Antifungal Activity of Synthetic Peptides Targeting Apoptosis in Candida auris

Abstract

Background: Nosocomial infections caused by the multidrug-resistant (MDR) yeast Candida auris pose a significant healthcare threat. This pathogen presents a major clinical challenge due to its resistance profile, virulence factors, including biofilm formation, and the overexpression of efflux pumps. Current antifungal treatments are often limited in efficacy against MDR fungal pathogens, particularly C. auris. Therefore, novel strategies to address these challenges are urgently needed. Apoptosis, a form of programmed cell death, represents a potential target for novel antifungal approaches. Furthermore, strategies targeting virulence and efflux pump activity could be a valuable approach to combat C. auris infections. Human antimicrobial peptides (AMPs) hold promises due to their broad-spectrum activity, including activity against various fungi. Thus, this study investigated the potential of selected human AMPs against C. auris and the role of apoptosis induction in their antimicrobial activity. These peptides were also evaluated for their ability to inhibit virulence and efflux pump in C. auris. Methods: Antifungal efficacy of seven distinct human AMPs, including HNP-1-3, hBD-1-3 and His 5, against ten C. auris clinical isolates, was determined adhering to the established CLSI guidelines for antifungal susceptibility testing. The antifungal activity of the AMPs was further assessed via time-kill curves and cell viability assays. The potential synergy with established antifungals (fluconazole, amphotericin B, and caspofungin) was explored using combination assay. To understand their antifungal mechanism, propidium iodide staining and confocal imaging were employed to evaluate their effects on C. auris membrane permeability. The three most active AMPs (HNP-1, hBD-3, and His 5) were further investigated for their ability to induce apoptosis in two C. auris isolates. Specific markers for apoptosis, including phosphatidylserine externalization (FITC Annexin-V/PI assay) and DNA fragmentation (TUNEL assay), were employed. Additionally, mitochondrial membrane potential (JC-10 staining), cytochrome c release (spectrophotometry), and reactive oxygen species (ROS) production (H2DCFDA staining) were assessed to understand potential intracellular triggers of apoptosis. Furthermore, the study investigated the impact of the most active AMPs on C. auris virulence factors and efflux pumps. Adherence and proteinase activity were assessed using adherence assays and bovine serum albumin (BSA) plates, respectively. The effects on biofilm formation, maturation and structure were evaluated using the MTT (3-(4, 5-dimethylthiazolyl- v 2)-2, 5-diphenyltetrazolium bromide) reduction test and confocal microscopy. The influence on efflux pump activity was determined by measuring rhodamine-6-G accumulation and efflux. Reverse transcription quantitative real-time PCR (RT-qPCR) was employed to investigate the effects on the expression of virulence and efflux pump-related genes. Finally, their safety was evaluated through a haemolytic assay. Results: All examined AMPs exhibited fungicidal activity against C. auris, with hBD-3 demonstrating the most potent effect. Time-kill curves confirmed the fungicidal activity of all tested peptides and viability assays demonstrated a significant decrease in viable cells upon exposure to AMPs. Notably, AMPs displayed synergistic interactions with conventional antifungals, particularly caspofungin. Mechanistically, AMPs disrupted C. auris membrane permeability, correlating with growth inhibition. Furthermore, FITC Annexin-V/PI staining and TUNEL assays demonstrated that HNP-1 and hBD-3 induced both early and late apoptosis in C. auris, while His 5 primarily caused necrosis. HNP-1 and hBD-3 triggered significant mitochondrial membrane depolarization and cytochrome c release. In contrast, His 5 displayed minimal impact on mitochondrial function. All peptides significantly increased ROS production. In addition, subinhibitory concentrations of HNP-1, hBD-3, and His 5 significantly reduced C. auris adherence and proteinase activity. All three peptides effectively inhibited biofilm formation and reduced the density of established biofilms. They also impaired efflux pump activity and downregulated genes associated with virulence and efflux pumps. Furthermore, haemolytic assays confirmed the safety of these peptides towards mammalian red blood cells. Conclusion: These findings highlight the potential of human AMPs as broad-spectrum antifungal agents against C. auris. Their ability to target multiple mechanisms, including direct killing, apoptosis induction, virulence inhibition and efflux pump reversal, suggests a multifaceted approach to combat C. auris infections. Additionally, the synergistic interaction with conventional antifungals suggests potential for combination therapy. Overall, this study findings provide a strong rationale for further investigation and development of human AMPs as novel antifungal agents against this MDR pathogen.