A synthetic peptide mimic kills Candida albicans and synergistically prevents infection

Abstract

More than two million people worldwide are affected by life-threatening, invasive fungal infections annually. Candida species are the most common cause of nosocomial, invasive fungal infections and are associated with mortality rates above 40%. Despite the increasing incidence of drug-resistance, the development of novel antifungal formulations has been limited. Here we investigate the antifungal mode of action and therapeutic potential of positively charged, synthetic peptide mimics to combat Candida albicans infections. Our data indicates that these synthetic polymers cause endoplasmic reticulum stress and affect protein glycosylation, a mode of action distinct from currently approved antifungal drugs. The most promising polymer composition damaged the mannan layer of the cell wall, with additional membrane-disrupting activity. The synergistic combination of the polymer with caspofungin prevented infection of human epithelial cells in vitro, improved fungal clearance by human macrophages, and significantly increased host survival in a Galleria mellonella model of systemic candidiasis. Additionally, prolonged exposure of C. albicans to the synergistic combination of polymer and caspofungin did not lead to the evolution of tolerant strains in vitro. Together, this work highlights the enormous potential of these synthetic peptide mimics to be used as novel antifungal formulations as well as adjunctive antifungal therapy.

Fig. 1. Chemical structures of polyacrylamides synthesised for this study.

R represents the different side chains and x indicates the targeted number of hydrophobic residues within the molecule.

Fig. 2. Antifungal polymers cause transcriptomic responses associated with impaired protein glycosylation, membrane stress, and cell wall damage in C. albicans.

A Gene Ontology (GO) term enrichment analysis, based on molecular function, and B KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis of RNA microarray data after treating C. albicans SC5314 for 1 h at 30 °C with sub-inhibitory concentrations of polymers and LL-37. Statistically significantly up- (red) or downregulated (green) gene groups associated with GO terms and KEGG pathways compared to the untreated control are shown. Diameter of circles reflects the percentage of genes differentially regulated in the associated pathway or term and the shading represents the adjusted p-value (calculated by hypergeometric distribution and adjusted by Benjamini–Hochberg correction for multiple testing). GO terms are additionally ordered by their assigned parental processes.

Fig. 3. Polymer LH causes membrane lysis in C. albicans.

Detection of fluorescence of C. albicans expressing cytoplasmic GFP (ADH1-GFP) after 6 h at MIC assay conditions for (A) untreated C. albicans cells, B cells treated with 1× MIC Amphotericin B (AmpB), C 1× MIC tunicamycin, D 0.25× MIC polymer LH, E 0.5× MIC polymer LH, and F 1× MIC polymer LH to investigate lytic activity of the compounds. Scale bars in (A–F) represent 10 µm. G The GFP signal was quantified from at least 50 C. albicans-GFP cells per biological replicate (n = 3), and then averaged and normalised to the respective untreated control. H Colony forming units (CFU) were determined by backplating and normalised to the inoculum (n = 3 biological replicates). Statistical significance in (G) and (H) was determined by Dunnett’s ordinary one-way ANOVA multiple comparisons analysis (compared to untreated (100%) in (G) or inoculum (100%) in (H), *p < 0.05 [G: p = 0.0148, H: p = 0.0296], **p <  0.01 [G: p = 0.0051], ***p < 0.0005 [H: p = 0.0002], ****p < 0.0001) with ns indicating non-significant [H: p = 0.4987]. Average conductance (G_m) of tethered membranes isolated from C. albicans yeast (black) or hyphae (grey), or from erythrocytes (red) after the addition of increasing concentrations (c) of (I) antifungal polymer LH and (J) non-toxic poly-HEA in RPMI medium at 37 °C (n = 3 biological replicates). Error bars in (G–J) represent the standard deviation (SD) around the mean. Source data are provided as a Source Data file.

Fig. 4. Cell wall changes in C. albicans and immune cell response to LH-treated C. albicans.

Transmission electron microscopy (TEM) micrographs of the C. albicans cell wall after 6 h incubation in SD medium at 30 °C with (A) no additives, B with poly-HEA, C with tunicamycin, or D antifungal polymer LH, at sub-inhibitory concentrations of the antifungal compounds. The scale bars in (A–D) represent 100 nm. E The amount of glucan and mannan in cell walls isolated from C. albicans wildtype (SC5314) and och1 mutant cells after 6 h of incubation in SD medium (untreated) and with sub-inhibitory concentrations of polymer LH, poly-HEA, tunicamycin, and caspofungin was determined by HPLC. Glucose (from glucan) and mannose (from mannan) represented 55% and 45% of the dry weight of the cell wall in wild-type untreated cells. The graph shows the proportions of glucan (grey bars) and mannan (striped bars) relative to those observed in untreated C. albicans wild-type cells. F Proportion of C. albicans cells not taken up over 15–30 min by human monocyte-derived macrophages. C. albicans cells were pre-treated with LH for one hour before putting them into contact with the macrophages (n = 6 biological replicates). Statistical significance in (E) was determined by Dunnett’s repeated measures ANOVA multiple comparisons analysis (*p <  0.05 [0 vs 8 µg/ml: p = 0.0173; 0 vs 16 µg/ml; p = 0.0192]). Error bars in (F) represent the standard deviation (SD) around the mean. Source data are provided as a Source Data file.

Fig. 5. Synergistic effects of LH in combination with selected antifungal drugs against C. albicans SC5314 during vaginal epithelial cell infections.

Damage to vaginal epithelial (A-431) cells (infected by C. albicans and uninfected) after treatment with polymer LH and A caspofungin (Cas) or B fluconazole (Flu) and their respective combinations was measured by LDH release (n = 4 biological replicates, SD around the mean). Damage to A-431 cells was normalised to untreated infection control (for infected samples) or a Triton-X-treated 100% lysis control (uninfected samples; each indicated by a dotted line). Source data are provided as a Source Data file. (C–J) show fluorescence microscopy images of the scenarios represented in (A) and (B) to visualise morphological changes and the viability of vaginal epithelial cells by staining with 1 µg/mL propidium iodide. Scale bars represent 100 µm.

Fig. 6. Treatment with polymer LH in combination with caspofungin (Cas) prolongs survival of C. albicans-infected Galleria mellonella larvae.

G. mellonella larvae were infected with 1 × 10⁵ C. albicans cells (except uninfected control, grey) and treated after 2 h with caspofungin (Cas; 5 mg/kg: blue; 100 mg/kg: blue dotted), polymer LH (250 mg/kg, green dashed), and the combination of 5 mg/kg Cas and 250 mg/kg LH (green and blue dashed). Untreated, infection controls are shown in black (C. albicans only) or black dashed (C. albicans, injected with water). Survival of G. mellonella was monitored over 14 d. Nineteen larvae per condition were tested. Statistical significance was determined for infected larvae treated with the combination (5 mg/kg Cas + 250 mg/kg LH) compared to infected larvae treated with the respective dose of a single drug by Log Rank (Mantel–Cox) pairwise comparison (**p < 0.01 [here: p = 0.003], ***p < 0.001). At the concentrations used here, the single compounds exhibited no toxicity against G. mellonella, as shown in Supplementary Fig. S30. Source data are provided as a Source Data file.

Fig. 7. In vitro evolution experiment of C. albicans challenged with 1× MIC of caspofungin (Cas), fluconazole (Flu), polymer LH, and the synergistic combinations.

A Experimental setup of the in vitro evolution experiment. B Growth of C. albicans was monitored by absorbance over 14 d and normalised to the untreated controls. Filled circles highlight strains that were selected for whole genome sequencing and empty circles highlight strains additionally analysed for their MIC against antifungal compounds in Supplementary Table S5. Source data are provided as a Source Data file. C Genomes of the isolated strains with the highest tolerance to antifungal drugs were sequenced and analysed for their relative copy number for Flu- and LH-evolved strains. Each point in (C) represents the mean normalised read depth compared to wild-type C. albicans strain SC5314 at t = 0 for a gene (Y-axis) on its chromosome position (X-axis), colour-coded by allele. Positions of the centromeres are indicated by red circles. The MICs for Flu and LH are indicated on the right, where MIC in SC5314 is depicted in green and higher values scale to red.