After Candida albicans, the most prevalent yeast species reported to have been isolated from humans is Candida glabrata (C. glabrata). The past two and a half decades have witnessed C. glabrata becoming one of the most predominant Candida species, causing fungal infections (Chakrabarti et al., 2009; Lockhart et al., 2012; Benaducci et al., 2014; Cleveland et al., 2015; Pappas et al., 2018; Naicker et., 2023). The reason behind this steep upward trend of C. glabrata infections may be associated with geographical variation and the rampant overuse of azoles (Richardson and Lass-Flörl, 2008). Given the prevalence of C. glabrata as the second leading cause of candidiasis infections, including oral candidiasis, candidemia, invasive candidiasis, and urinary candidiasis (Esfandiary et al., 2012; Nash et al., 2016), it is essential to understand the alarming 40% mortality rate associated with C. glabrata infection underscores the seriousness of this opportunistic human pathogen, especially in immunocompromised hosts (Rodrigues et al., 2014; Mota et al., 2015; Nagayoshi et al., 2017). It has been recently reported that C. glabrata has developed several strategies to survive and thrive in its host cells and found ways to overcome antifungal resistance to commonly used antifungals, which also contribute to increased virulence (Pappas et al., 2018; Salazar et al., 2018). Flow cytometry (FCM) has been one of the major techniques that has successfully demonstrated its utility in determining the effect of antimycotics on yeast cells, cell membranes, or mechanisms of action when used with appropriate dye or fluorescent probes and experimental conditions (Pore, 1994). In our previous study (Ramachandran et al., 2018b), the antifungal susceptibility test (AFST) analyses of the two lipopeptides were done according to the CLSI guidelines. In this current investigation, the two lipopeptide fractions (AF₄ and AF₅) coproduced by Bacillus subtilis RLID 12.1 were purified, and the main objective of the study was to determine their membrane-permeabilizing effects on the yeast cell membrane and metabolic activity at two different peptide concentrations using the membrane-impermeant propidium iodide (PI) dye and two-colour FUN-1 stain.

The MICs of two lipopeptides and AMB (used as the standard antifungal) against C. glabrata were determined to be 4 mg/L and 1 mg/L, respectively. The results agree with the previously determined MIC values (Ramachandran et al., 2018b; Ramchandran et al., 2020). To gain more insights into lipopeptides’ membrane permeabilizing potential, we performed PI uptake assays using 8 mg/L (×2) and 16 mg/L (×4) MICs as determined by the AFST for C. glabrata ATCC 2001. FC results were expressed as the fluorescence intensity (FI) of PI-stained yeasts. AF₄ and AF₅ treatments caused cell membrane disruption in C. glabrata cells, as seen by the enhanced fluorescence that resulted from PI uptake. In cells treated with AMB, AF₄, AF₅, and 70% ethanol, the increase in PI-fluorescence is manifested as a distinct shift of the peak along the x-axis (Figures 1A, B) and (Figures 2A, B). The histograms pertaining to all flow cytometry assays where C. glabrata ATCC 2001 cells were stained with PI after being exposed to AMB for 5 h (Gokahmetoglu et al., 2003) at 1 and 2 mg/L and AF₄/AF₅ (8 and 16 mg/L) for 18 h have been shown in Figures 1A, 2A. All the events of FC population density plots have been shown in Figures 1B, 2B.

Significant congruence was found when comparing the proportion of PI uptake with the drop in CFU/mL among AF₄ treatments. A substantial association was observed between the percentage PI uptake estimated in flow cytometry and the percentage reduction in plate counts, presented in tabular form (Table 1) to summarize the findings. Cells unexposed to lipopeptides showed very negligible fluorescence, as evidenced by the PI uptake percentage graph (Figure 3). At 8 mg/L, the AF₄-treated C. glabrata showed an average of 81.92% PI-positive cells and 99.90% CFU reduction, better was the observation in AF₄-(16 mg/L)-treated C. glabrata cells, where 99.51% PI-positive cells were noted and a CFU reduction of 99.97% recorded (Figure 3; Table 1). At 8 mg/L, the AF₅-treated C. glabrata showed an average of 92.14% PI-positive cells and 99.4% CFU reduction. However, AF₅-(16 mg/L)-treated C. glabrata cells showed 81.35% PI-positive cells, and the CFU reduction of 99.92% was recorded, indicating that AF₅ even at a 2-fold concentration, did not show more membrane permeabilization and increased fungicidal effects. This may be due to the hydrophobic aggregation of AF₅ lipopeptide at higher concentrations (Ramchandran et al., 2020). In AMB (1 and 2 mg/L)-treated cells for 5 h, only 20.02% and 26.06% PI-positive cells were detected, respectively. The increase in the fluorescence intensity percentage observed in yeast cells treated with higher AF₄ and AF₅ (8 mg/L) concentrations agreed with the positive control values, indicating potential antifungal activity. In comparison, untreated cells showed negligible fluorescence.

Candida cells exposed to AF₄/AF₅ (8 mg/L) displayed PI uptake as evident from red fluorescence, indicating cell membrane damage and hence permeabilization [Figure 1C (×120) and Figure 1D (×200)] magnifications. Since the formation of cylindrical intravacuolar structures (CIVS) needs metabolically active cells with undamaged plasma membrane, the presence of yellow-green fluorescence (Figure 2C) in FUN-1 stained cells instead of red fluorescence in lipopeptide-treated cells clearly indicates the loss of membrane integrity, metabolic activity, and cell death (Millard et al., 1997). The cell deaths are quite evident from the log reductions of 3.03 and 3.64 CFU/mL by AF₄ (8 and 16 mg/L). In the lipopeptide-treated and AMB-treated cells CIVS were not observed. Collectively, it may be surmised that the prime targets of AF₄ and AF₅ might be the components of the cell membrane (Ramesh et al., 2023). The presence of a long acyl chain in antifungal cyclic lipopeptides such as iturin and fenzycin enables oligomer formation and subsequent insertion into the plasma membrane (Malina and Shai, 2005) and the organisation of aggregates in biological membranes triggering early membrane damage and membrane disintegration was also reported by Horn et al. (2013). The preliminary mechanism of action on the yeast cell membrane has been schematically depicted in the Supplementary Figure S2. Taken together, the results revealed that two lipopeptides demonstrated remarkable fungicidal effects on C. glabrata cells with enhanced cell membrane permeability, and damage. Consequently, the findings positively indicate that the lipopeptides AF₄ and AF₅ hold considerable promise as prospective antifungal agents.