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Communication

Phage Display-Derived Peptides Have Neutralizing Activities Against Biofilm Formation by Candida albicans, Candidozyma auris and Candida parapsilosis

by
Grigory Bolotnikov
1,†,
Daniel Gruber
1,†,
Jan-Christoph Walter
1,
Kim Kühnel
1,
Turgay Kemal
1,
Armando Rodriguez
2,3,
Nico Preising
2,
Ludger Ständker
2,
Carolina Firacative
4,
Barbara Spellerberg
5,
Steffen Stenger
5,
Frank Rosenau
1,* and
Ann-Kathrin Kissmann
1,6,*
1
Institute of Pharmaceutical Biotechnology, Ulm University, 89081 Ulm, Germany
2
Core Facility for Functional Peptidomics (CFP), Faculty of Medicine, Ulm University, 89081 Ulm, Germany
3
Core Unit of Mass Spectrometry and Proteomics, Faculty of Medicine, Ulm University, 89081 Ulm, Germany
4
Studies in Translational Microbiology and Emerging Diseases (MICROS) Research Group, School of Medicine and Health Sciences, Universidad de Rosario, Bogota 111221, Colombia
5
Institute for Medical Microbiology and Hygiene, University Hospital Ulm, 89081 Ulm, Germany
6
Laboratory for Life Sciences and Technology (LiST), Faculty of Medicine and Dentistry, Danube Private University, 3500 Krems, Austria
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Pharmaceuticals 2026, 19(2), 286; https://doi.org/10.3390/ph19020286
Submission received: 17 December 2025 / Revised: 14 January 2026 / Accepted: 6 February 2026 / Published: 8 February 2026
(This article belongs to the Special Issue Peptide-Based Drug Discovery: Innovations and Breakthroughs)
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Abstract

Background/Objectives: Infections caused by Candida albicans, Candidozyma auris, and Candida parapsilosis increasingly challenge current treatment options as resistance to currently used antifungals is continuously developing. Neutralizing antimicrobial peptides (nAMPs), which modulate pathogenic behavior rather than inducing cell death, represent a promising approach to fighting against fungal infections. Methods: This study established a whole-cell phage display workflow to identify novel nAMPs, and therefore three independent biopanning processes with the Ph.D.-12 phage display library against C. albicans, C. auris, and C. parapsilosis cells were conducted. Results: Phage display produced species-selective, high-affinity peptides that were non-cytotoxic to human cells and did not affect planktonic Candida viability. These peptides inhibited early biofilm formation, and several also slowed early biofilm maturation down. Conclusions: These findings demonstrate that whole-cell phage display as a powerful and adaptable discovery tool is suitable for identifying nAMPs that neutralize biofilm development without toxicity towards human cells. Beyond the peptides described here, this approach expands the methodological toolbox for antifungal research and provides a sustainable approach for generating targeted peptides.

Graphical Abstract

1. Introduction

Fungal infections caused by the genus Candida and Candida-related genera have shaped medical history far longer than the concept of “fungal pathogens” itself [1]. Descriptions of thrush (historically termed muguet) appear in the early medical literature and became frequent in eighteenth- and nineteenth-century French medical texts particularly in reports from foundling hospitals and treatises on neonatal disease [2]. “Muguet” is the French term for lily-of-the-valley, which possibly refers to the characteristic white patches on the tongue and palate that are now recognized as oral candidiasis. Physicians and early pathologists carefully recorded these afflictions, which were regarded as a common danger among newborns and the weak [3]. In 1853, the French physician Charles Philippe Robin provided the first microscopic identification of the causative agent, naming it Oidium albicans, later reclassified as Candida albicans [4]. In the following decades, oral and systemic Candida infections were increasingly documented in hospitals, prisons, and military barracks, especially under poor hygienic conditions [5]. With the onset of the HIV/AIDS epidemic, in the 1980s, Candida infections reached pandemic proportions, as immunocompromised patients worldwide suffered from severe mucosal and systemic candidiasis [6,7].
Today, Candida species remain the fourth most common cause of nosocomial bloodstream infections, responsible for over 400,000 infections annually, with mortality rates exceeding 40% in the global south [8,9,10,11]. Driven in part by global warming [12,13], the epidemiological landscape continues to develop: C. albicans [14,15,16] remains the most prevalent species, yet Candida parapsilosis [17,18,19] and Nakaseomyces glabrata (formerly Candida glabrata) [20,21,22] have become major contributors to hospital-acquired infections. Candidozyma auris (formerly Candida auris), first identified in 2009 in Japan, has caused hospital infections across all continents [23]. The World Health Organization now lists C. auris among the critical-priority pathogens due to its multidrug resistance and environmental persistence [24,25]. These characteristics underline how a disease once observed in XVIII century nurseries and hospitals has become a global medical challenge, intensified by modern antifungal resistance.
Traditional antifungal treatments, primarily azoles, echinocandins, and polyenes, face increasing resistance due to their fungicidal or fungistatic mechanisms, which exert strong evolutionary pressure on fungal populations [26,27,28]. This ongoing resistance crisis requires alternative therapeutic concepts that target fungal virulence rather than survival [29]. Natural and synthetic antimicrobial peptides (AMPs) [30] are a potent group of drug molecules with broad activity against bacteria [31,32,33,34,35,36], viruses [37,38,39,40,41,42] and fungi [43]. Classical AMPs typically act through one of two principal mechanisms: they are either membrane-active, integrating into microbial membranes to form various types of pores, or they act intracellularly by inhibiting essential metabolic pathways [44,45,46,47]. Membrane-active AMPs disrupt the structural and functional integrity of the pathogen’s membrane, leading to loss of viability. Numerous mechanistic models have been proposed to describe how AMPs bind to and insert into biological membranes [48,49,50,51,52]. We have explored both natural mollusk-derived peptides and rationally designed compounds to address some of these limitations. Notably peptides, such as Cm-p5 from Cenchritis muricatus [53,54,55], Nv-p1 from Nerita versicolor [56], and the synthetic peptide C14R [57,58,59], have demonstrated multifunctional antimicrobial and anti-biofilm activity with low cytotoxicity, illustrating how both natural and designed peptides can be used to modulate microbial behavior rather than simply kill cells.
Pom-1, derived from the freshwater snail Pomacea poeyana [60,61,62,63], exemplifies a neutralizing AMP (nAMP): it inhibits Candida biofilm formation, particularly when used in combination with conventional antifungals, while showing minimal activity against planktonic cells [64]. nAMPs have recently emerged as a promising alternative, and in contrast to classical antimicrobial peptides, which disrupt membranes and kill cells, nAMPs interfere with microbial behavior, blocking adhesion and preventing biofilm initiation. As demonstrated previously [64], such peptides can effectively suppress early biofilm formation in Candida without harming human cells. By neutralizing rather than killing, nAMPs impose minimal selective pressure, show species specificity, and preserve beneficial microbiota, offering a sustainable approach to managing resistant fungal infections.
The identification of novel nAMPs benefits from technologies that can efficiently screen large molecular spaces for peptides with specific biological functions [65,66,67,68]. One of the most powerful of these is phage display, a technology introduced by George P. Smith in 1985 [69], which allows peptide sequences to be displayed on the surface of filamentous bacteriophages such as M13 [70]. This innovation established a physical link between genotype and phenotype, enabling the screening of peptide libraries containing up to 109 variants for binding to virtually any target. Building on Smith’s foundation, Gregory Winter and colleagues adapted the method for antibody selection in the 1990s [71], culminating in the development of therapeutic antibodies such as adalimumab [72]. The impact of this work was recognized with a “half share” of the 2018 Nobel Prize in Chemistry that was awarded jointly to Smith and Winter [73]. Since then, phage display has become a versatile method not only for antibody engineering but also for identifying peptides that bind to proteins, cells, or even intact microbial surfaces [74]. In recent decades, the adaptation of phage display to complex biological targets has been recognized, including bacterial biofilms and fungal cell walls, demonstrating its exceptional capacity to uncover ligands that modulate adhesion and colonization processes [75,76,77,78]. Early selections against intact C. albicans cells demonstrated that short random peptides can be enriched for high-affinity surface binders, establishing the feasibility of peptide-based antifungal targeting [79]. Subsequent methodological advances, such as magnetic particle-assisted functional panning [80], expanded the ability to recover peptides with genuine antimicrobial activity rather than mere surface affinity. More recent applications, including protective phage-displayed peptides in murine Candida models, further underscore the translational potential of display-derived antifungal sequences [81,82].
Based on this knowledge, we present here an approach for screening and identifying nAMPs using phage display. We applied this method to assess three clinically relevant Candida species—C. albicans, C. auris, and C. parapsilosis (Figure 1), with the objective of identifying species-specific, non-lytic peptides that are able to selectively bind fungal cells and interfere with early formation of biofilms.

2. Results

To identify peptide ligands capable of selectively recognizing C. albicans, C. auris, or C. parapsilosis, three independent whole-cell phage displays were performed using the PhD™ Phage Display Peptide Library v2 (New England Biolabs, Ipswich, MA, USA). Over the course of three consecutive biopanning cycles, the recovered phage titers increased substantially for all three species, indicating a progressive enrichment of clones with enhanced binding affinity (Figure 2). This upward trend in PFU/mL reflects the gradual focusing of the initially highly diverse library, containing approximately 109 unique phage variants, toward subpopulations capable of interacting with the characteristic surface of each Candida species. The most pronounced enrichment occurred between the first and second rounds of selection, with an average overall titer increase of 436%. Between rounds two and three, enrichment continued but at a reduced rate, reaching 170%. Notably, between the third and fourth rounds, the titers dropped by almost 90%, and white plaques began to appear on IPTG/X-gal plates, indicating a loss of insert-bearing phage clones. Taken together, these dynamics suggest that maximal enrichment of high-affinity binders was achieved by the third round and that additional panning cycles may have introduced counter-selection against insert-containing phage. Thus, the third round captures the most relevant and highest-affinity phage populations for subsequent sequencing and characterization.
To verify that biopanning generated species-selective phage pools, enriched phage libraries from Round 3 were incubated with each of the three Candida species and compared against the Round 1 libraries. qPCR-based quantification of recovered genomes demonstrated pronounced binding specificity (Figure 3). The C. albicans-enriched pool bound preferentially to C. albicans, while the C. auris and C. parapsilosis pools showed highest recovery on their respective targets. Notably, cross-recognition between the C. auris and C. parapsilosis pools was detected, indicating potential shared surface-exposed ligands between these species.
From the third round of biopanning, 50 individual plaques from each enriched library were isolated, amplified, and sequenced. Analysis of the resulting sequences using CLC Workbench revealed a diverse set of peptide variants (Table 1, Appendix A Table A1).
Within each species-specific library, several peptide motifs appeared repeatedly, indicating convergent enrichment toward preferred binding sequences. Based on (i) enrichment frequency and (ii) exclusivity to a particular Candida library, six peptides—Can-1, AuPan-1, Aln-1, Aln-2, Aun-1, and Pan-1—were selected as candidates for synthesis and functional characterization. To validate binding at the phage level, the six selected phage clones were amplified and fluorescently labeled with Atto 665 NHS. Incubation with C. albicans, C. auris, and C. parapsilosis revealed strong, surface-associated fluorescence for clones enriched against their respective target species (Figure 4A–C). Specifically, Aln-1 and Aln-2 displayed selective binding to C. albicans, Aun-1 showed exclusive affinity toward C. auris and Pan-1 bound specifically to C. parapsilosis. AuPan-1 exhibited dual specificity toward C. auris and C. parapsilosis, while Can-1 demonstrated broader affinity across all three Candida species. In contrast, non-enriched control clones showed minimal fluorescence signal. Brightfield images confirmed uniform cell density across all conditions, whereas fluorescence micrographs displayed distinct red fluorescence corresponding to phage particles localized at the fungal surface. In summary, these data confirm specific and robust clone-to-cell binding for the enriched phage populations.
All six peptides were synthesized via microwave-assisted Fmoc-SPPS with a three-glycine linker and a cysteine residue N-terminal display position of the pIII protein. The peptides were tested for cytotoxicity in human dermal fibroblasts (HDFs) at 100 μg·mL−1. Viability remained above the 70% cytotoxicity threshold for all peptides (Figure 5), whereas Triton X-100 produced the expected pronounced toxicity. No statistically significant reduction in HDF viability was observed for any peptide, indicating suitable tolerability for further antifungal assessment.
To determine whether biofilm inhibition might arise from fungicidal effects on non-adherent cells, planktonic viability assays were performed for C. albicans, C. auris, and C. parapsilosis following incubation with 100 μg·mL−1 of each peptide. None of the peptides caused a meaningful reduction in viability compared to untreated controls (Figure 6). These findings demonstrate that the peptides do not exhibit classical antimycotic activity and suggest a biofilm-specific mechanism of action.
A conceptual schematic (Figure 7) illustrates the proposed mode of action: peptides interfere with early-stage adherence or growth initiation, preventing maturation into structured biofilms. Representative crystal violet staining visually demonstrates the reduction of surface-attached biomass in peptide-treated wells.
We next assessed whether the binding observed in earlier experiments translates into functional interference with biofilm formation. Biofilm inhibition was quantified using the crystal violet assay, which demonstrated that all six peptides reduced biofilm biomass in a concentration-dependent manner in at least one Candida species (Figure 8A–C). Dose–response fitting using a four-parameter variable-slope Hill model further confirmed clear inhibitory gradients and revealed distinct potency profiles among the peptides (Figure 8D). Can-1 and AuPan-1 exhibited broad-spectrum inhibition across all three species, whereas Aln-1, Aln-2, Aun-1, and Pan-1 showed more selective activity. Importantly, all effects were observed under conditions where peptides were present continuously from the start of incubation, indicating interference with early adhesion or initial growth—the developmental stages at which nAMPs are expected to act—rather than disruption of established biofilms.
To determine whether peptide activity extends beyond initial adherence and can prevent biofilm maturation, we established 24 h biofilms before adding peptides for an additional 24 h. Several peptides significantly reduced further biomass accumulation compared to untreated controls (Figure 9), indicating partial interference with early maturation stages. Notably, in C. albicans, the Aln-2 peptide led to a clear reduction in biomass increase between the 24 and 48 h incubation periods. In C. auris, both Aln-2 and Pan-1 similarly attenuated the continued biomass increase, suggesting that these peptides retain inhibitory activity after the initial adhesion phase and can modulate early biofilm maturation. In contrast, none of the tested peptides produced significant inhibition of biofilm maturation in C. parapsilosis under the conditions examined.

3. Discussion

In this study, we identified and characterized a set of phage display-derived neutralizing peptides with selective binding activity toward C. albicans, C. auris, and C. parapsilosis. Like Pom-1 and its derivatives, previously described biofilm-interfering antifungal peptides [64], the six peptides here characterized exhibited inhibition of early biofilm formation while exerting no measurable effect on the viability of planktonic Candida cells. Importantly, several peptides not only impaired the initiation of biofilm formation but also limited biofilm maturation, indicating that the peptide activity extends beyond the earliest attachment steps and affects subsequent biofilm proliferation stages. Another major challenge in the development of peptide-based antifungals lies in their pharmacokinetic limitations, proteolytic sensitivity, and potential cytotoxicity. Classical natural AMPs frequently fail in clinical translation due to short half-lives, toxicity, and insufficient therapeutic windows [83]. While Candida bloodstream infections are clinically severe, the biofilm formation is predominantly a surface-associated process occurring on tissues rather than within circulating blood [84]. Fibroblasts were therefore used as the mammalian cell model to assess host cell compatibility in the context of biofilm prevention. Encouragingly, our peptides did not reduce human fibroblast viability at concentrations relevant to their active concentrations, fulfilling the ISO 10993-5 threshold that viability reductions below 30% constitute cytotoxicity [85]. Even at high concentrations (100 µg/mL), no toxicity was observed, underscoring their biocompatibility. Pom peptides were shown to interfere with biofilm establishment while only marginally affecting planktonic viability [61,62]. Their behavior challenged the traditional AMP concept where antimicrobial activity is typically mediated through membrane permeabilization and cytolysis [44,51]. Instead, we proposed a mechanism analogous to antiviral neutralizing peptides [86] or therapeutic monoclonal antibodies [87,88] targeting viral fusion machinery where bioactivity arises from the blockade of highly specific surface epitopes rather than general lytic activity. The results of this study parallel these findings: the peptides identified in the phage display selections bind strongly and selectively to their respective Candida targets, as confirmed by fluorescence microscopy and qPCR, but do not impair planktonic metabolic activity. This indicates that, much like Pom-derived nAMPs, the peptides identified in this study likely interfere with early biofilm-associated processes rather than causing cell death. It should be noted that biofilms formed under in vitro conditions may differ from in vivo, which may influence peptide activity and should be considered when translating these findings to physiological settings [89]. In vivo biofilms are shaped by environmental factors such as host-derived proteins, immune pressure, nutrient limitation and competition/synergy with other microorganisms [90]. Proteomic analysis of in vivo C. albicans biofilm implies that in contrast to in vitro biofilms, the extracellular matrix is partially composed of host-derived proteins [91,92]. Even clinically relevant antifungal agents demonstrate the limited predictive power of in vitro biofilm models. In vitro Candida biofilms show resistance against azoles, yet are more susceptible in vivo, likely due to host immune contributions and altered biofilm architecture [93]. Multiple studies have demonstrated that compounds that are active against in vitro biofilms often display reduced or context dependent activity in animal models [94]. Furthermore, the biofilm extracellular matrix itself contributes to immune evasion by inhibiting neutrophil activity and suppressing the formation of neutrophil extracellular traps, thereby promoting persistence in the host environment [94]. Despite these challenges, there is evidence that targeting early biofilm-associated process can be biologically relevant in vivo. Several anti-biofilm peptides have demonstrated efficacy in reducing biofilm formation or persistence in animal infection models [95]. The Candida cell surface is a composite and dynamic structure containing over twenty major cell wall proteins (CWPs) [96], including adhesins, Als-family proteins, and hypha-associated factors, all of which contribute to adhesion, aggregation, hyphal morphogenesis, and biofilm maturation. Several CWPs including Csa1, Eap1, Hwp1, Pga10, and Rbt5 play essential roles in cell–cell adhesion and biofilm integrity, as loss-of-function mutants exhibit fragile or severely impaired biofilms [97,98,99]. Because the peptides bind intact cells strongly but fail to influence cell viability, it is possible that they interact with one or several of such surface-exposed proteins. The strong enrichment of these peptides during phage display supports the idea that selection converged on physiologically exposed membrane motifs. In line with this, the strongest biofilm-inhibitory effects were observed when peptides were administered at the onset of cultivation [100,101], precisely the stage at which CWP-mediated adhesion, aggregation, and morphogenesis initiate biofilm formation [101]. Whether these peptides interfere with adhesion domains (e.g., Eap1 N-terminus), bud-localized proteins (e.g., Csa1p), amyloid-prone Als domains, or other biofilm-relevant determinants remains an open question. The specific molecular targets of the peptides have not been confirmed. However, domain-specific mutants in combination with biochemical peptide–protein interaction assays can be employed to elucidate the targets. Our study focused exclusively on the early developmental phase of biofilm formation, aiming to evaluate the preventive potential of these peptides rather than their ability to eradicate mature biofilms. Although prevention of biofilm formation is essential and clinically relevant for catheter locks, implant coatings, and prophylactic antifungal strategies, the ability to disassemble mature Candida biofilms remains a particularly difficult objective due to their dense matrix, heterogeneous architecture, and elevated antifungal tolerance. Whether the peptides identified here retain activity against established biofilms or exhibit synergistic interactions with classical antifungals in that context remains to be explored. Building on these findings, further investigation will focus on optimizing the identified peptides, using the currently characterized sequences as lead structures for rational design. Cyclization and/or dimerization as well as sequence alterations will be employed to improve proteolytic resistance and pharmacokinetic properties, as previously demonstrated for other antimicrobial peptides. Structural insights gained from this study will guide modifications aimed at enhancing biofilm inhibitory activity and stability in the future.

4. Materials and Methods

4.1. Materials

Agar-agar, crystal violet, 3-(N-morpholino)propanesulfonic acid (MOPS), acetic acid, peptone, and yeast extract were obtained from Carl Roth GmbH (Karlsruhe, Germany). RPMI-1640 medium supplemented with L-glutamine was sourced from Thermo Fisher Scientific (Waltham, MA, USA), and resazurin sodium salt was purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Amphotericin B was obtained from Carl Roth GmbH. Sodium iodide was acquired from BD Biosciences (Franklin Lakes, NJ, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin, Accutase®, and Eagle’s minimum essential medium non-essential amino acids (MEM NEAAs) were purchased from Life Technologies (Carlsbad, CA, USA). Phosphate-buffered saline (PBS) was obtained from VWR International (Radnor, PA, USA).
Lysogeny (LB) broth and LB agar were prepared using tryptone, yeast extract, and sodium chloride (Carl Roth GmbH (Karlsruhe, Germany)). Isopropyl β-D-1-thiogalactopyranoside (IPTG) and X-gal (Carl Roth GmbH (Karlsruhe, Germany) were dissolved in dimethylformamide (DMF) to prepare IPTG/X-gal plates. The following buffers and reagent solutions were prepared: Tris-buffered saline (TBS; 50 mM Tris-HCl, 150 mM NaCl, pH 7.3), TBST (TBS supplemented with 0.1%, 0.3%, or 0.5% Tween-20), a PEG/NaCl precipitation solution containing 20% (w/v) PEG-8000 and 2.5 M NaCl, and SM buffer composed of 5.8 g NaCl, 2.0 g MgSO4·7H2O, and 50 mL of 1 M Tris-HCl (pH 7.4) per liter. Resazurin solution for viability assays was prepared from resazurin sodium salt at 0.15 mg/mL. Antifungal agents fluconazole, caspofungin, and amphotericin B were diluted from 2 mg/mL stock solutions to final assay concentrations as required (suppliers to be added).
The Ph.D.-12 Phage Display Library Kit v2 (NEB #E8110) was obtained from New England Biolabs (Ipswich, MA, USA). PEG-8000, sodium chloride, bovine serum albumin (BSA), glycine, and Tris-HCl for phage preparation and purification were purchased from (Carl Roth GmbH (Karlsruhe, Germany)). For fluorescent labeling of phage coat proteins, Atto-665-NHS ester (Sigma-Aldrich) was used.
For all PCR reactions, the Herculase II Fusion DNA Polymerase Kit from Agilent Technologies, Inc. (Santa Clara, CA, USA) was used. SYBR Green was acquired from Carl Roth GmbH. Master mixes were prepared in accordance with the Herculase II Fusion DNA Polymerase Kit instructions. Oligonucleotide primers were synthesized by Biomers.net GmbH (Ulm, Germany). Microscopy was performed on a Leica DMi8 coded microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany). Fluorescence and absorbance measurements were conducted on a Tecan Infinite F200 microplate reader (Tecan Group Ltd., Männedorf, Switzerland).

4.2. Strains and Growth Conditions

4.2.1. Microbial Strains

C. albicans (ATCC90028), C. auris (DSMZ-No. 21092), and C. parapsilosis (ATCC22019) were cultured in RPMI-1640 supplemented with 0.165 M MOPS at 37 °C with orbital shaking. E. coli K12 ER2738 was cultured in LB medium at 37 °C on a shaker supplemented with tetracycline (20 µg/mL) when required.

4.2.2. Cell Culture

Human dermal fibroblasts (HDFs) obtained from tebu-bio GmbH (Offenbach, Germany) were used for all experiments [102]. Cells were cultured in DMEM supplemented with 10% or 15% (w/v) fetal bovine serum, 1% (w/v) MEM non-essential amino acids, and penicillin–streptomycin (100 U/mL, 1% (w/v)). Cultures were grown at 37 °C in a humidified incubator containing 5% CO2. Before splitting the cultures, the growth medium (DMEM + 10% FBS for A549 cells; DMEM + 15% FBS for HDFs) was prewarmed to 37 °C. After aspirating the spent medium, 3 mL of Accutase® was added to the flask to detach the adherent cells. The flask was incubated for approximately 5–10 min until the cells rounded up. To achieve complete detachment, the flask was gently tapped. The required cell number was then transferred into a fresh culture flask containing the prewarmed medium, followed by incubation at 37 °C and 5% CO2.

4.3. Biopanning Against Yeasts

Phage display biopanning experiments were conducted against C. albicans, C. auris, and C. parapsilosis following the manufacturer’s protocols provided by New England Biolabs for the Ph.D.-12 Phage Display Peptide Library Kit v2 [103], with minor adjustments as described below. Overnight cultures of each Candida strain were inoculated into fresh RPMI-1640 supplemented with 0.165 M MOPS and grown to an OD600 of approximately 0.1 (~5 × 106 cells/mL). Cells were washed twice with TBS, blocked in 2% BSA/TBS for 1 h at room temperature, and washed three additional times with TBS to remove unbound blocking agent. For the binding step, 5 × 106 Candida cells were incubated with 1011 plaque-forming units (pfu) of phage in 1.5 mL tubes for 1 h at room temperature under continuous rotation. To control binding stringency, cells were washed three times per round with TBST containing increasing concentrations of Tween-20: 0.1% for round 1, 0.3% for round 2, and 0.5% for rounds 3–4. Between washes, cells were pelleted by centrifugation at 4000× g for 5 min at 4 °C. Bound phages were eluted by incubation with 80 µL of 0.1 M glycine (pH 2.0) for 10 min and subsequently neutralized using 20 µL of 1 M Tris-HCl (pH 8.0). Cellular debris was removed by centrifugation at 5000× g for 5 min at 4 °C. The neutralized eluates were then added to 20 mL of mid-log-phase E. coli ER2738 cultures and incubated at 37 °C with shaking at 250 rpm for 4.5 h to amplify the phages. Cultures were centrifuged (5000× g, 15 min), and one-sixth volume of PEG/NaCl solution was added to the supernatant. Phages were precipitated overnight at 4 °C, pelleted at 12,000× g for 45 min, dissolved in TBS, clarified briefly by centrifugation, and re-precipitated using PEG/NaCl. Final phage pellets were resuspended in SM buffer to achieve a concentration of ~2 × 1011 pfu/mL for subsequent selection rounds. Phage titration was performed by infecting 200 µL of log-phase E. coli ER2738 with 10 µL serially diluted phage samples. After 3 min at room temperature, the mixtures were added to 3 mL of prewarmed soft agar and overlaid onto IPTG/X-gal LB plates containing tetracycline and amphotericin B. Plates were incubated for 16 h at 37 °C, and blue plaques representing active pIII-displaying phages were counted. Individual phage clones were isolated by picking blue plaques with sterile pipette tips into 1 mL LB + ER2738 and amplifying for 4.5–5 h at 37 °C. Phages were precipitated using PEG/NaCl, and single-stranded DNA was purified using iodide/ethanol precipitation. DNA was resuspended in TE buffer and quantified by 1% TBE agarose gel electrophoresis. Sanger sequencing of the inserts was performed using the –96gIII primer (Eurofins Genomics). Sequences were reverse-complemented, inserts were identified between KpnI (GGTACC) and EagI (CGGCCG) restriction sites, and translated peptides were analyzed for physicochemical properties and BLAST 2.17.0 similarity.

4.4. Fluorescent Labeling of Phage Coat Proteins

Bacteriophage M13 clones (titer 1 × 1013 PFU·mL−1) were labeled with Atto-665-NHS (Sigma-Aldrich) (MW 820 g·mol−1; stock concentration 2.5 mg·mL−1) to target an average labeling density of ~300 dye molecules per phage (≈11% of the ~2700 major coat protein sites per particle). Labeling reactions were performed in PBS-NaHCO3 adjusted to pH 8.3. Reaction stoichiometry was chosen based on the calculated molar ratio of dye molecules to coat protein sites. The labeling reaction was performed at room temperature in the dark for 60 min at constant shaking. Following conjugation, the phage was precipitated using the previously described PEG/NaCl precipitation method. Labeled phage preparations were stored at 4 °C for further usage.

4.5. Fluorescence Microscopy

To visualize specific phage binding, cells were examined using a Leica DMi8 micro-scope (Leica Microsystems CMS GmbH, Wetzlar, Germany). Prior to imaging, 1013 phage particles of each selected clone were amplified and ATTO 655 labeled as described above. Separately, 2.5 × 104 cells of C. albicans, C. auris, and C. parapsilosis were collected by centrifugation and washed once with PBS. Each cell population was incubated with the corresponding phage solution for 15 min at 37 °C. Following incubation, cells were pelleted at 9000× g for 3 min and washed three times with TBST containing 0.5% Tween-20 to remove unbound phages. For fluorescence imaging, the washed cells were resuspended in 100 µL PBS and transferred to a 96-well plate for microscopic observation.

4.6. qPCR-Based Phage Quantification

Phage genome copy numbers were determined using SYBR Green real-time PCR. Primers specific to the phage insert sequence were as follows: Phage_Fw 5′-tcg caa ttc ctt tag tgg tac ct-3′ and Phage_Rv 5′-aag ttt tgt cgt ctt tcc aga c-3′. For each qPCR run, a calibration curve was generated using ten-fold serial dilutions of purified phage, enumerated by plaque assay (or an alternative standard), covering a range from 1 × 1011 to 1 × 103 phage particles per reaction. All standards, samples, and controls were assayed in technical triplicates, with no-template controls (NTCs) and a negative extraction control included on every plate. Reactions were performed on an Analytik Jena qTower3 G, and the data analysis was performed using Jena Analytik qPCR software 4.0. Thermal cycling conditions included an initial denaturation at 94 °C for 3 min, followed by 40 cycles of 94 °C for 30 s, 64 °C for 30 s, and 72 °C for 10 s. Melt-curve analysis was conducted at the end of each run to confirm amplicon specificity. Before amplification, phage preparations were thermally treated to release nucleic acids. Quantification was based on plotting the cycle threshold (Ct) values against the logarithm of the known phage quantities in the calibration series and applying linear regression. Phage copy numbers in samples were interpolated from the standard curve, and reaction efficiency (E) was calculated from the slope using the formula E = 10^((−1/slope) − 1). The coefficient of determination (R2) was reported for each curve, and the lower limit of quantification (LLOQ) was defined by the smallest standard reliably detected (1 × 103 phage particles). All reported values represent the mean ± SD of technical triplicates unless stated otherwise.

4.7. Peptide Synthesis

Peptides were prepared on a 0.10 mmol scale using standard Fmoc solid-phase peptide synthesis (SPPS) on a microwave-assisted synthesizer (CEM Corporation, Matthews, NC, USA). The synthesis was performed on an arginine-preloaded resin, which was initially washed with dimethylformamide (DMF). Fmoc protecting groups were removed using 20% (v/v) piperidine in DMF under microwave irradiation, followed by additional DMF washes. During each coupling cycle, five equivalents of the appropriate Fmoc-protected amino acid and five equivalents of HBTU were added along with ten equivalents of N,N-diisopropylethylamine (DIEA). Couplings were conducted under microwave irradiation for several minutes, and the resin was thoroughly washed with DMF between steps. This cycle was repeated until the entire peptide sequence was assembled, concluding with a final Fmoc deprotection. Cleavage from the resin was achieved using a trifluoroacetic acid (TFA) cleavage cocktail (95% TFA, 2.5% triisopropylsilane (TIS), 2.5% H2O) for 1 h. The crude peptides were precipitated with cold diethyl ether (DEE), collected by centrifugation, washed with DEE, and dried under vacuum. Purification was carried out via preparative reversed-phase HPLC (Waters Corporation, Milford, MA, USA) using a Phenomenex Luna C18 column (250 × 21.2 mm, 5 μm, 100 Å) with an acetonitrile/water gradient under acidic conditions. Purified fractions were combined and lyophilized (Labconco Corporation, Kansas City, MO, USA). Peptide identity and molecular mass were confirmed using LC-MS and MALDI-TOF mass spectrometry (Waters Corporation, Milford, MA, USA). All synthesized peptides exhibited a purity of more than 95%.

4.8. Cell Viability Assay

HDF Cell viability was evaluated using a resazurin-based metabolic assay. For this purpose, 2 × 104 cells were seeded per well in 96-well plates and cultured in 200 µL supplemented DMEM at 37 °C and 5% CO2. After medium removal, each well received 100 µL fresh medium and 100 µL of the respective peptide (final concentration: 100 µg/mL). Following a 24 h incubation, 20 µL of a resazurin solution (0.15 mg/mL) was added. Plates were incubated for another 24 h under identical conditions. Fluorescence from the resorufin product (λ_ex = 535 nm, λ_em = 595 nm) was measured using a Tecan Infinite F200 microplate reader (Tecan Group Ltd., Männedorf, Switzerland).

4.9. Resazurin Reduction Viability Assay

The viability of C. albicans, C. auris and C. parapsilosis exposed to the peptides was investigated using a protocol adapted from CLSI M27-A3 standards [104]. Yeast suspensions containing 2.5 × 104 cells/mL were cultured in 200 µL of RPMI-1640 medium supplemented with 100 µg/mL of Can-1, AuPan-1, Aln-1, Aln-2, Aun-1, or Pan-1 in 96-well flat-bottom microplates (Sarstedt AG & Co. KG, Nümbrecht, Germany). Incubation was carried out at 37 °C with shaking at 900 rpm on an Eppendorf orbital shaker. Viable cells were quantified using a resazurin-based metabolic assay. After peptide exposure, 20 µL of a 0.15 mg/mL resazurin solution was added to each well, and plates were incubated for an additional 2 h. Fluorescence originating from resorufin (λ_ex = 535 nm, λ_em = 595 nm) was measured using a Tecan Infinite F200 microplate reader. The fluorescence intensity correlated with the number of metabolically active cells.

4.10. Biofilm Formation and Crystal Violet Staining

To assess how the peptides Can-1, AuPan-1, Aln-1, Aln-2, Aun-1, and Pan-1 influence biofilm development, a crystal violet-based quantification assay was carried out [105,106]. Yeast cultures were adjusted to 2.5 × 104 cells/mL and distributed into 96-well flat-bottom polystyrene plates, with each well containing 200 µL RPMI-1640 medium supplemented with 0–100 µg/mL of the respective peptide. Plates were incubated without shaking for 24 h at 37 °C to allow biofilm formation. After incubation, the supernatant containing planktonic cells was carefully discarded. The adherent cells were rinsed twice with 200 µL demineralized water and then stained with 200 µL of 0.1% (w/v) crystal violet for 15 min. Excess dye was removed, followed by two additional washing steps with demineralized water. The plates were allowed to dry for 24 h at 25 °C. Bound crystal violet was subsequently solubilized by adding 200 µL of 30% acetic acid, and after 15 min, the eluate was transferred to a fresh microplate. Absorbance at 560 nm was recorded using a Tecan Infinite F200 microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Comparison with untreated controls enabled evaluation of the biofilm-inhibitory activity of each peptide.

4.11. Decay of Mature Biofilms

The capacity of the peptides to disrupt mature Candida biofilms was examined in a two-step assay. First, Candida cells were seeded at 2.5 × 104 cells per well in 200 µL RPMI-1640 medium in flat-bottom 96-well polystyrene plates (Sarstedt AG & Co. KG, Nümbrecht, Germany). Plates were incubated for 24 h at 37 °C without shaking to permit biofilm development. Following incubation, biofilm biomass was quantified in triplicate for each strain using a crystal violet staining procedure, providing baseline values for 24-h biofilm formation. In the subsequent treatment phase, the supernatants from the remaining wells were gently removed and replaced with 200 µL RPMI-1640 medium containing either Amphotericin B (2 µg/mL) or one of the peptides at a concentration of 100 µg/mL. Treatments were applied in triplicate. The plates were incubated for an additional 24 h at 37 °C. After this second incubation, the crystal violet assay was performed again to determine the extent of biofilm reduction relative to the untreated reference biofilms.

5. Conclusions

In summary, the peptides identified in this study represent a promising class of phage display-derived nAMP-like antifungal agents that selectively bind Candida cells and inhibit the early stages of biofilm formation without affecting planktonic viability or human cell growth. Such peptides may, in principle, offer a complementary way to influence early biofilm development. Based on their low cytotoxicity, species-selective binding, and inhibition of biofilm initiation, we believe they may offer valuable components for next-generation antifungal formulations, catheter lock solutions, or antifungal peptide-coated biomaterials. Further research will need to determine their precise molecular targets, assess synergistic effects with fluconazole or amphotericin B, and explore their stability and pharmacological behavior in vivo. Despite these open questions, our findings provide a starting foundation for the development of new anti-biofilm strategies against C. albicans, C. auris, and C. parapsilosis, pathogens of growing clinical importance and increasing resistance.

Author Contributions

Conceptualization: A.-K.K. and F.R.; validation, G.B., J.-C.W. and D.G.; formal analysis, G.B. and D.G.; investigation, G.B., D.G., J.-C.W., K.K., T.K., A.R., N.P., L.S., C.F., B.S., S.S., F.R. and A.-K.K.; resources, F.R., A.R., N.P., L.S., B.S. and S.S.; data curation, A.-K.K. and F.R.; writing—original draft preparation, G.B. and D.G.; writing—review and editing, J.-C.W., K.K., T.K., A.R., N.P., L.S., C.F., B.S., S.S., F.R. and A.-K.K.; visualization, G.B. and D.G.; supervision, A.-K.K. and F.R.; project administration, A.-K.K. and F.R.; funding acquisition, A.-K.K., F.R. and L.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Gesellschaft für Forschungsförderung (GFF) of Lower Austria as part of the project “Aptamers and Odorant Binding Proteins—Innovative Receptors for Electronic Small Ligand Sensing” (FTI22-G-012) and the Förderstelle Wirtschaft, Tourismus und Technologie (WST) (WST-F-5035462/004-2024). This work was also supported by the Austrian Research Promotion Agency (FFG) within the COMET Project “PI-SENS” (project no. 915477) as well as by the Federal Provinces of Lower Austria and Tirol. It was also supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) project 465229237 and the Anschubfinanzierung A 2025 of Ulm University.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are openly available at [Cloudstore Uni Ulm] [https://cloudstore.uni-ulm.de/s/r7kbNRHNH7xDkKc, accessed on 15 December 2025].

Conflicts of Interest

The authors declare no conflicts of interest.

Appendix A

Table A1. Properties of peptides identified from phage display against Candida species. Columns indicate peptide sequence, assigned name (if applicable), number of hits against C. albicans (C. alb.), C. auris (C. au.), and C. parapsilosis (C. para.), total occurrences (Prop.), predicted isoelectric point (pI), net charge at pH 7 (Net Q), secondary structure content predicted by the Chou–Fasman method (α-helix [%], β-sheet [%]), and overall predicted structure.
Table A1. Properties of peptides identified from phage display against Candida species. Columns indicate peptide sequence, assigned name (if applicable), number of hits against C. albicans (C. alb.), C. auris (C. au.), and C. parapsilosis (C. para.), total occurrences (Prop.), predicted isoelectric point (pI), net charge at pH 7 (Net Q), secondary structure content predicted by the Chou–Fasman method (α-helix [%], β-sheet [%]), and overall predicted structure.
PeptideNameC. alb.C. au.C. para.Prop.MW (Da)H (%)pINet Q (pH 7)α-Helix (CF) [%]β-Sheet (CF) [%]Pred. Struct.
ACNNLAMSSCTM 1102124541.672.84−0.1012.66Coil
ASVLTPMHFGAT 0011123158.337.880.11.759.16Coil and Helix
DENELRAMLTLN 1102141841.673.69−215.334.25Helix
DIISTVTGAIKF 01011264506.660015.83Helix
DLHKFNLSATHN 0022139633.337.980.22.330Helix
DVFVLQRVKEIR 010115015010.14111.515.08Helix
FFDYAWLVQESY 01011567500.67−27.257.91Coil and Helix
FSLSHNSQLNISAln-12002134633.337.560.100Coil and Helix
FTLNDSIAHVLTAUn-102021330504.87−0.94.1611.75Helix
GAVGYGDVWARM 10011281506.590010.08Coil and Helix
GFLKFPDELASL 1023133658.333.93−111.410Coil
GHGGMAIGPAQP 01011092507.810.100Coil
GMIDRNAINWNR 1034145941.6710.68107.1Coil
GVGPHRNLSGTG 100111512510.841.100Coil
HMPWETTNARRFPAn-10044154541.6710.441.12.330.41Helix
IPYSQITYNPIF 00111455503.650013.91Coil
SDIPTSITIGPV 01011199500.72−106.08Coil
SDLTPIRFTGNL 0011133341.676.38000.83Coil
SHFGSSHEIHGS 3058128116.676.34−0.700Coil
SIFDITREGGMH 1001136233.335.17−0.902.32Helix
SNSNQLIFNGNRAln-2200213632510.57100Helix
SNSNQLIFNWNRAUPAn-1015520149233.3310.57102.67Helix
SRLPYGLLNDYT 1102141133.336.34000Coil
SSLPQKLKVMFG 160713345010.6920.413.42Helix
SYSDSYVYPFDN 00111456250.62−200Coil
TGTLLVSNKLLTCan-12518125941.679.821010.67Helix
TTYVGWDRTIDI 0101143933.333.71−1014.5Beta sheet
VEYHRSPLSLDS 0011140233.335.17−0.900Coil and Helix
VITHHDSVATEH 0011134533.335.76−1.755.08Coil and Helix
VSDPANWVTTRS 0011133241.676.61002.58Coil
VTNLETVKNWSI 0101140341.676.8200.2511.83Helix
WEGGELGILLRH 0101137941.675.26−0.97.920Coil and Helix
YEDFSLSAMDPL 01011387500.51−36.330Coil
YEDFSLSPMDPL 00111413500.51−300Coil
YVTQITGKTRLG 010113362510.412014Beta sheet

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Figure 1. Overview of the phage display-based development and characterization workflow for identification of neutralizing antimicrobial peptides. (A) Schematic representation of M13 phage displaying foreign peptide sequences on the pIII coat protein, enabling the construction of a diverse phage display peptide library. (B) Target Candida species used for biopanning: C. albicans, C. auris, and C. parapsilosis. (C) Four-cycle biopanning process. Binding of phages from the initial library to fungal cells (1), unbound phages are removed by washing (2), bound phages are eluted (3), and infectious particles are amplified in E. coli K12 ER2738 (4), generating a focused library enriched for peptide sequences with affinity toward biofilm-forming cells. (D) Insert sequencing of enriched phage clones and subsequent solid-phase peptide synthesis followed by HPLC purification to obtain individual peptide candidates. (E) Fluorescent labeling of selected phage clones using Atto 665 NHS-ester for microscopy-based binding validation on fungal cells. (F) Evaluation of peptide cytotoxicity using human dermal fibroblasts (HDFs). (G) Biofilm inhibition assays performed with purified peptides against C. albicans, C. auris, and C. parapsilosis to identify neutralizing antimicrobial peptides capable of reducing biofilm formation.
Figure 1. Overview of the phage display-based development and characterization workflow for identification of neutralizing antimicrobial peptides. (A) Schematic representation of M13 phage displaying foreign peptide sequences on the pIII coat protein, enabling the construction of a diverse phage display peptide library. (B) Target Candida species used for biopanning: C. albicans, C. auris, and C. parapsilosis. (C) Four-cycle biopanning process. Binding of phages from the initial library to fungal cells (1), unbound phages are removed by washing (2), bound phages are eluted (3), and infectious particles are amplified in E. coli K12 ER2738 (4), generating a focused library enriched for peptide sequences with affinity toward biofilm-forming cells. (D) Insert sequencing of enriched phage clones and subsequent solid-phase peptide synthesis followed by HPLC purification to obtain individual peptide candidates. (E) Fluorescent labeling of selected phage clones using Atto 665 NHS-ester for microscopy-based binding validation on fungal cells. (F) Evaluation of peptide cytotoxicity using human dermal fibroblasts (HDFs). (G) Biofilm inhibition assays performed with purified peptides against C. albicans, C. auris, and C. parapsilosis to identify neutralizing antimicrobial peptides capable of reducing biofilm formation.
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Figure 2. Phage titers measured after each round of biopanning (Rounds 1–4) against three target strains: (A) C. albicans, (B) C. auris, (C) C. parapsilosis. Each bar indicates the recovered phage titer in PFU (plaque-forming units) mL−1 following the indicated biopanning round, demonstrating progressive enrichment of phage clones specific to the respective Candida strain. The steady increase in titers across successive rounds is consistent with successful selection and amplification of strain-binding phage populations.
Figure 2. Phage titers measured after each round of biopanning (Rounds 1–4) against three target strains: (A) C. albicans, (B) C. auris, (C) C. parapsilosis. Each bar indicates the recovered phage titer in PFU (plaque-forming units) mL−1 following the indicated biopanning round, demonstrating progressive enrichment of phage clones specific to the respective Candida strain. The steady increase in titers across successive rounds is consistent with successful selection and amplification of strain-binding phage populations.
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Figure 3. Cross-specificity analysis of phage libraries enriched against (A) C. albicans, (B) C. auris, and (C) C. parapsilosis. Phage pools obtained after three rounds of biopanning against C. albicans, C. auris, and C. parapsilosis were incubated with each of the three fungal species to assess binding specificity. qPCR quantification of recovered phage genomes revealed distinct specificity profiles: the library enriched against C. albicans showed strong and preferential binding to C. albicans; the library enriched against C. auris displayed highest recovery on C. auris; and the library enriched against C. parapsilosis also exhibited specific binding to C. parapsilosis. These data demonstrate successful enrichment of species-selective phage pools and uncover an unexpected cross-recognition between the C. auris-selected library and C. parapsilosis. Significances calculated by student-t-test (*** p < 0.001).
Figure 3. Cross-specificity analysis of phage libraries enriched against (A) C. albicans, (B) C. auris, and (C) C. parapsilosis. Phage pools obtained after three rounds of biopanning against C. albicans, C. auris, and C. parapsilosis were incubated with each of the three fungal species to assess binding specificity. qPCR quantification of recovered phage genomes revealed distinct specificity profiles: the library enriched against C. albicans showed strong and preferential binding to C. albicans; the library enriched against C. auris displayed highest recovery on C. auris; and the library enriched against C. parapsilosis also exhibited specific binding to C. parapsilosis. These data demonstrate successful enrichment of species-selective phage pools and uncover an unexpected cross-recognition between the C. auris-selected library and C. parapsilosis. Significances calculated by student-t-test (*** p < 0.001).
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Figure 4. Specific binding of enriched phage clones to C. albicans, C. auris, and C. parapsilosis. (A–C) qPCR determined phage titer (grey) and quantification and microscopic visualization (red) using n = 10 micrographs of binding interactions between Atto 665-labeled phage clones and C. albicans (A), C. auris (B), and C. parapsilosis (C). Bar graphs display mean fluorescence intensities obtained after incubation of each Candida species with individual phage clones. Enriched binders identified during biopanning are highlighted in red and consistently show the strongest fluorescence signals. For each species, representative brightfield images (middle panels) illustrate cell distribution and morphology during binding assays. Corresponding representative fluorescence micrographs (bottom panels) show Atto 665 signal associated with phage particles bound to the fungal cell surface. Strong fluorescence is detected for the enriched phage clones, whereas background and non-binding clones exhibit minimal signal. All images were acquired under identical exposure and gain settings to allow direct comparison.
Figure 4. Specific binding of enriched phage clones to C. albicans, C. auris, and C. parapsilosis. (A–C) qPCR determined phage titer (grey) and quantification and microscopic visualization (red) using n = 10 micrographs of binding interactions between Atto 665-labeled phage clones and C. albicans (A), C. auris (B), and C. parapsilosis (C). Bar graphs display mean fluorescence intensities obtained after incubation of each Candida species with individual phage clones. Enriched binders identified during biopanning are highlighted in red and consistently show the strongest fluorescence signals. For each species, representative brightfield images (middle panels) illustrate cell distribution and morphology during binding assays. Corresponding representative fluorescence micrographs (bottom panels) show Atto 665 signal associated with phage particles bound to the fungal cell surface. Strong fluorescence is detected for the enriched phage clones, whereas background and non-binding clones exhibit minimal signal. All images were acquired under identical exposure and gain settings to allow direct comparison.
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Figure 5. Effects of the phage display-derived peptides on the viability of human dermal fibroblasts after addition of 100 µg/mL peptide. Triton X-100 was included as a control. All the experiments were performed in triplicate, and the error bars depict the standard deviations. Statistical analysis was performed with a t-test; p-values  <  0.05 were considered significant (*** p  <  0.001). The columns without specific labeling show no significant differences (ns). A cell viability lower than 70% was considered cytotoxic according to ISO 10993-5 and is marked as a dashed line.
Figure 5. Effects of the phage display-derived peptides on the viability of human dermal fibroblasts after addition of 100 µg/mL peptide. Triton X-100 was included as a control. All the experiments were performed in triplicate, and the error bars depict the standard deviations. Statistical analysis was performed with a t-test; p-values  <  0.05 were considered significant (*** p  <  0.001). The columns without specific labeling show no significant differences (ns). A cell viability lower than 70% was considered cytotoxic according to ISO 10993-5 and is marked as a dashed line.
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Figure 6. Schematic overview of the crystal violet biofilm inhibition assay and the proposed nAMP mode of action of phage display-derived peptides. (A) Model of Candida biofilm development and peptide intervention. Biofilm formation progresses through four stages: (1) initial adherence of cells to the surface, (2) growth initiation, (3) biofilm maturation, and (4) dispersal of progeny cells. The identified peptides are proposed to interfere primarily with the early adhesion or growth initiation phases, thereby preventing stable biofilm development and reducing total biomass accumulation. (B) Crystal violet staining after 24 h of incubation. Wells containing untreated cells served as controls, while wells supplemented with synthetic peptides were used to assess inhibitory activity. Representative stained plates illustrate the reduction in surface-attached biomass in peptide-treated samples.
Figure 6. Schematic overview of the crystal violet biofilm inhibition assay and the proposed nAMP mode of action of phage display-derived peptides. (A) Model of Candida biofilm development and peptide intervention. Biofilm formation progresses through four stages: (1) initial adherence of cells to the surface, (2) growth initiation, (3) biofilm maturation, and (4) dispersal of progeny cells. The identified peptides are proposed to interfere primarily with the early adhesion or growth initiation phases, thereby preventing stable biofilm development and reducing total biomass accumulation. (B) Crystal violet staining after 24 h of incubation. Wells containing untreated cells served as controls, while wells supplemented with synthetic peptides were used to assess inhibitory activity. Representative stained plates illustrate the reduction in surface-attached biomass in peptide-treated samples.
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Figure 7. Planktonic viability of Candida spp. after peptide exposure (100 μg·mL−1). Relative viability of planktonic C. albicans (left), C. auris (center) and C. parapsilosis (right) following incubation with 100 μg·mL−1 of each synthetic peptide (conditions as described in Section 4). Bars show mean viability relative to untreated control (normalized to 100%) from three independent experiments (n = 3). No peptide produced a meaningful reduction in planktonic viability at this concentration; results were not significantly different from control. Error bars indicate SD and were small relative to the mean. These data indicate that the anti-biofilm activity reported in other assays is not attributable to direct fungicidal effects on planktonic cells. Statistical analysis was performed with a t-test; p-values  <  0.05 were considered significant (*** p  <  0.001; ns = non significant).
Figure 7. Planktonic viability of Candida spp. after peptide exposure (100 μg·mL−1). Relative viability of planktonic C. albicans (left), C. auris (center) and C. parapsilosis (right) following incubation with 100 μg·mL−1 of each synthetic peptide (conditions as described in Section 4). Bars show mean viability relative to untreated control (normalized to 100%) from three independent experiments (n = 3). No peptide produced a meaningful reduction in planktonic viability at this concentration; results were not significantly different from control. Error bars indicate SD and were small relative to the mean. These data indicate that the anti-biofilm activity reported in other assays is not attributable to direct fungicidal effects on planktonic cells. Statistical analysis was performed with a t-test; p-values  <  0.05 were considered significant (*** p  <  0.001; ns = non significant).
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Figure 8. Dose-dependent biofilm inhibition by phage display-derived (Can-1, AuPan-1, Aln-1, Aln-2, Aun-1, Pan-1) peptides. (A–C) Dose-dependent inhibition of C. albicans, C. auris and C. parapsilosis de novo biofilm formation by the identified peptides. Biofilms were quantified after 24 h using crystal violet staining. Inhibitors were present throughout growth. All experiments were performed in triplicate. The curves were fitted by non-linear regression using a dose–response Hill equation ([Inhibitor] vs. response—variable slope, four-parameter fit) in GraphPad Prism V8.0.1. (D) Summary of peptide activities, illustrating differences in inhibitory potency based on the fitted inhibition curve tested on Candida species. (E) Representative brightfield micrographs of C. albicans, C. auris, and C. parapsilosis biofilms after 24 h in the presence or absence of selected peptides, illustrating the characteristic reduction in surface-associated biomass and structural integrity upon peptide treatment. Scale bars: 500 µm.
Figure 8. Dose-dependent biofilm inhibition by phage display-derived (Can-1, AuPan-1, Aln-1, Aln-2, Aun-1, Pan-1) peptides. (A–C) Dose-dependent inhibition of C. albicans, C. auris and C. parapsilosis de novo biofilm formation by the identified peptides. Biofilms were quantified after 24 h using crystal violet staining. Inhibitors were present throughout growth. All experiments were performed in triplicate. The curves were fitted by non-linear regression using a dose–response Hill equation ([Inhibitor] vs. response—variable slope, four-parameter fit) in GraphPad Prism V8.0.1. (D) Summary of peptide activities, illustrating differences in inhibitory potency based on the fitted inhibition curve tested on Candida species. (E) Representative brightfield micrographs of C. albicans, C. auris, and C. parapsilosis biofilms after 24 h in the presence or absence of selected peptides, illustrating the characteristic reduction in surface-associated biomass and structural integrity upon peptide treatment. Scale bars: 500 µm.
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Figure 9. Inhibition of biofilm maturation by phage display-derived peptides during the incubation phase (24–48 h). Biofilms of C. albicans (A), C. auris (B), and C. parapsilosis (C) were first established for 24 h without peptide (phase 1) and subsequently incubated for an additional 24 h in the presence of 100 µg/mL of each peptide or amphotericin B (AmB) (phase 2). The increase in biofilm biomass during phase 2 was compared to the peptide-treated samples to assess inhibition of further maturation. Significant reductions in biomass relative to the untreated baseline are indicated (* p < 0.05, ** p < 0.01, *** p < 0.001; n.s. = non significant).
Figure 9. Inhibition of biofilm maturation by phage display-derived peptides during the incubation phase (24–48 h). Biofilms of C. albicans (A), C. auris (B), and C. parapsilosis (C) were first established for 24 h without peptide (phase 1) and subsequently incubated for an additional 24 h in the presence of 100 µg/mL of each peptide or amphotericin B (AmB) (phase 2). The increase in biofilm biomass during phase 2 was compared to the peptide-treated samples to assess inhibition of further maturation. Significant reductions in biomass relative to the untreated baseline are indicated (* p < 0.05, ** p < 0.01, *** p < 0.001; n.s. = non significant).
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Table 1. Overview of the six lead peptides identified from species-specific whole-cell phage display selections. The table lists each peptide sequence together with the calculated molecular weight (MW), the hydrophobicity without linker (H) and its selection origin. Can-1 was detected across all three Candida selections, whereas AaPan-1 was the overall highest enriched peptide but only appeared in the C. auris and C. parapsilosis pannings. Aln-1, Aln-2, Aun-1, and Pan-1 represent the most strongly enriched clones within their respective C. auris, C. albicans, or C. parapsilosis panning.
Table 1. Overview of the six lead peptides identified from species-specific whole-cell phage display selections. The table lists each peptide sequence together with the calculated molecular weight (MW), the hydrophobicity without linker (H) and its selection origin. Can-1 was detected across all three Candida selections, whereas AaPan-1 was the overall highest enriched peptide but only appeared in the C. auris and C. parapsilosis pannings. Aln-1, Aln-2, Aun-1, and Pan-1 represent the most strongly enriched clones within their respective C. auris, C. albicans, or C. parapsilosis panning.
NameSequenceMW (Da)H (%)Reason for Selection
Can-1TGTLLVSNKLLTGGGC1533.78942Found independently in all three Candida pannings
AuPan-1SNSNQLIFNWNRGGGC1766.89333Highest enrichment overall and in C. auris and C. parapsilosis pannings
Aln-1FSLSHNSQLNISGGGC1620.74233Highest enrichment in C. albicans panning
Aln-2SNSNQLIFNGNRGGGC1637.73225Highest enrichment in C. albicans panning
Aun-1FTLNDSIAHVLTGGGC1604.78350Highest enrichment in C. auris panning
Pan-1HMPWETTNARRFGGGC1820.02142Highest enrichment in C. parapsilosis panning
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MDPI and ACS Style

Bolotnikov, G.; Gruber, D.; Walter, J.-C.; Kühnel, K.; Kemal, T.; Rodriguez, A.; Preising, N.; Ständker, L.; Firacative, C.; Spellerberg, B.; et al. Phage Display-Derived Peptides Have Neutralizing Activities Against Biofilm Formation by Candida albicans, Candidozyma auris and Candida parapsilosis. Pharmaceuticals 2026, 19, 286. https://doi.org/10.3390/ph19020286

AMA Style

Bolotnikov G, Gruber D, Walter J-C, Kühnel K, Kemal T, Rodriguez A, Preising N, Ständker L, Firacative C, Spellerberg B, et al. Phage Display-Derived Peptides Have Neutralizing Activities Against Biofilm Formation by Candida albicans, Candidozyma auris and Candida parapsilosis. Pharmaceuticals. 2026; 19(2):286. https://doi.org/10.3390/ph19020286

Chicago/Turabian Style

Bolotnikov, Grigory, Daniel Gruber, Jan-Christoph Walter, Kim Kühnel, Turgay Kemal, Armando Rodriguez, Nico Preising, Ludger Ständker, Carolina Firacative, Barbara Spellerberg, and et al. 2026. "Phage Display-Derived Peptides Have Neutralizing Activities Against Biofilm Formation by Candida albicans, Candidozyma auris and Candida parapsilosis" Pharmaceuticals 19, no. 2: 286. https://doi.org/10.3390/ph19020286

APA Style

Bolotnikov, G., Gruber, D., Walter, J.-C., Kühnel, K., Kemal, T., Rodriguez, A., Preising, N., Ständker, L., Firacative, C., Spellerberg, B., Stenger, S., Rosenau, F., & Kissmann, A.-K. (2026). Phage Display-Derived Peptides Have Neutralizing Activities Against Biofilm Formation by Candida albicans, Candidozyma auris and Candida parapsilosis. Pharmaceuticals, 19(2), 286. https://doi.org/10.3390/ph19020286

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