Antibiofilm strategies targeting Candida glabrata increasingly focus on membrane-active compounds capable of disrupting biofilm formation and mature biofilm structure. Building on previous work identifying alkylamidobetaine C9 (AAB C9) - [(3-decanoylmethylamino)propyl]dimethylammonium acetate - and β-escin as antibiofilm agents against C. glabrata, this study examined the biochemical and physiological basis of their efficacy in a reference strain (ATCC 90030) and a clinical isolate (2586). Oxidative stress was assessed microscopically after two hours of exposure, and proteome changes were quantified after six hours, for each compound alone and in combination. C. glabrata cells retained membrane integrity under all treatment conditions, indicating that antibiofilm activity does not stem from membrane lysis. β-Escin induced the strongest mitochondrial and superoxide ROS response in the reference strain, whereas the AAB C9–β-escin combination was most effective in the clinical isolate, revealing strain-dependent sensitivity. Proteomic analysis identified altered abundance of proteins involved in protein quality control (FES1), transcriptional regulation (SPT4), DNA replication initiation (SLD3), and glucose transport (HXT10). Notably, β-escin reduced abundance of the virulence-associated β-mannosyltransferase, and AAB C9 reduced the multidrug transporter TPO1_1, linked to azole and antimicrobial peptide resistance. These findings suggest that AAB C9 and β-escin act through selective, strain-dependent remodeling of membrane-associated proteins coupled with oxidative and proteostatic stress, supporting their potential as antibiofilm and antifungal agents against C. glabrata.

Candida biofilms associated with medical devices represent a major clinical challenge due to their high resistance to conventional antifungal therapies. In this study, we investigated the structure–activity relationship of a homologous series of dicephalic cationic surfactants branched on carbon atom (Cn-DCNMe3Br, n= 10, 12, 14), with a focus on the effect of alkyl chain length on antifungal, antibiofilm, and cytotoxic properties. Planktonic susceptibility assays revealed a non-linear “cut-off” effect, with the C12 homologue exhibiting maximal fungicidal efficacy across the tested strains (C. albicans, C. glabrata, C. krusei, and C. tropicalis). In contrast, adhesion and biofilm-related assays demonstrated that the less hydrophobic C10 homologue showed superior performance in inhibiting initial cell attachment and disrupting mature biofilms. These findings highlight the critical role of early-stage surface interactions in determining overall antibiofilm efficacy. Mechanistically, the activity of these amphiphiles appears to be strongly associated with their ability to modulate cell–surface interactions, likely through a combination of physicochemical effects, including surface adsorption, alteration of wettability, and electrostatic interactions with microbial cell envelopes. While oxidative stress was observed as a secondary effect, it is not considered the primary determinant of antifungal activity. Crucially, biocompatibility assessment based on hemolysis assays demonstrated a clear chain-length-dependent toxicity profile. While C10 and C12 exhibited concentration-dependent hemolytic activity above 0.5 mM, the C14 derivative showed negligible hemolysis across the tested concentration range (up to 5 mM). These findings underscore the importance of fine-tuning alkyl chain length to balance antimicrobial efficacy, anti-adhesive performance, and host cytotoxicity, positioning this novel class of dicephalic surfactants as promising candidates for surface-functionalization strategies aimed at preventing device-associated infections.

Antibiofilm strategies targeting Candida glabrata increasingly focus on membrane-active compounds capable of disrupting biofilm formation and mature biofilm structure. Building on previous work identifying alkylamidobetaine C9 (AAB C9)—[(3-decanoylmethylamino)propyl]dimethylammonium acetate—and β-escin as antibiofilm agents against C. glabrata, this study examined the biochemical and physiological basis of their efficacy in a reference strain (ATCC 90030) and a clinical isolate (2586). Oxidative stress was assessed microscopically after two hours of exposure, and proteome changes were quantified after six hours, for each compound alone and in combination. Fluorescence microscopy showed that AAB C9 and β-escin interacted differently depending on the ROS compartment and strain: in the strain ATCC 90030, the combination antagonized β-escin-induced mitochondrial superoxide, whereas in the multidrug-resistant isolate 2586 it exceeded either agent alone for both cytosolic and mitochondrial superoxide, indicating a strain-dependent, additive interaction. Quantitative proteomics showed that strain genetic background, rather than treatment, was the dominant source of proteome variance. Preranked gene set enrichment analysis revealed that AAB C9 significantly suppressed accumulation of biofilm-formation-associated proteins specifically in isolate 2586, corroborating its phenotypic antibiofilm activity, while β-escin and the combination produced opposing transmembrane-transport signatures, pointing to compound-specific mechanisms underlying their differential antifungal and antibiofilm effects.