Polymer-driven neutralization of candidalysin induced cytotoxicity

Introduction: Candida albicans is an opportunistic fungal pathogen, causing diseases ranging from mucosal, localized infections to more severe infections, with mortality rates as high as 40%1. This fungal species can exist in three morphological forms: yeast, pseudohyphae, and hyphae. The yeast-to-hyphal transition is essential for C. albicans pathogenicity, as hyphal cells can escape host immune defenses and physically penetrate host cells through the formation of invasion pockets2,3. However, this transition alone is not enough to cause damage to host cells. Within the invasion pocket, hyphae release virulence factors that contribute to the progression of the infection4,5. Candidalysin, a recently discovered peptide toxin, has gained recognition as one of the main virulence factors responsible for C. albicans pathogenicity by enhancing epithelial cell damage during invasion6. Candidalysin self-assembles into a polymeric loop-like structure before insertion into the host cell membrane, leading to pore formation and cell membrane damage7. Given the major role of candidalysin in C. albicans infections, neutralizing this peptide toxin has the potential to reduce infection severity. We hypothesized that biopolymer interactions with candidalysin may be used to mitigate the damaging effects of candidalysin. There are limited studies exploring the interaction of biological polymers with candidalysin8,9, although none focus on the potential mechanisms involved or feasibility of future biomaterials development using the polymers studied. In this work, we identified anionic, synthetic and natural polymers that inhibit candidalysin loop formation and reduce mammalian cell toxicity.

Materials and

Methods: Synthetic candidalysin (SIIGIIMGILGNIPQVIQIIMSIVKAFKGNK)6 was synthesized using microwave-assisted solid-phase peptide synthesis with fluorenylmethoxycarbonyl (Fmoc) chemistry. Quartz crystal microbalance with dissipation monitoring (QCM-D) was used to monitor interactions of the selected polymers (Table 1) and synthetic candidalysin by monitoring real-time frequency and dissipation changes on gold-coated quartz crystals. First, poly-L-lysine (PLL) was adsorbed to the sensor to create a positive priming layer. Then, the polyanion of interest (100 μg/mL) and candidalysin (100 μg/mL) were introduced sequentially to monitor adsorption and interaction. Candidalysin loop formation with and without exposure to the polyanions was evaluated using atomic force microscopy (AFM). Briefly, candidalysin (250 μg/mL) or the candidalysin and polyanion mixture (450 μg/mL) in ultrapure water was filtered (0.22 μm). A 100 μL aliquot was added onto a clean glass cover slip and dried at 22°C. A silicon cantilever with aluminum reflective coating (stiffness ≈ 40 N/m) was utilized for topographical height and phase scans over a range of scan sizes (10 μm to 100 nm). Candidalysin cytotoxicity against NIH 3T3 mouse fibroblasts, human umbilical vein endothelial cells (HUVECs), and VK2/E6E7 vaginal epithelial cells was assessed using Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) release assays. Mammalian cells were exposed to 250 μg/mL of candidalysin and polymers at various concentrations (450, 225, 112.5, and 56.25 μg/mL). Neutralization of candidalysin was assessed using two methods: simultaneous exposure of the cells to the peptide and polymer (method 1) and pre-incubation of the peptide and polymer in 0.1× phosphate buffered saline before cell exposure (method 2).

Results, Conclusions, and Discussions: Successful synthesis of synthetic candidalysin was confirmed using liquid chromatography-mass spectrometry (data now shown). All polymers were found to successfully adsorb to the PLL-coated sensor (Figure 1a), as evidenced by the decrease in ∆F following PLL adsorption (step C), remaining stable during washing (step D). A decrease in ∆F was observed for polymer-coated sensors after introducing candidalysin (step E), indicating interaction between each polymer and candidalysin. For a thorough assessment of mass deposition and polymer-candidalysin interaction, we analyzed overall ∆F (Figure 1b) and ∆D (Figure 1c) post-buffer rinse. HA, albumin, and CS showed similar ∆F differences between polymer and candidalysin addition, with values of approximately −21 Hz, −14 Hz, and −18 Hz, respectively. PAA exhibited the highest candidalysin adsorption (−100 Hz), while heparin showed the lowest candidalysin adsorption (−13.4 Hz). Variations in candidalysin mass deposition may stem from differences in polymer-candidalysin interaction affinity or polymer molecular weight, causing stripping of previously formed polymer layers. All polymers formed tightly coupled layers with consistently low ∆D values. PAA, due to its hygroscopic nature, exhibited the highest ∆D values, indicating less rigid layer formation. AFM was used to study candidalysin loop formation in the presence of polymers. Candidalysin formed heterogeneous loops in water, with diameters ranging from ~150 nm to ~400 nm (Figure 1d, 1e). Loop formation was still observed after mixing candidalysin with albumin and PLL (Figure 1f, 1g), but not with the other polymers of interest. Samples with polymer alone also showed no loop-like structures. After confirming candidalysin-polymer interaction via QCM-D and loop formation using AFM, we explored whether interaction with polymers could neutralize the peptide toxin in vitro. Both heparin and PAA decreased candidalysin-induced cytotoxicity across all cell types with LDH release (indicating membrane damage) comparable to untreated controls. Albumin and PLL did not inhibit candidalysin induced cytotoxicity. Neutralization induced by CS and HA varied across cell type, suggesting that electrostatic interactions may not be the only mechanism responsible for candidalysin neutralization (Figure 1h, 1i, 1j). Overall, this study offers novel insights into neutralizing candidalysin using biopolymers, which could potentially be used to decrease pathogenicity of C. albicans during infection.

Acknowledgements (Optional): We would like to thank Lisa Chen (Brown University) for assistance with LC-MS data collection.