Multiplex antimicrobial activities of the self-assembled amphiphilic polypeptide β nanofiber KF-5 against vaginal pathogens

Abstract

Background: Vaginal infections caused by multidrug-resistant pathogens such as Candida albicans and Gardnerella spp. represent a significant health challenge. Current treatments often fail because of resistance and toxicity. This study aimed to synthesize and characterize a novel amphiphilic polypeptide, KF-5, and evaluate its antibacterial and antifungal activities, biocompatibility, and potential mechanisms of action.

Results: The KF-5 peptide was synthesized via solid-phase peptide synthesis and self-assembled into nanostructures with filamentous and hydrogel-like configurations. Characterization by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) confirmed the unique nanostructural properties of KF-5. KF-5 (125, 250, or 500 µg/ml) demonstrated potent antibacterial and antifungal activities, with significant inhibitory effects on drug-resistant Candida albicans and Gardnerella spp. (P < 0.05). In vitro assays revealed that 500 µg/ml KF-5 disrupted microbial cell membranes, increased membrane permeability, and induced lipid oxidation, leading to cell death (P < 0.05). Cytotoxicity tests revealed minimal toxicity in human vaginal epithelial cells, keratinocytes, and macrophages, with over 95% viability at high concentrations. Molecular dynamics simulations indicated that KF-5 interacts with phospholipid bilayers through electrostatic interactions, causing membrane disruption. In vivo studies using a mouse model of vaginal infection revealed that 0.5, 1, and 2 mg/ml KF-5 significantly reduced fungal burden and inflammation, and histological analysis confirmed the restoration of vaginal mucosal integrity (P < 0.01). Compared with conventional antifungal treatments such as miconazole, KF-5 exhibited superior efficacy (P < 0.01).

Conclusions: KF-5 demonstrates significant potential as a safe and effective antimicrobial agent for treating vaginal infections. Its ability to disrupt microbial membranes while maintaining biocompatibility with human cells highlights its potential for clinical application. These findings provide a foundation for further development of KF-5 as a therapeutic option for combating drug-resistant infections.

Keywords: Antibacterial; Bacterial biofilm; Self-assembled peptide nanoscale; Vaginal infections.

Fig. 1

Synthesis and physicochemical properties of KF-5. A High-performance liquid chromatography (HPLC) analysis of the KF-5 polypeptide showing a peak at a 9.35 min retention time with 98.7029% purity. B Mass spectrometry (MS) identification of the KF-5 polypeptide confirming the molecular weight of KF-5

Fig. 2

Characterization of KF-5 self-assembled nanostructures. A Scanning electron microscopy (SEM) image showing larger-scale filamentous structures formed by the self-assembly of KF-5. B Low-magnification transmission electron microscopy (TEM) image showing a dense network of interconnected KF-5 nanofibers, indicating uniform self-assembly. C High-magnification TEM image of individual KF-5 nanofibers, revealing a fiber width of approximately 26.97 nm. (D-F) TEM images illustrating increased filament density and structural complexity at different KF-5 concentrations: D 1 mg/mL, E 2 mg/mL, and F 5 mg/mL. G TEM image of KF-5 nanostructures negatively stained with 3% uranyl acetate, highlighting the intricate arrangement of fibers. H, I Atomic force microscopy (AFM) images showing the 3D topography of KF-5 nanostructures, which form a staggered grid-like structure with a height of approximately 2.4 nm. J Fourier transform infrared spectroscopy (FTIR) analysis of KF-5 nanofibers, indicating that they predominantly consist of β-sheets and antiparallel β-sheets, with minimal α-helical content

Fig. 3

Antifungal and antibacterial activities of KF-5 against Candida albicans and Gardnerella vaginalis. A Time-concentration gradient of KF-5 at concentrations of 0 (μg/mL) (control), 125 (μg/mL) (μg/mL), 250 and 500 (μg/mL) on Candida albicans. B Antifungal effects of 0 (μg/mL) (control), 125 (μg/mL), 250 (μg/mL) and 500 (μg/mL) KF-5 on Candida albicans spores. C Antibacterial effect of KF-5 at concentrations of 0, 125, 250 and 500 μg/ml on Gardnerella spp. D, E Confocal microscopy images showing the increased proportion of dead D Candida albicans spores and E mycelia after KF-5 treatment. Scale bar: 50 μm. The data are presented as the means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

KF-5 causes membrane perturbations and oxidative stress in Candida albicans. A Zeta potential of KF-5 measured at different pH values, showing positive charges at pH 3, 5, and 7 and negative charges at pH 9 and 11. B KF-5-induced membrane depolarization in Candida albicans, as measured by the increase in fluorescence of the diSC3(5) probe (Triton X-100 was used as a positive control). C KF-5-induced membrane permeabilization was measured via an 8-anilonapthalene-1-sulfonic acid (ANS) uptake assay (melittin was used as a positive control). D Lipid peroxidation in Candida albicans detected via C11-BODIPY581/591 after KF-5 treatment, indicating increased oxidative stress. The data are presented as the means ± SDs. ****P < 0.0001

Fig. 5

Biocompatibility of KF-5 with mammalian cells. CCK-8 assays were performed to evaluate the cytotoxicity of KF-5 at concentrations of 125 (μg/mL), 250 (μg/mL), 500 (μg/mL), and 1000 (μg/mL) on A VK2 (human vaginal epithelial cells), B HaCaT (keratinocytes), and C RAW264.7 (rat macrophages) cells. Over 95% cell viability was maintained even after treatment with 1000 µg/mL KF-5

Fig. 6

Simulation of the interaction between the KF-5 peptide and the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipid membrane. A Root-mean-square deviation (RMSD) in the molecular simulation of KF-5 and POPC phospholipid membranes. B The interaction energy between the KF-5 polypeptide molecule and the whole phospholipid membrane molecule, showing primarily electrostatic interactions. C, D Density distributions of the KF-5 polypeptide and POPC phospholipid in the initial and final complex structures. E, F Spatial distribution projection of the KF-5 polypeptide molecule and POPC phospholipid membrane in the initial and final complex structures. G, H KF-5 polypeptide molecule and POPC phospholipid membrane mechanism of the initial and final complex structure simulation diagrams. (I) Enlarged view of the interaction region between the KF-5 polypeptide molecule and the POPC phospholipid membrane in the final complex structure, showing local embedding of KF-5 on the membrane surface

Fig. 7

KF-5 effectively moderates vaginal infection in mice. A Vaginal fungal burden analysis via colony counting in the vaginal lavage fluid of control, model, 0.5 (mg/mL) KF-5, 1 (mg/mL) KF-5, and 2 (mg/mL) KF-5 mice from days 1–14. Treatment was administered daily for 5 days starting on Day 0. B The formation of hyphae in the vaginal excreta of the mice in the control group, model group, 0.5 (mg/mL) KF-5 group, 1 (mg/mL) KF-5 group, and 2 (mg/mL) KF-5 group was determined by Gram staining on the 0th and 7th days. Scale bar: 50 μm. C Histopathological analysis of vaginal tissues from mice in the control group, model group, 0.5 (mg/mL) KF-5 group, 1 (mg/mL) KF-5 group, and 2 (mg/mL) KF-5 group was performed via hematoxylin and eosin (H&E) staining (top row) and periodic acid Schiff (PAS) staining (bottom row). Scale bar: 100 μm. (D) ELISA was performed to detect TNF-α and IL-6 levels in the vaginal tissues of mice in the control group, model group, 0.5 (mg/mL) KF-5 group, 1 (mg/mL) KF-5 group, and 2 (mg/mL) KF-5 group. The data are presented as the means ± SDs. +  + indicates P < 0.01 compared with the control group. + indicates P < 0.05 compared with the control group. * indicates P < 0.05 compared with the model group. ** indicates P < 0.01 compared with the model

Fig. 8

Biosafety characteristics of KF-5. Toxicological evaluation of KF-5 following a repeated intravaginal daily dose for 28 days. A HE staining of vaginal tissues from mice in the control, 0.5 (mg/mL) KF-5, 1 (mg/mL) KF-5, and 2 (mg/mL) KF-5 groups revealed no significant epithelial damage or inflammatory cell infiltration. Scale bar: 100 μm. B ELISA results showing TNF-α and IL-6 levels in the vaginal tissues of different mice. No statistically significant differences were detected between the control group, 0.5 (mg/mL) KF-5 group, 1 (mg/mL) KF-5 group, and 2 (mg/mL) KF-5 group (P > 0.05). The data are presented as the means ± SDs