
57	25	0.06	2
59	12.5	0.125	2
68	25	0.25	4
72	25	0.5	8
83	12.5	0.125	4
88	25	0.5	4

Then, we performed a time-kill assay (n = 6 replicates/group/time point) to investigate the efficacy of iturin A in killing C. neoformans strain H99 cells after 72 h (Fig. 1A). Iturin A at 25 μg/mL (mean ± STDEV: 3.03 Log₁₀ CFU/mL ± 0.22) and AmpB at 1 μg/mL (2.59 Log₁₀ CFU/mL ± 0.35) treatment for 24 h demonstrated considerable reduction (>3 Log₁₀) in cryptococcal viability relative to untreated cells (6.47 Log₁₀ CFU/mL ± 0.13; Fig. 1A). Similarly, iturin A-treated cryptococci (2.38 Log₁₀ CFU/mL ± 0.09) evinced substantial killing after 72 h compared to untreated cells (7.01 Log₁₀ CFU/mL ± 0.27; 4.63 Log₁₀) but less marked than AmpB (0.65 Log₁₀ CFU/mL ± 0.08; 1.73 Log₁₀ vs. iturin A; 6.36 Log₁₀ vs. untreated; Fig. 1A).

Fig. 1. Iturin A kills Cryptococcus neoformans cells and shows low toxicity in A549 human pulmonary epithelial cells and HepG2/C3A liver cells.

A The kinetics of iturin A-mediated killing of Cryptococcus neoformans H99 strain cells were performed to evaluate this lipopeptide's time-dependent antifungal activity. Amphotericin B (AmpB) was used as a positive control. Each symbol represents an average of 6 replicates per time point. The cytotoxicity of iturin A was assessed on B A549 cells [percentage (%) of A549 cell viability] and C HepG2/C3A liver cells to determine this lipopeptide's safety profile across a range (8–64 μg/mL) of concentrations. Each symbol represents a single replicate (n = 3–4 replicates per group). Bars and error bars denote means and standard deviations (SDs), respectively. For A and B, each experiment was performed twice, and similar results were obtained.

Since we showed that iturin A is effective in inhibiting the growth of multiple C. neoformans strains, we then investigated the toxicity of this cyclic lipopeptide compound in human A549 lung epithelial cells and HepG2/C3A liver cells (Fig. 1B and C). A549 and HepG2/C3A cells are widely utilized as an in vitro system to assess the toxicity of experimental drugs and therapeutics. Iturin A demonstrated low toxicity in A549 and HepG2/C3A cells, with detectable cytotoxicity only observed at 64 μg/mL (Fig. 1B and C).

These results demonstrate that iturin A is efficacious in reducing the cryptococcal viability and has minimal cytotoxicity in a validated human lung and liver cell models.

Iturin A and AmpB combination is synergistic against C. neoformans

We investigated the in vitro interaction between iturin A and AmpB against C. neoformans strain H99 cells using the checkerboard assay (Fig. 2). First, cryptococci were exposed to various concentrations of iturin A (0.78–25 μg/mL) and AmpB (0.0039–2 μg/mL) combination for 72 h (Fig. 2A and B). Then, we calculated the fractional inhibitory concentration index (FICI) of 0.37 indicating that iturin A has synergistic interaction with AmpB against C. neoformans (Fig. 2A). To assess fungicidal activity of iturin A + AmpB, aliquots from the checkerboard assay were plated onto Sabouraud dextrose agar, confirming enhanced killing with the drug combination (Fig. 2B). Moreover, using combinations of sub-inhibitory concentrations of iturin A (3 μg/mL) and AmpB (0.25 μg/mL) and comparing them to each drug alone or untreated cells (Fig. 2C, D), we confirmed that the antifungal drug combination against C. neoformans H99 cells greatly reduces fungal viability after 12 h (2.51 Log₁₀ CFU/mL ± 0.28) relative to the other conditions (untreated, 4.11 Log₁₀ CFU/mL ± 0.24; iturin A, 3.64 Log₁₀ CFU/mL ± 0.25; AmpB, 4.07 Log₁₀ CFU/mL ± 0.19). Cryptococcal cell viability is further reduced by combination treatment after 24 h (0.85 Log₁₀CFU/mL ± 0.01) whereas complete eradication of cryptococci is further shown after 48 h when compared to each antifungal drug (24 h: iturin A, 4.65 Log₁₀CFU/mL ± 0.24; AmpB, 5.14 Log₁₀ CFU/mL ± 0.87; 48 h: iturin A, 5.13 Log₁₀CFU/mL ± 0.12; AmpB, 6.63 Log₁₀ CFU/mL ± 0.2) and cells with no treatment (24 h: untreated, 6.52 Log₁₀ CFU/mL ± 0.13; 48 h: untreated, 6.87 Log₁₀ CFU/mL ± 0.04). In addition, fungal cells exposed to iturin A alone showed lower viability (~1 log) than untreated and AmpB-treated cells after 72 h. Together, these findings indicate that iturin A and AmpB have a synergistic interaction and markedly reduce the viability of C. neoformans cells.

Fig. 2. Susceptibility of C. neoformans cells to iturin A and AmpB combination therapy, as measured by the checkerboard assay.

A A density of 10³ C. neoformans cells/mL was exposed to various combinations of iturin A (0.78–25 μg/mL) and AmpB (0.0039–2 μg/mL) concentrations in RPMI 1640 medium and incubated at 35 °C for 72 h. The fractional inhibitory concentration index (FICI) was calculated to evaluate therapeutic combination synergy (FICI ≤ 0.5). B To evaluate the fungicidal activity of the combination, aliquots from the checkerboard assay were plated onto Sabouraud dextrose agar. C The kinetics of iturin A and AmpB-mediated killing of C. neoformans H99 cells (time-kill assay) were performed to evaluate this therapeutic combination's time-dependent antifungal activity. D Spotting images of H99 cultures treated with iturin A (3 μg/mL), AmpB (0.25 μg/mL), or a combination of both drugs were captured after 72 h of incubation. Each symbol represents an average of 6 replicates per time point. For A–D, each experiment was performed twice, and similar results were obtained.

Iturin A causes membrane permeabilization in C. neoformans cells

Iturin A penetrates the cell wall and causes disruption of the plasma membrane in yeast cells²⁶. Hence, we investigated cell membrane permeabilization using propidium iodide (PI) as a potential mechanism of action (MOA) by which this lipopeptide antibiotic kills C. neoformans cells. Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI. We used AmpB as a positive control, considering that this drug acts by irreversibly binding to ergosterol in fungal cell membranes, creating transmembrane pores/channels, leading to increased membrane permeability, leakage of essential ions (e.g., K⁺, Na⁺, H⁺), and subsequent cell death. Using flow cytometry, we observed that iturin A-treated cryptococci showed 89.96% PI incorporation compared to 23.8% in untreated cells (Fig. 3A). Furthermore, given that AmpB binds to ergosterol to disrupt the C. neoformans plasma cell membrane and our findings that iturin A causes plasma cell membrane permeabilization, we hypothesized that iturin A has a similar MOA to AmpB in killing cryptococcal cells. Therefore, we supplemented the cryptococcal culture in RPMI 1640 medium with 200 μg/mL ergosterol and assessed whether this mycosterol reduced iturin A activity. By measuring fluorescence intensity (Fig. 3B) and imaging (Fig. 3C), we observed that cryptococci treated with 25 μg/mL iturin A (mean: 209.8%) have significantly higher relative percentage (%) of PI incorporation, which is indicative of cell membrane permeabilization, than untreated cells (mean: 100%; P < 0.0001). In addition, ergosterol supplementation reduced the PI incorporation of iturin A-treated cryptococci to 104.5%. As expected, AmpB-treated cells produced strong PI incorporation, with a mean relative PI incorporation of 585.7%. Consistent with our findings, ergosterol supplementation increased iturin A minimum fungicidal concentration (MFC) by 4-fold (Fig. 3D). Thus, our results indicate that iturin A causes C. neoformans cell plasma membrane permeabilization by potentially attaching to ergosterol, thereby affecting its integrity and flexibility.

Fig. 3. Iturin A causes cryptococcal plasma membrane permeabilization.

A Propidium iodide (PI; red) incorporation was quantified to measure the extent of fungal cell membrane permeabilization by flow cytometry analysis. Representative plots of permeabilized PI-labeled fungal cells are shown in untreated and iturin A-treated cells. The % of live (green) and dead (red) cells is shown. Each plot was generated after 10,000 events were analyzed. B Relative PI incorporation was quantified by fluorescence intensity in untreated and iturin A (25 μg/mL)-, iturin A supplemented with ergosterol (200 µg/mL)-treated, and AmpB (1 μg/mL)-treated cells. Each symbol represents a single replicate (n = 4 replicates per group). Bars and error bars denote means and standard deviations (SDs), respectively. P-value significance (****P < 0.0001) was calculated by analysis of variance (ANOVA) and adjusted using Tukey’s post hoc analysis. C Representative fluorescence microscopy images of C. neoformans cells treated with vehicle, AmpB, iturin A, and iturin A + ergosterol. 10⁶ untreated and treated C. neoformans cells were grown on RPMI 1640 medium for 6 h at 30 °C and then stained for PI uptake, which indicates plasma membrane integrity disruption and cell death. The pictures were taken at a magnification of ×40. Scale bar, 20 μm. D Growth of C. neoformans cells after exposure to iturin A (0.78–25 μg/mL) or iturin A supplemented with ergosterol (200 µg/mL) for 72 h at 35 °C. For A–D, each experiment was performed twice, and similar results were obtained.

Iturin A enhances C. neoformans intracellular ROS accumulation

Iturin A crosses the plasma membrane and associates with both the nuclear membrane and potentially the membranes of various other organelles within the cytoplasm²⁶. Also, iturin A causes the accumulation of ROS, reduction of the mitochondrial membrane potential, and the induction of nuclear condensation in Candida albicans, resulting in apoptosis³⁰. Likewise, AmpB induces the oxidative burst affecting fungal cell damage of macromolecules [e.g., proteins, nucleic acids, lipids³³]. Therefore, we investigated the impact of iturin A in promoting C. neoformans intracellular ROS production and accumulation using the ROS indicator dihydrofluorescein diacetate (DHFDA) dye and flow cytometry analysis (Fig. 4). We compared ROS generation in untreated and treated cryptococci with 10 μM hydrogen peroxide (H₂O₂; positive control) or 25 μg/mL iturin A. Iturin A treatment resulted in 42.8% cryptococci showing intracellular ROS generation compared to 67.7% in H₂O₂-treated and 0.046% in untreated control cells (Fig. 4A). Then, we calculated the relative % fluorescent intensity in these experimental groups and showed that iturin A-treated fungal cells (mean: 188%) generated higher intracellular ROS than untreated cells (mean: 100%; P < 0.0001). AmpB-treated cells (mean:124.8%) also increased ROS production relative to untreated cells (P < 0.01), although to a lesser extent than iturin A (P < 0.0001). As expected, H₂O₂-treated cells (mean: 416.7%) produced the highest ROS signal, increasing fluorescence intensity to 416.7% relative to untreated cells (P < 0.0001). Our findings suggest that iturin A leads to elevated intracellular ROS levels in C. neoformans cells.

Fig. 4. Iturin A promotes C. neoformans intracellular reactive oxygen species (ROS) generation.

A The levels of fungal intracellular ROS were quantified by flow cytometry analysis in untreated and iturin A-treated C. neoformans cells to measure oxidative stress. Cryptococci were treated with hydrogen peroxide (H₂O₂) as a positive control. Representative plots of Dihydrofluorescein diacetate (DHFDA)-labeled fungal cells are shown. The DHFDA dye is a cell-permeable, non-fluorescent probe used to detect intracellular ROS. The % of typical (fuchsia) and oxidative-stressed (blue) cells is shown. Each plot was generated after 10,000 events were analyzed. B The percentage of relative DHFDA fluorescence is reported. Each symbol represents a single replicate (n = 4 replicates per group). Bars and error bars denote means and SDs, respectively. P-value significance (**P < 0.01; ****P < 0.0001) was calculated by ANOVA and adjusted using Tukey’s post hoc analysis.

Iturin A inhibits C. neoformans capsule size

Exposure to serum³⁴ and cerebrospinal fluid³⁵ has been demonstrated to induce C. neoformans capsular enlargement. This capsule expansion, which occurs during infection, diminishes phagocytic uptake by host immune cells³⁶, thereby functioning as a critical virulence mechanism. To evaluate the impact of iturin A on cryptococcal capsular growth response, we grew H99 cultures in Dulbecco′s modified Eagle′s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; capsular induction medium) at 37 °C for 72 h in absence or presence sub-inhibitory concentrations (3, 6, 12 μg/mL) of the lipopeptide (Fig. 5). Light microscopy imaging using India ink staining showed that the capsules (white halo) of iturin A-treated cryptococci were smaller than those of untreated cells (Fig. 5A). We analyzed the capsule volume (Fig. 5B), body size (Fig. 5C), and capsule size (Fig. 5D) of each strain after growth in capsular inducing medium. We did not observe differences in capsule volume between untreated (mean: 688.2 µm³) and 3 µg/mL iturin A-treated cryptococci (mean: 680.7 µm³), although the capsule volume of 6 (mean: 204.4 µm³; P < 0.0001) and 12 µg/mL (mean: 193.2 µm³; P < 0.0001) iturin A-treated cryptococcal cell evinced significant > 2-fold reduction (Fig. 5B). The body of cryptococcal cells treated with 3 µg/mL iturin A (mean: 6.31 µm) was similar to the cell body of fungi treated with 6 µg/mL iturin A (mean: 5.74 µm) and significantly larger than untreated (mean: 5.55 µm; P < 0.05) and 12 µg/mL iturin A (mean: 5.08 µm; P < 0.0001)-treated cells (Fig. 5C). Additionally, the reduction in the capsule size of iturin A-treated fungal cells was inversely proportional to the lipopeptide concentration (Fig. 5D). Iturin-A treated cells had significantly smaller capsule size (3 µg/mL, mean: 3.24 µm, P < 0.05; 6 µg/mL, mean: 2.46 µm, P < 0.0001; 12 µg/mL, mean: 2.37 µm, P < 0.0001) than untreated cells (mean: 5.29 µm), demonstrating a concentration-dependent reduction in capsule thickness. There is significant difference in the capsule size between iturin A-treated cells at 3 and 6 µg/mL (P < 0.05), whereas no significant difference was observed between cells treated with iturin A at 6 and 12 µg/mL. These findings show that sub-inhibitory concentrations of iturin A interfere with C. neoformans cell capsular enlargement.

Fig. 5. Iturin A reduces C. neoformans capsule size.

A Representative light microscopy images of untreated and iturin A-treated (sub-inhibitory concentrations: 3, 6, and 12 µg/mL) C. neoformans cells stained with India ink are shown. A density of 10⁶ cryptococci/mL was grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in an atmosphere containing 5% CO₂ for 72 h. The pictures were taken using a ×40 power field. Scale bar: 50 µm. B Capsule volume (V = 4/3πr³), C cell body size (whole cell diameter−capsule diameter), and D capsule size (whole cell diameter–cell body diameter) for cells grown under each condition were determined. For B–D, violin plots show the averages (n = 50 cells per group; dashed lines) and SDs of the results. P-value significance (****P < 0.0001; *P < 0.05) was calculated by ANOVA and adjusted using Tukey’s post hoc analysis.

Iturin A interferes with C. neoformans biofilm formation

C. neoformans can form biofilms on medical devices in a process that requires capsular polysaccharide release¹³. Biofilm formation makes C. neoformans more resistant to commonly used antifungal drugs¹⁵ and may diminish their efficacy during cryptococcal infection. Using confocal microscopy, CFU determinations, and crystal violet staining, we examined the ability of iturin A in inhibiting C. neoformans biofilms (Fig. 6). Confocal microscopy images of untreated biofilms showed cryptococci distributed across the visualization field with higher biomass deposition in the upper right region (Fig. 6A). In contrast, 3 and 6 µg/mL iturin A-treated cryptococcal cells exhibited similar scattered depositions of biofilm clusters that were substantially reduced at the highest concentration (12 µg/mL). CFU determinations demonstrated that iturin A-treated biofilm-derived cells had lower viability (3 µg/mL, 7.56 Log₁₀ CFU/mL, P < 0.05; 6 µg/mL, 7.39 Log₁₀ CFU/mL, P < 0.01; 12 µg/mL, 6.4 Log₁₀ CFU/mL, P < 0.0001) than cryptococci-derived from untreated biofilms (8.07 Log₁₀ CFU/mL) (Fig. 6B). This reduction in cryptococcal biofilm formation was directly proportional to an increase in iturin A concentration especially at the highest concentration of 12 µg/mL, which demonstrated increased significance when compared to 3 and 6 µg/mL (P < 0.001). Similar trend was observed when the % of biofilm biomass (cryptococci and extracellular matrix) was calculated using crystal violet staining (Fig. 6C). Iturin A-treatment at 3 (mean: 61.7%; P < 0.001), 6 (mean: 48.4%; P < 0.0001), and 12 µg/mL (mean: 27%; P < 0.0001) resulted in significant reduction in the % of C. neoformans biomass compared to untreated biofilms. In addition, 12 µg/mL iturin A-treated biofilms exhibited significantly lower % of biofilm biomass than 3 (P < 0.01) and 6 (P < 0.05) µg/mL, whereas no difference was observed between the lower concentrations. These results suggest that iturin A interferes with cryptococcal biofilm formation during the adhesion and biofilm development phase.

Fig. 6. Iturin A inhibits C. neoformans biofilm formation.

A Representative confocal microscopic images of untreated and iturin A-treated (sub-inhibitory concentrations: 3, 6, and 12 µg/mL) C. neoformans biofilms [green; stained with monoclonal antibody (mAb) 18B7-Alexa Fluor 488-conjugated goat anti-mouse IgG₁] grown at 35 °C for 48 h. Biofilm formation was compared and determined using B colony-forming units (CFU) determination and C crystal violet staining [% of biofilm biomass] assays. For B and C each symbol represents a single replicate (n = 3–4 replicates per group). Bars and error bars denote means and SDs, respectively. P-value significance (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05) was calculated by ANOVA and adjusted using Tukey’s post hoc analysis.

Iturin A decreases C. neoformans phagocytosis and stimulates phagocyte intracellular killing

C. neoformans is a facultative intracellular pathogen that survives and replicates within host macrophages by inhibiting phagolysosome maturation³⁷ and utilizing urease to neutralize acidic environments³⁸. To assess the impact of iturin A on C. neoformans interactions with phagocytes, we first examined the phagocytosis of the fungus by J774 macrophage-like and NR-9460 microglia-like cells after 4 h at 37 °C (Fig. 7). We chose macrophages and microglia due to their involvement in combating cryptococcal infection in the lungs and brain, respectively. Representative light microscopy images revealed a noticeable reduction in internalized yeast cells (arrows) by macrophage-like cells treated with 6 μg/mL iturin A when compared to untreated cells (Fig. 7A). Quantitative analysis of the phagocytic index (n = 100 cells per group) confirmed a significant decrease in fungal uptake relative to untreated phagocytes (J774, mean: 4.25; NR-9460, mean: 4.19), as indicated by fewer intracellular cryptococci in both macrophage (mean: 3.62, P < 0.05; Fig. 7B)-like and microglia (mean: 3.53, P < 0.01; Fig. 7D)-like cells following iturin A treatment (6 µg/mL). In contrast, 3 μg/mL of iturin A did not significantly reduce phagocytosis in either cell type. These results suggest that iturin A decreases fungal uptake by phagocytes, which may have important implications in disease progression and control.

Fig. 7. Iturin A reduces C. neoformans phagocytosis and induces intracellular killing by phagocytes.

A Representative light microscopy images of untreated and iturin A (6 μg/mL)-treated J774 macrophage-like cells show intracellular C. neoformans (yellow arrows). The phagocytic indices (ratio of number of intracellular yeast cells to number of microglia counted) were determined after 4 h of incubation of 10⁵ B J774 macrophage-like or D NR-9460 microglia-like cells with 10⁶ cryptococci in the absence or presence of iturin A at 37 °C and 5% CO₂. Violin plots show the averages (n = 100 cells per group; dashed lines) and SDs of the results. P-value significance (**P < 0.01; *P < 0.05) was calculated by Student’s t-test analysis. CFU determinations were performed after 48 h of incubation of engulfed fungi by C macrophages or E microglia with iturin A (3 and 6 μg/mL). Each symbol represents a single replicate (n = 3 replicates per group). Bars and error bars denote means and SDs, respectively. P-value significance (****P < 0.0001; ***P < 0.001; **P < 0.01) was calculated by ANOVA and adjusted using Tukey’s post hoc analysis. This experiment was performed twice, and similar results were obtained.

Nevertheless, despite reduced cryptococcal phagocytic uptake by macrophage- and microglia-like cells, iturin A treatment resulted in a significant decrease in phagocyte intracellular fungal load (Fig. 7C and E). CFU determinations performed on lysed macrophages (3 µg/mL, 6.45 Log₁₀ CFU/mL, P < 0.01; 6 µg/mL, 5.99 Log₁₀ CFU/mL, P < 0.0001; Fig. 7C) and microglia (3 µg/mL, 6.41 Log₁₀ CFU/mL, P < 0.001; 6 µg/mL, 6.03 Log₁₀ CFU/mL, P < 0.0001; Fig. 7E) demonstrated a substantial reduction in viable C. neoformans compared with untreated cells (6.82 and 6.92 Log₁₀ CFU/mL, respectively). Moreover, macrophage (P < 0.01)- and microglia (P < 0.01)-like cells treated with 6 µg/mL demonstrated lower fungal load than those treated with 3 µg/mL. These findings indicate that iturin A enhances intracellular fungal clearance, likely by inhibiting fungal growth within host cells.

Iturin A improves the survival rate of the nematode C. elegans by lowering the fungal load

C. elegans is an effective model organism for studying drug efficacy in several fungi^39,40 and bacteria^41,42. We infected C. elegans with C. neoformans, treated with iturin A (3, 12, or 25 μg/mL), AmpB (0.25 or 1 μg/mL), or a combination of sub-inhibitory concentrations of iturin A (3 μg/mL) and AmpB (0.25 μg/mL), and incubated for 2 or 10 days at 25 °C, for CFU determinations per worm and worm survival, respectively (Fig. 8A). Untreated C. elegans worms were used as a negative control. We found no difference in worm survival between untreated (mean: 6 days) and iturin A (3 μg/mL; mean: 3 days)-treated or AmpB (mean: 6 days)-treated C. elegans (Fig. 8B). However, iturin A-treated worms with ≥12 μg/mL demonstrated 100% survival after 10 days. Sub-inhibitory concentrations of iturin A and AmpB combination-treated C. elegans exhibited delayed killing (>7 days) and 80% survival. Then, we determined the fungal burden on C. elegans worms after treatment with either AmpB (1 μg/mL) or iturin A (12–25 μg/mL) for 2 days (Fig. 8C). Iturin A (12 µg/mL) significantly reduced the fungal load per worm by 3.35 log₁₀ relative to the untreated worms (P < 0.0001) and completely eradicated C. neoformans cells at the highest (25 µg/mL) concentration tested (Fig. 8C). Then, untreated and treated C. elegans with sub-inhibitory concentrations of iturin A (3 μg/mL) or AmpB (0.25 μg/mL) and their combination was infected with C. neoformans cells and the number of CFU per worm was compared after 2 days (Fig. 8D). The combination of iturin A and AmpB resulted in clearance of the fungal burden in C. elegans (P < 0.0001). In contrast, iturin A (3 µg/mL; mean: 4.16 Log₁₀ CFU/worm) and AmpB (0.25 µg/mL; mean: 4.12 Log₁₀ CFU/worm) alone showed similar fungal load as the untreated cells (mean: 4.07 Log₁₀ CFU/worm). Collectively, these findings provide strong in vivo evidence that iturin A, both as monotherapy and in combination with AmpB, effectively eradicates C. neoformans infection.

Fig. 8. Iturin A enhances Caenorhabditis elegans survival by reducing fungal burden.

A Experimental timeline for the C. neoformans infection and treatment of the C. elegans worm model. Survival and CFU determinations studies were performed in separate groups of worms. The diagram was created with BioRender.com by Dr. Yehia Elgammal. B Survival differences (average n = 35 per group) of C. elegans alone (untreated) or with AmpB (0.25 μg/mL), iturin A (3–25 μg/mL), or a combination of sub-inhibitory concentrations of iturin A (3 μg/mL) and AmpB (0.25 μg/mL) co-incubated with 10⁶ C. neoformans strain H99 cells/mL at 25 °C. Significance (P < 0.05) was calculated by log-rank (Mantel–Cox) analysis. Fungal burden in worms treated with (C) iturin A (12 and 25 μg/mL) or (D) combination of sub-inhibitory iturin A and AmpB concentrations after a 2-day co-incubation. Quantification of viable yeast cells from infected worms was determined by CFU per worm (n = 3–6 replicates) in sterile phosphate-buffered saline (PBS). Bars and error bars denote means and SDs, respectively. P-value significance (****P < 0.0001) was calculated by ANOVA and adjusted using Tukey’s post hoc analysis.

Discussion

Cryptococcosis, primarily CME, remains a leading cause of mortality in HIV/AIDS patients, particularly in sub-Saharan Africa, accounting for ~20% of AIDS-related deaths globally^9,10. Current treatment options remain severely limited, relying heavily on AmpB and flucytosine, drugs developed decades ago that are associated with significant toxicity, including nephrotoxicity and bone marrow suppression^43,44. Moreover, these regimens often require prolonged intravenous administration in resource-limited settings where the disease burden is highest²⁰. The emergence of antifungal resistance, coupled with poor CNS penetration of existing agents and high treatment failure rates (mortality can exceed 70% in some regions), underscores the critical need for novel therapeutics with improved safety profiles, better bioavailability, and enhanced efficacy^45–47. The development of new antifungal drugs targeting cryptococcosis is therefore an urgent public health priority to reduce the devastating morbidity and mortality associated with this life-threatening disease. In this study, we demonstrate that Bacillus species-derived iturin A exhibits robust antifungal activity against multiple C. neoformans clinical strains and minimal toxicity in A549 and HepG2/C3 cells, two validated cell culture models^48,49. Similarly, iturin A has shown a broad-spectrum and potent antifungal activity against yeast-like²⁶ and filamentous fungi^50,51.

We observed that iturin A exhibits synergistic antifungal activity with AmpB against C. neoformans and enhances survival in the C. elegans infection model, highlighting its potential as a promising approach for improving treatment outcomes in cryptococcosis. This synergy is particularly significant because it may allow for reduced AmpB doses, potentially mitigating the severe nephrotoxicity and infusion-related adverse effects that limit its clinical use, while maintaining or even enhancing fungicidal activity. Although iturin A showed substantial antifungal activity, resistance development was not evaluated in the current study. Therefore, serial-passage studies will be needed to determine whether repeated exposure to iturin A leads to reduced susceptibility in C. neoformans.

Whether iturin A can cross the blood–brain barrier (BBB) is currently unknown and was not evaluated in this study. Given its cyclic lipopeptide structure and relatively large molecular size, passive CNS penetration cannot be assumed. Accordingly, pharmacokinetic studies and BBB permeability experiments will be required before iturin A can be considered a candidate for CME treatment. Nevertheless, the iturin A–AmpB combination may represent a promising strategy for cryptococcal infection, though its direct relevance to CME remains to be established.

The current standard of care for cerebral cryptococcosis combines AmpB with flucytosine for induction therapy, but flucytosine is often unavailable in resource-limited settings where the disease burden is highest, and this combination still carries substantial toxicity and suboptimal treatment success rates²⁰. If the iturin A–AmpB combination demonstrates similar or superior efficacy to the AmpB–flucytosine regimen in mammalian models and eventual clinical trials, with improved safety profiles and better accessibility, it could offer a transformative alternative for managing this life-threatening infection, particularly in settings where flucytosine is unavailable or treatment failure rates remain unacceptably high.

We also confirmed that iturin A’s MOA is consistent with previous studies that have established fungal cell membrane disruption as a primary antifungal mechanism for this lipopeptide²⁶. Iturin A interacts with ergosterol, the predominant sterol in fungal cell membranes, leading to membrane permeabilization, pore formation, and ultimately cell lysis^52–54. Supporting this, supplementation of the culture medium with ergosterol increased iturin A’s MFC, suggesting an interaction between the lipopeptide and this mycosterol. In addition, iturin A activates the voltage-gated potassium channels, stimulating K⁺ efflux from fungal cells²⁹. Moreover, iturin A exposure induces the accumulation of intracellular ROS in fungal cells, triggering oxidative stress-mediated cell death pathways^55,56. Our work builds upon this established mechanistic understanding by validating that both ergosterol-mediated membrane disruption and ROS generation are operative against C. neoformans, and further demonstrates how these effects contribute to the observed synergy with AmpB, which also targets ergosterol, potentially amplifying membrane damage and oxidative stress to achieve enhanced fungicidal activity.

Our findings that iturin A at sub-inhibitory concentrations reduce C. neoformans capsule size and inhibits biofilm formation have profound implications for combating this infection. The polysaccharide capsule is the primary virulence factor of C. neoformans, enabling immune evasion by inhibiting phagocytosis, suppressing inflammatory responses, and protecting the fungus from oxidative stress imposed by host immune cells¹⁴; thus, capsule reduction could significantly enhance host immune clearance and improve the efficacy of both innate and adaptive immune responses. Furthermore, biofilm formation by C. neoformans contributes to antifungal tolerance, chronic infection, and treatment failure, as cells within biofilms exhibit reduced susceptibility to conventional antifungal agents compared to their planktonic counterparts¹⁵. By inhibiting biofilm formation and attenuating capsule synthesis at concentrations below those required for direct fungicidal activity, iturin A could serve as an adjunctive therapy that enhances the penetration and effectiveness of standard antifungals, reduces the fungal burden, prevents dissemination, and potentially shortens treatment duration. Of note, the reduction in capsule size, capsule volume, and cell body size observed at similar concentrations may also contribute to decreased biofilm crystal violet staining and biomass measurements. These anti-virulence properties position iturin A as a promising candidate for combination therapy strategies aimed at not only killing the fungus directly but also disarming its key defense mechanisms to facilitate more effective infection control and improved clinical outcomes.

Interestingly, our observation that iturin A reduces C. neoformans phagocytosis by macrophages and microglia while simultaneously enhancing intracellular fungal killing presents a complex but potentially beneficial scenario for controlling cryptococcal dissemination. C. neoformans employs a “Trojan horse” mechanism of dissemination, whereby the fungus survives and replicates within macrophages that then traffic across the BBB, facilitating CNS invasion and the establishment of meningoencephalitis^57,58. By reducing the rate of phagocytosis, iturin A may limit the fungal hijacking of immune cells for dissemination, potentially decreasing the fungal burden. Simultaneously, the enhanced intracellular killing observed in phagocytes that do engulf C. neoformans suggests that iturin A treatment may shift the balance from permissive intracellular replication to effective fungal clearance, possibly by attenuating capsule-mediated resistance to phagolysosomal killing or by sensitizing the fungus to oxidative and nitrosative stress within the phagosome^59,60. This dual effect could be particularly advantageous in preventing early dissemination from pulmonary sites of infection while ensuring that any internalized fungi are efficiently eliminated. However, the reduced phagocytosis must be carefully balanced, as effective immune clearance ultimately requires phagocyte engagement; thus, the net therapeutic benefit would depend on whether the enhanced killing efficiency outweighs any potential delays in overall fungal clearance from extracellular spaces.

In conclusion, iturin A represents a promising therapeutic candidate against cryptococcosis through its multifaceted mechanism: disrupting fungal membranes, inducing oxidative stress, and demonstrating potent synergy with AmpB that could reduce nephrotoxicity while maintaining efficacy. Its ability to attenuate C. neoformans’ virulence factors may disarm immune evasion mechanisms and limit CNS dissemination and invasion. The demonstrated efficacy of iturin A against C. neoformans in the C. elegans infection model provides preliminary validation, warranting further investigation in mammalian models and eventual clinical trials to potentially transform treatment outcomes for cryptococcal infections, particularly in resource-limited settings.

Materials and methods

C. neoformans

C. neoformans strain H99 was obtained from Arturo Casadevall (Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA) and isolated by John Perfect (Duke University School of Medicine, Durham, NC, USA). In addition, a total of 7 clinical isolates (C. neoformans strains: 55, 57, 59, 68, 72, 83, 88; a gift from Arturo Casadevall) were included in this study. The fungi were inoculated in Sabouraud dextrose broth (Difco) and incubated at 30 °C for 24 h in an orbital shaker incubator set at 150 rpm.

Reagents and chemicals

Iturin A (MedChemExpress) was dissolved in dimethyl sulfoxide (DMSO; Fisher Chemical) to prepare a 10 mg/mL stock solution, which was stored at 4 °C until use. Also, the following chemicals and reagents were used in this study: RPMI 1640 medium powder (Gibco) ; 3-(N-morpholino) propanesulfonic acid (MOPS, Sigma-Aldrich); DMEM (ATCC); Fetal bovine serum (FBS, ATCC); Tris-buffered saline (TBS, Thermo Fisher Scientific); PI (Thermo Fisher Scientific); the ROS-sensitive probe DHFDA (MedChemExpress); AmpB deoxycholate (Gibco); 5-fluorocytosine (Fisher Scientific); XTT sodium salt (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) and phenazine methosulfate (Sigma-Aldrich); Alexa Fluor 488-conjugated goat anti-mouse IgG₁ (Invitrogen).

Antifungal susceptibility testing

Antifungal susceptibility was determined by MIC, the standard metric for evaluating antifungal activity, enabling direct comparison with established agents, including AmpB and fluconazole. The MICs of iturin A, AmpB, and fluconazole against eight C. neoformans isolates were determined using the broth microdilution method according to Clinical Laboratory Standards Institute⁶¹. Drugs were serially diluted two-fold in RPMI 1640 medium in a sterile 96-well microtiter plate. Each well contained 150 μL of RPMI 1640 medium inoculated with 10³ C. neoformans cells/mL. Plates were incubated at 35 °C for 72 h. The MIC was defined as the lowest drug concentration that produced complete (100%) growth inhibition, based on visual turbidity assessment.

Time-kill assay

To evaluate the killing kinetics of iturin A against C. neoformans H99 cells, fungal suspensions (10⁴ CFU/mL) were prepared in RPMI 1640 medium and exposed to iturin A at a final concentration of 25 μg/mL [corresponding to 2×MIC⁶²]. AmpB (1 μg/mL, 2×MIC) was used as a positive control. The cultures were incubated at 35 °C. At pre-determined time points (0, 2, 4, 6, 12, 24, 36, 48, and 72 h), 100 μL aliquots were withdrawn, serially diluted in sterile phosphate-buffered saline (PBS), and plated onto Sabouraud dextrose agar. For killing assays, fungal burden was reported as log₁₀ CFU/mL because CFU recovery directly measures viable fungal burden following drug exposure. Plates were incubated at 35 °C for 48 h, after which CFUs were counted. Fungal viability was expressed as Log₁₀ CFU/mL over time. Additionally, sub-inhibitory concentrations of iturin A (3 μg/mL), AmpB (0.25 μg/mL) or their combination were added to test their synergistic fungicidal activity over time.

Synergy testing

The synergistic activity between iturin A and AmpB was assessed using the checkerboard method⁶³. Aliquots of iturin A and AmpB were serially diluted in RPMI 1640 medium in various concentration combinations. A fresh RPMI 1640 broth culture of C. neoformans H99 was added to each well of a 96-well microtiter plate (Falcon) at a final concentration of 10³ cells/mL, followed by an incubation at 35 °C for 72 h. Then, 4 μL from each dilution was spotted on a Sabouraud dextrose agar plate, containing each drug alone or multiple concentrations of iturin A and AmpB as indicated, and incubated at 35 °C. Scanning images were captured after 48 h. Moreover, the fractional inhibitory concentration index (FICI) was calculated to determine the synergistic activity between iturin A and AmpB. The FICI of an antimicrobial was determined by establishing the MIC of the antimicrobial (A or B) in the combination (MICcomb) divided by the MIC of the antimicrobial acting alone (MICalone). The formula used to calculate the FICI is as follows: FICI = FICA + FICB = MICAcomb/MICAalone + MICBcomb/MICBalone. The following parameters were considered: synergy (FICI ≤ 0.5), additivity (0.5 < FICI ≤ 1.0), indifference (1.0 < FICI ≤ 4.0) or antagonism (FICI > 4.0).

Cytotoxicity evaluation

Human lung epithelial cells (A549; ATCC) and liver cells (HepG2/C3A; acquired from Dr. Christopher Vulpe at the Center for Environmental and Human Toxicology, University of Florida) were used to assess the toxicity of iturin A as described previously⁶⁴. A density of 10⁶ A549 and HepG2/C3A cells/well was seeded in 96-well microtiter culture plates with DMEM and 10% FBS and incubated at 37 °C and 5% CO₂ for 24 h to allow for cell adhesion. Then, the cells were exposed to a wide range of iturin A concentrations (0.5–64 µg/mL) in duplicate/triplicate for 24 h, while untreated cells served as a negative control. The cell viability was assessed using the XTT reduction assay. Briefly, the culture medium was aspirated and replaced with 100 µL of fresh DMEM medium, followed by the addition of 50 µL of XTT solution (1 mM) with 1% phenazine methosulfate to each well, including the untreated control. The treated, cell culture 96-well microtiter plate (Falcon) was then incubated at 37 °C and 5% CO₂ for 4 h. The optical density (OD) was measured at a wavelength of 450 nm with a reference wavelength of 660 nm using a BioTek Synergy H1 microplate reader. The XTT reduction was calculated by subtracting the OD 660 nm from the OD 450 nm of each sample. The 50% cytotoxicity concentration (CC₅₀) was defined as the drug concentration that reduced the viability of the intact cells by 50%.

Cell membrane permeabilization assay

The cell membrane integrity of C. neoformans cells was investigated by flow cytometry and fluorescent microscopy using the propidium iodide (PI) staining after iturin A treatment⁶⁵. For flow cytometry analysis, C. neoformans cells were adjusted to 10⁶ CFU/mL in RPMI 1640 medium and treated with iturin A (2×MIC, 25 μg/mL) for 6 h at 30 °C. After incubation, PI (10 μg/mL) was added, incubated for 30 min in the dark at room temperature. Then, cells were washed once with PBS and analyzed by flow cytometry. A minimum of 10,000 events per sample was collected, and the data were analyzed to determine the percentage of cells stained with PI. To validate the fungal membrane permeability after ITA treatment in the presence and absence of ergosterol, C. neoformans cells stained with PI were visualized under a Leica DMi8 inverted microscope at a magnification of ×40, prior to imaging with a Leica DFC7000T digital camera using the Leica software platform LAS X. Untreated and AmpB-treated (2×MIC, 1 μg/mL) cells were used as negative and positive controls, respectively. The fluorescence intensity was measured at excitation/emission 535/617 nm, and results were calculated as a percentage of PI incorporation relative to the untreated control.

Ergosterol rescue assay

To confirm whether iturin A permeabilizes the fungal membrane through the interaction with ergosterol, we performed the ergosterol supplementation assay⁶⁶. Briefly, C. neoformans cells were exposed to 0.78–25 µg/mL iturin A in RPMI 1640 medium with or without ergosterol (200 μg/mL) at 35 °C for 72 h. Then, aliquots of each iturin A concentration were plated on Sabouraud dextrose agar to determine the MFC of iturin A in the presence or absence of ergosterol.

ROS assay

To investigate the impact of iturin A on the generation of intracellular cryptococcal ROS, a density of 10⁶ C. neoformans cells/mL was treated with 25 μg/mL iturin A and 10 μM H₂O₂ as a positive control⁶⁷. After 6 h of incubation at 30 °C, cells were stained with the ROS dye DHFDA for 30 min in the dark, and the fluorescent intensity was measured using a microplate reader at excitation/emission 485/535 nm. ROS levels were further analyzed using flow cytometry, and the results were reported as relative fluorescence intensity percentage (%).

Capsule inhibition assay

To evaluate the impact of iturin A on C. neoformans capsule production, overnight (o/n) cultures of C. neoformans H99 cells were collected by centrifugation, washed once with sterile PBS, diluted to 10⁶ CFU/mL in DMEM supplemented with 10% FBS, and incubated at 37 °C for 72 h in the presence of sub-inhibitory concentrations of ITA [3, 6, and 12 µg/mL]⁶⁸. To visualize the capsule, 5 μL aliquots of the cell suspension were mixed with one drop of India ink, and the capsule was visualized using light microscopy. The images were captured for at least 50 cells per concentration tested, and the statistical analyses for the capsule volume, cell body size, and capsule size (whole cell diameter–cell body diameter) among samples were conducted.

Biofilm formation

The activity of iturin A against C. neoformans biofilms was evaluated according to the method described by Kumari et al. ⁶⁹ with slight modifications. Briefly, a density of 10⁶ C. neoformans cells/mL was suspended in RPMI in a treated polystyrene 96-well plate (Falcon). Iturin A was then added at final concentrations of 3, 6, and 12 μg/mL, while the drug-free wells were set as a negative control. The plate was incubated at 35 °C for 48 h for biofilm formation. Then, biofilms were gently washed 3× with sterile PBS to remove planktonic yeast cells. Biofilm viability and biomass were examined using CFU determination and crystal violet staining, respectively. Following PBS washing, biofilms were disrupted by adding 100 µL of 0.01% triton X-100, followed by vigorous pipetting to ensure complete detachment. The suspension was transferred to sterile microcentrifuge tubes and vortexed for 1 min to achieve uniform dispersion. Serial tenfold dilutions were prepared in sterile PBS, and aliquots of the dilutions were plated onto Sabouraud dextrose agar plates. Plates were incubated at 35 °C for 48 h, after which colonies were counted. Results were expressed as log₁₀ CFU/mL, representing viable biofilm-associated cells. For total biofilm biomass determination, washed biofilms were stained with 0.1% (w/v) crystal violet solution for 15 min at room temperature. Excess stain was removed by gently washing the wells 3× with sterile PBS, and plates were air-dried completely. Stained cells were solubilized by adding 100 µL of 70% (v/v) ethanol to each well and incubating for 10–15 min with gentle shaking. The absorbance was measured at 600 nm using a BioTek Synergy H1 microplate reader. Biomass values were normalized to untreated controls and expressed as a percentage of control biofilm formation.

Confocal microscopy

Confocal microscopy was used to visualize the inhibitory effect of iturin A on cryptococcal biofilms⁷⁰. Glass-bottom plates were coated with poly-L-lysine (0.1 mg/mL; Sigma) for 4 h at room temperature to promote cell adhesion and air-dried under sterile conditions. C. neoformans cells were grown in minimal medium (20 mg/mL thiamine, 30 mM glucose, 26 mM glycine, 20 mM MgSO₄·7H₂O, and 58.8 mM KH₂PO₄; pH 5.5; Sigma) and incubated at 30 °C for 48 h. Following biofilm formation, the wells containing C. neoformans biofilms were gently washed 3× with PBS to remove non-adhered cryptococci using a multichannel pipette. The primary monoclonal antibody GXM-specific 18B7 (2 µg/mL) was added, followed by the incubation of the secondary goat anti-mouse IgG₁ Alexa Fluor 488 (1 µg/mL) prepared in 1% bovine serum albumin/HEPES. Each incubation was performed at 30 °C for 1 h, and the wells were washed with TBS containing 0.05% tween 20 between steps. Biofilms were imaged using a Nikon Eclipse Ti2 inverted confocal laser scanning microscope equipped with a ×40 objective lens. A series of horizontal (xy) optical sections was acquired throughout the full thickness of the biofilm to generate z-stack images. Green fluorescence corresponding to GXM labeling (18B7-Alexa Fluor 488) was recorded, and biofilm architecture and fluorescence intensity were analyzed using NIS-Elements software.

Phagocytosis assay

Monolayers of J774 macrophage- or NR-9460 microglia-like cells were incubated in DMEM feeding medium (10% FBS and 1% penicillin/streptomycin) supplemented with IFN-γ (100 U/mL), LPS (0.5 ng/mL), mAb 18B7, and 6 μg/mL iturin A, followed by the addition of H99 cryptococci in a 1 (phagocyte):10 (yeast) ratio⁷¹. For phagocytosis, the plates were incubated for 4 h at 37 °C and 5% CO₂. The monolayer coculture was washed thrice with PBS to remove nonadherent cells, fixed with cold methanol, Giemsa (5%) stained, and visualized with a Leica DMi8 inverted microscope, and images were captured with a Leica DFC7000 digital camera using LAS X digital imaging software. The number of internalized yeast cells per phagocyte was reported after counting 100 phagocytic cells per field. Internalized cells were differentiated from attached cells by the presence of a well-defined phagocytic vacuole.

Intracellular killing by phagocytic cells

After Ab-mediated phagocytosis, each well containing interacting phagocyte-cryptococci was gently washed with feeding medium three times to get rid of fungal cells that were not phagocytized. Then, cryptococcus-engulfed macrophage or microglia were incubated in the absence or presence of iturin A (3 and 6 μg/mL) for 48 h at 37 °C and 5% CO₂. Phagocytic cells were lysed by adding 100 µL of 0.01% triton X-100, followed by vigorous pipetting to ensure complete lysis of phagocytes. A 100-µL volume of suspension containing cryptococci was aspirated from the wells and transferred to a microcentrifuge tube with 900 µL PBS. For each well, serial dilutions were performed and plated in triplicate onto Sabouraud dextrose agar plates, which were incubated at 30 °C for 48 h. Viable cryptococcal cells were quantified as CFU. Although it is plausible that noninternalized cryptococci could replicate for several generations in feeding medium during a 24-h period, wells for each condition were microscopically monitored after phagocytosis to reduce the possibility of obtaining confounding results.

Caenorhabditis elegans infection model

C. elegans AU37 [sek-1(km4); glp-4(bn2) I] was used to evaluate the in vivo efficacy of iturin A against C. neoformans⁷². Worms were maintained on solid nematode growth medium (NGM) agar plates at 15 °C. For the experiments, worms were synchronized and co-cultured with Escherichia coli OP50 from the L1–L4 developmental stages at 25 °C. In parallel, C. neoformans H99 cells were cultured on Sabouraud dextrose broth at 30 °C. Synchronized L4-stage worms were then transferred to 15 mL tube with 10⁶ C. neoformans H99 cells/mL and incubated at 25 °C for 6 h. Following incubation, nematodes were collected and washed 5× with PBS and transferred to a 96-well microtiter plate. Iturin A (12 and 25 μg/mL) and iturin A (3 μg/mL)–AmpB (0.25 μg/mL) combination treatments were added to the worms in triplicate for 48 h. For survival, worms were monitored daily for 10 days, and survival was expressed as the % of live worms. For CFU determinations, the nematodes were collected, washed, homogenized using zirconium beads to produce fungal suspensions, serially diluted, and plated on Sabouraud dextrose agar plates. The results were reported as Log₁₀ CFU per worm.

Statistical analysis

All data were subjected to statistical analysis using Prism 10.6 (GraphPad). Differences in survival rates were analyzed by the log-rank test (Mantel–Cox). P-values for multiple comparisons were calculated by one-way analysis of variance (ANOVA) and were adjusted by use of Tukey’s post-hoc analysis. P-values for individual comparisons were calculated using Student’s t-test analyses. P-values of <0.05 were considered significant.

Author contributions

Y.E., R.I.G.M., and C.L.C-N. designed and/or conducted the experiments and collected and/or analyzed the data. Y.E. and L.R.M. wrote the manuscript and participated in its editing.

Funding

Y.E., C.L.C-N., and L.R.M. were supported by the National Institute of Allergy and Infectious Diseases (NIAID award # AI187391) of the US National Institutes of Health (NIH). Y.E. and L.R.M. were also supported by the FAILSAFE Funding: Accelerating Global Solutions to Antifungal Resistance. L.R.M. is supported by the UF Center for Translational Research in Neurodegenerative Disease Bridge Funding. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data availability

All data generated or analyzed during this study are included in this published article.

Code availability

Not applicable.

Declarations

Competing interests

L.R.M. reports a patent (issued and pending) assigned to the University of Florida. All the other authors declare no competing interests.