The antifungal lipopeptide AF₄ was isolated and fractionated using a process that was previously described in Ramchandran et al., 2019. B. subtilis RLID 12.1 cultured in shake flasks for 60 h at 30°C in a media comprised of dextrose, malt extract, peptone, sodium chloride and MnSO₄. Using HCl (6N) of appropriate volume, the crude lipopeptide was precipitated from the cell-free supernatant after 60 h. In a 1:1 ratio, n-butanol and sodium phosphate buffer (pH 8.0, 50 mM) were used to dissolve the crude precipitate and solvent extraction was performed with overnight mixing followed by centrifugation. The crude extract in butanol, was then evaporated completely and dry-loaded onto an adsorption column packed with silica gel (230–400 mesh, particle size 37–63 µm). A gradient of chloroform and methanol was used to fractionate the crude extract. The eluted fractions were tested for activity against C. albicans SC5314 by spot-on lawn method. Fractions exhibiting clear zones of inhibition were taken up for purification by reverse-phase (RP) HPLC using a Luna 5 μm -C18 column (250 mm × 10 mm, particle size 5 μm) at a semi-preparative scale. The fractions that showed anti-Candida activity were dissolved in 200 µL methanol and injected into the HPLC. Lipopeptides were purified using a gradient solvent system comprising acetonitrile and water with 0.1% trifluoroacetic acid. The gradient of acetonitrile used for purification were as follows: 0%–45% for 0–10 min at the flow rate of 1 mL/min, 45%–54% from 10 to 20 min at 0.5 mL/min, 54%–60% from 20 to 48 min at 0.5 mL/min, 60%–100% from 48 to 65 min at 1 mL/min and 100%–5% from 65 to 75 min at 1 mL/min and monitored at 210 nm. To ensure that pure fractions with single peaks were obtained, AF₄ fractions were collected and re-chromatographed using a Luna 5 μm C18 250 mm × 4.6 mm, particle size 5 μm). Purified fractions were pooled, lyophilised and resuspended in autoclaved 10 mM sodium phosphate buffer. The concentration of the lipopeptide was estimated by the Bicinchonnic acid (BCA) method (Ramachandran et al., 2018) (Pierce ™ BCA-Protein assay kit ThermoFisher, United States). The minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of AF₄ and amphotericin B (AMB) were determined for C. albicans SC5314 and clinical isolates of C. albicans (obtained from NCCPF, Mycology Division, PGIMER, Chandigarh India) according to CLSI guidelines (Clinical and Laboratory Standards Institute CLSI, 2017).

Cells suspensions with an inoculum size of ∼10⁶ CFU/mL were prepared and exposed to the antifungal compound AF₄ at a concentration of 8 mg/L (2X MIC) overnight. As controls, Candida cells were cultivated without any antifungal treatment and treated with conventional antifungal amphotericin B (AMB) at 1 mg/L (2X MIC) for 3 h. The samples were prepared using glutaraldehyde as a fixative, followed by osmium tetroxide staining. Excess fixative and osmium tetroxide were washed off after each step using sodium cacodylate buffer and sterile water as described in (Semis et al., 2013; Ramesh et al., 2023). The samples were dried using gradient ethanol dehydration and subjected to critical point drying, sputter coated with gold and visualised at a magnification of 20000X using a Quanta FEG 250 (Thermo Fisher Scientific, United States) instrument.

Confocal microscopy was employed to observe the impact of pure fractions of AF₄ on membrane integrity (Kuhn et al., 2002; Chan et al., 2011; Zhang et al., 2018). Cell suspensions were prepared as described above. Acridine orange (AO) (20 µM) and propidium iodide (PI) (5 µg/mL) were used to stain the harvested Candida cells for 15 min each in the dark. Excess stain was removed by phosphate buffered saline (PBS) washes and trapped between a glass slide and cover slip. The coverslips were fixed using enamel and imaged at 120X magnification (Ramesh et al., 2023). The FUN-1 dye (5 µM) was applied to a cell suspension resuspended in a glucose-supplemented HEPES solution. The cells were incubated at 30°C in the dark for 30 min (Pina-Vaz et al., 2001a). The unbound stain was washed off with the same solution, and samples were fixed on glass slides and imaged as described above. Images were acquired using the Olympus Fluoview 3000. Excitation at 490 nm and emission at 520 nm were used for AO, while PI was used with excitation and emission maxima of 535 nm and emission maximum of 615 nm respectively. A fluorescein filter set with excitation at 480 nm and emission at 530 nm was used to image cells with FUN-1.

The ability of AF₄ to permeabilize membranes was determined by PI uptake in cells treated with AF₄ (Ramani and Chaturvedi, 2000; Lee and Lee, 2015). The antifungal lipopeptide AF₄ at concentrations of 4 and 8 mg/L and AMB at 0.5 mg/L and 1.0 mg/L were added to cell suspension (∼5 × 10⁶ CFU/mL) in RPMI-1640 and incubated for 18 h and 3 h, respectively. Cells grown in the absence of antifungals and 70% ethanol-treated cells were used as negative and positive controls for PI uptake, respectively. Cells were harvested after treatment and resuspended in PBS. The suspensions were stained with PI (5.0 µg/mL) and incubated in the dark for 20 min. The stained cells were washed with PBS and suspensions were analysed using a FACScan flow cytometer (Becton Dickinson FACS Melody) using a 488 nm laser line and 586 nm filter for PI. The forward scatter, side scatter and percentage of PI-stained cells were recorded and analysed (Hwang et al., 2011a; Hao et al., 2013; Jia et al., 2019; Li et al., 2020). Aliquots of treated and untreated samples were serially diluted, plated on Sabouraud Dextrose (SD) agar plates and incubated for 24 h at 37°C (Green et al., 1994). The colonies formed were counted to determine the reduction in CFU/mL across treatments.

Changes to the membrane dynamics due to antifungal action were assessed by labelling fungal cell membranes with 1,6-diphenyl-1,3,5-hexatriene (DPH). A decrease in DPH fluorescence is proportional to the extent of disruption in the membrane lipid bilayer (Lee et al., 2018). Cells were treated with 4 and 8 mg/L of AF₄ overnight and AMB (0.5 mg/L). Cells were then fixed with 0.37% formaldehyde and incubated for 30 min at 28°C. Cells were then washed and flash-frozen by dipping them in liquid nitrogen. Cells were subsequently thawed, resuspended, and labelled with 0.6 mM DPH in PBS and incubated for 45 min at 28°C. Post-incubation cells were washed with PBS and homogenized by sonication on ice (Park and Lee, 2009; Lee et al., 2018). Cells were centrifuged, and the fluorescence intensity of the supernatant was measured using a spectrofluorophotometer (JASCO FP-8500, Japan) with 350 nm excitation and 425 nm emission.

ROS produced upon treatment with the antifungal was determined using 2′-7′ dichloro-dihydro-fluorescein diacetate (DCFH-DA) (Sigma-Aldrich, United States) fluorescence by flow cytometry (Da Silva et al., 2014; Seyedjavadi et al., 2020). Cells suspensions prepared in RPMI-1640 were incubated overnight with AF₄ (4, 8 and 16 mg/L) and AMB at 0.5, 1.0 and 2.0 mg/L at 37°C in shaking condition. Hydrogen peroxide (10 mM) (H₂O₂)-treated cells were used as positive control and untreated cells as negative control. After incubation cells were harvested by centrifugation, washed, and resuspended in PBS with 10 µM of DCFH-DA (Li et al., 2020). The cells were stained for 30 min in the dark at 37°C. The cell suspension was washed and resuspended in PBS, and DCF fluorescence was recorded using a BD FACScan Flow cytometer. The increase in the percentage of ROS-positive cells was determined by the fluorescence of treated samples in comparison with that of untreated samples.

C. albicans cells treated with AF₄ at 2, 4, and 8 mg/L overnight were harvested and washed in 1X PBS. The pellet was resuspended in 1 mL of 0.1 M potassium phosphate buffer (50 mM K₂ HPO₄, 5 mM EDTA, 50 mM DTT, 50 mM KH₂PO₄, and 40 mM β-mercaptoethanol) with 2.4 M sorbitol and at pH 7.2 (Hao et al., 2013; Jia et al., 2019). To the suspension, Zymolyase 20T (0.02 mg/mL) (US Biologics, United States) was added and incubated for 20 min at 35°C. The protoplasts were washed and resuspended in 100 μL permeabilization solution (0.1 M sodium citrate (pH 6.0) with 0.1% Triton X- 100), and placed on ice for 5 min. The protoplasts were washed again and stained with Annexin V- FITC/PI apoptosis detection kit (MedChem Express, United States) according to the manufacturer’s instructions (Hwang et al., 2011a; Hwang et al., 2011b; Hao et al., 2013). The cell staining data was acquired using flow cytometry and analysed using the software FlowJo V10.

Fragmentation and condensation of nucleic acid due to the antifungal activity of lipopeptide was assessed using 4′,6-diamidino-2-phenylindole (DAPI) (HiMedia). Cells treated with lipopeptide at 2, 4 and 8 mg/L as described above were harvested and stained with 1 µg/mL DAPI for 20 min at 37°C. Cells were imaged at 200X using an Olympus FV3000 microscope (Hwang et al., 2011a; Hwang et al., 2011b; Hao et al., 2013).

SEM was performed to elucidate the ultrastructural alterations of C. albicans SC5314 cells treated with the antifungal lipopeptide AF₄ and the standard polyene AMB. Results of SEM after overnight treatment with antifungal AF₄ and 3 h treatment with AMB (Figure 3A) revealed that cells treated with the lipopeptide AF₄ showed damages on the cell surface, comparable to that caused by antifungal AMB, observed as indents, dimples, and concavities indicated in Figure 3A by yellow arrows, whereas cells grown in the absence of antifungals maintained their ovoid cell shape with smooth surface and bud scars.

Confocal microscopy was used to examine the influence of the antifungals AF₄ and AMB on the permeability and metabolic state of C. albicans SC5314 cells. When treated with AO/PI, as seen in Figure 3B, only green fluorescence was observed in the control panels, indicating intact membranes permeable only to AO (Zhang et al., 2018; Fiołka et al., 2021). However, cells exposed to AMB (used as a positive control) and AF₄ showed reddish-orange fluorescence due to PI uptake, indicating cell membrane damage and permeabilization.

FUN-1 is a two-colour fluorescent probe that utilizes the metabolic pathways that are common among yeasts. The biochemical conversion of FUN-1 dye’s diffuse green fluorescence into condensed orange-red aggregates within the vacuoles (CIVS) in healthy cells is proof of intact cell membranes and active metabolism (Millard et al., 1997). In contrast, cells affected by the antifungal molecule show green-yellow fluorescence distributed throughout the cell. As depicted in Figure 3C, untreated cells remained viable and demonstrated the formation of red fluorescent CIVS. On the contrary, cells treated with AMB and AF₄ showed yellow-green fluorescence, signifying a loss of metabolic activity and increased membrane permeability.

Flow cytometry was utilised to measure the altered permeability of the cell membrane using the vital stain PI (Pina-Vaz et al., 2001b), following treatment with lipopeptide AF₄. Treatments with AF₄ and 70% ethanol resulted in a greater fluorescence of PI uptake than untreated cells, which showed only negligible PI fluorescence (Figure 4A). Scatter plots in Figure 4A describe the side scatter (pink dots) increase corresponding to antifungal treatment, indicating an increase in the granularity of the cells analysed. Colony counts of the lipopeptide treatment aliquots plated correlated well with the PI uptake percentages. For AF₄, the percentages of PI uptake were recorded as 72.5% ± 2.14% and 97% ± 1.47% when treated with 4 and 8 mg/L of AF₄, respectively. For AMB, the uptake percentage was observed to be 31.84% ± 0.65% and 48.86% ± 10.07% at 0.5 mg/L and 1 mg/L after 3 h of treatment. In comparison, cells treated with ethanol showed near-complete membrane damage, with 99% PI uptake. The number of colonies from the aliquots of the lipopeptide treatments correlated well with the percentages of PI uptake (Figure 4B).

Changes to cell surface granularity were observed from the FSC-A vs. SSC-A contour plots, an increase in SSC-A indicated increased membrane granularity (Figure 4C). Increased distribution in quadrant one (Q1-FSC-ve/SSC + ve) was seen in cells treated with AF₄ (Figures 4C(ii), (iii)) in comparison to untreated cells (Figure 4C(i)) indicating an increase in the granularity. For the cells treated with AMB (Figures 4C(iv), (v)) a reduction in distribution was observed in Q3(FSC +ve/SSC-ve) indicating reduced cell size and shrinkage and an increase in Q1 (Q1-FSC-ve/SSC+) was observed indicating increased granularity.

The reduction in cell numbers after treatments with AF₄ and AMB was estimated from the colony counts obtained by plating serial dilutions of aliquots of the samples treated with 4 and 8 mg/L of AF₄ and 0.5 and 1 mg/L of AMB on SD agar plates. The number of colonies was recorded after a 24-h incubation, and percentage reduction and log reduction values were determined. The percentage reduction in colony count of Candida cells treated with 4 and 8 mg/L of the AF₄ lipopeptide was found to be 80.59% (log reduction of 0.71 ± 0.14) and 99.98% (log reduction of 3.99 ± 0.015), respectively (Figure 4D). Treatment with AMB resulted in a 99% reduction in CFU numbers at both concentrations tested showing CFU log reduction of 2.41 ± 0.175 and 3.54 ± 0.095 for 0.5 and 1 mg/L respectively.

When the membrane is bound with DPH, the reduction in fluorescence intensity indicates the membrane perturbation of the C. albicans plasma membrane, mediated by the lipopeptide AF₄. The antifungal effect results in the disruption of lipid bilayer organisation and restricts the binding and subsequent fluorescence intensity of DPH. Treatment with AF₄ at 4 mg/L and 8 mg/L led to a reduction in relative fluorescence intensity to 59.06% ± 5.31% and 10.16% ± 2.76%, respectively in comparison to the fluorescence intensity observed in untreated cells (Figure 4E). Treatment with AMB at 0.5 mg/L showed a relative fluorescence intensity of 9.93% ± 5.22%.

The generation of ROS is an important marker of antifungal activity and of cells undergoing early apoptosis (Hao et al., 2013; Da Silva et al., 2014; Jia et al., 2018). To assess the influence of antifungal lipopeptide AF₄ on endogenous ROS levels, the intensity of fluorescence caused by the oxidation of the cell-permeable fluorescent probe DCFH-DA to DCF was estimated. Results showed that AF₄-treated C. albicans cells displayed significantly elevated levels of ROS (Figure 5A) in comparison to untreated cells, with values of 53.57, 78.44, and 77.30% (Figure 5B) for concentrations of 4, 8, and 16 mg/L, respectively. Additionally, AMB-treated cells showed a significant increase in fluorescence (Figure 5A), with more than 70% ROS-positive cells observed at all concentrations of AMB used (Figure 5B). ROS is considered a key factor in the induction of apoptosis in yeast, and hence exposure to phosphatidylserine (PS) and DNA damage were investigated.

Induction of apoptosis results in the translocation of PS from the inner to the outer leaflet of the plasma membrane (Da Silva et al., 2014; Crowley et al., 2016; Li et al., 2019). Apoptosis induced by lipopeptide treatment was measured by flow cytometry using Annexin V-FITC and PI double staining. Annexin V-FITC stain binds to externalised PS and is a marker for early apoptosis, and PI binds to membrane-compromised cells, indicating necrotic cells. C. albicans SC5314 cells after treatment with lipopeptide AF₄ were mostly detected in quadrant 2-corresponding to Annexin V-FITC, and PI-positive (Figure 6A) (Crowley et al., 2016), which indicates late-stage apoptosis or cell death. Untreated cells were distributed in quadrant 4, negative for the uptake of both stains and hence indicating the absence of PS exposure and membrane damage. A shift towards necrosis (Figure 6A) was observed at the fungicidal concentration of 8 mg/L, consistent with previously obtained results.

DAPI, a membrane-penetrable dye that fluoresces upon binding to nucleic acids, enabled the visualisation of nuclear morphology and DNA damage, fragmentation, or condensation. Cells treated with AF₄ at different concentrations exhibited abnormal nuclei possessing tubular, tear-drop shaped or distorted structures, as seen in Figure 6B, denoted by yellow arrows. Meanwhile, untreated cells, indicated by white arrows, demonstrated spherical nuclei with normal morphology.