Use of Self-Assembling Antimicrobial Peptides Against Pathogenic Candida Biofilms

Introduction: In certain circumstances, Candida albicans, a commensal fungus in the human oral microbiome, can overgrow, causing life-threatening systemic infections. The ability of C. albicans to form biofilms is one of the major virulence factors that allows it to become dangerous, hiding from the immune system while also resisting antifungal treatment. Another species in the Candida Genus of rising concern, Candida auris, is responsible for deadly nosocomial infections. This species is particularly dangerous because of its multidrug resistance, with some strains having resistance to all currently available classes of antifungals. C. albicans and C. auris are two of the four species listed as critical by the first fungal pathogens list (FPPL). This list was developed by WHO to prioritize research globally and development for new antifungal treatments considering the public health importance and unmet research needs of these fungi. There is a limited armament of drugs effective against fungi, and many of these existing drugs are toxic to human cells. Considering this unmet need, we have examined the efficacy of an antimicrobial self-assembled peptide hydrogel (AMP6) against biofilms of both Candida albicans and Candida auris. AMP6 has shown efficacy against bacteria biofilms but had not been evaluated against fungal biofilms. The self-assembling peptide AMP6 is poly-cationic and can potentially associate with fungal membranes, disrupting them. To test this, we will computationally evaluate the specificity of AMP6 for fungal membranes, test AMP6 against both C. albicans and C. auris biofilms, and then evaluate the biocompatibility of the highest effective concentration of AMP6.

Materials and

Methods: Cellular membrane stability was evaluated computationally by atomistic simulations of AMP6 peptide fibers and their effects on model yeast cell membranes. Results were analyzed for membrane destabilization based on lipid density and other parameters. To investigate the efficacy of AMP6 against Candida species, biofilms of Candida albicans and Candida auris were grown in a 96 well plate, then media was replaced with varying concentrations of the AMP6 peptide, varying concentrations of antifungals, and vehicle controls. After 24 hours of incubation, metabolic activity was measured and wells were sonicated to assess viability (CFU). In a murine model, biocompatibility of AMP6 was assessed by subcutaneous implantation assessing degradation and long term stability as well as repeat dose toxicity. Dorsal subcutaneous implants of 200 μL AMP6 or PBS sham control were injected into adult female Wistar rats and explanted at 7 days and 28 days. A repeat dose toxicity study was also performed in adult female Wistar rats, with 200 μL of AMP6 or PBS sham controls injected subcutaneously every three days for 30 days accompanied by blood collection. All explanted tissue was fixed, sectioned, and stained with H&E and Masson’s Trichrome to assess cellular infiltration, bolus degradation, and collagen deposition. To evaluate changes in systemic humoral immune function after repeated administration, serum samples from the AMP6 experimental group were analyzed for rat IgG titers at each timepoint and compared to PBS control serum by ELISA.

Results, Conclusions, and Discussions: Computational simulations of AMP6 have shown membrane stability disruptions against bacterial membranes, with fungal membrane simulations still ongoing. AMP6 showed efficacy against preformed biofilms of C. albicans and C. auris in millimolar concentrations. AMP6 performed better than the antifungal fluconazole at the same concentrations. The antifungal nystatin was more effective at the same concentration as AMP6. However, nystatin is highly toxic systemically and even topically, while AMP6 was shown to have good biocompatibility at high concentrations without signs of toxicity. The repeat dose testing also did not show any development of an immune response against AMP6.

The antimicrobial assay results indicate that AMP6 is efficacious against Candida species in their most pathogenic state. This peptide also shows no acute toxicity in vivo, which is vital as many antifungals are toxic, especially at high concentrations. This is promising as there are a limited number of drugs that are effective against pathogenic fungi, especially as those associated with nosocomial infections can often be resistant to one or more of the few classes of antifungals available. Expanding the number of available therapeutics effective against these pathogens is critical. Future work will explore the combination of AMP6 with other antifungals for added synergistic effects.

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