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. 2025 May 2;8:100399. doi: 10.1016/j.crmicr.2025.100399

A cell wall-targeting urease-derived peptide as a potential antifungal agent against Candida species

Ana Paula A Perin a, Julia CV Reuwsaat a, Heryk Motta a, Fernanda Cortez Lopes a, Matheus VC Grahl a, Andrea G Tavanti a, Marilene H Vainstein a, Charley C Staats a, Célia R Carlini a,b,c, Rodrigo Ligabue-Braun d, Lívia Kmetzsch a,⁎
PMCID: PMC12134608  PMID: 40469484

Highlights

  • •

    PmUreβ has broad-spectrum antifungal activity against Candida species.

  • •

    A higher concentration of PmUreβ (18 µM) was able to reduce C. albicans biofilm formation.

  • •

    The PmUreβ mechanism of action involves damaging the yeast cell wall.

  • •

    PmUreβ has additive and synergistic effects with fluconazole in treating resistant C. auris.

  • •

    PmUreβ did not exhibit any harmful effects on mammalian cells.

Keywords: PmUreβ, Candida albicans, Candida auris, Cell wall integrity, Antimicrobial peptide, Antifungal resistance

Abstract

Candida species are the most common opportunistic fungi that cause cutaneous and systemic infections, mainly in immunocompromised patients. The emergence of antifungal resistance has underscored the urgent need for new antifungal drugs, as highlighted by the World Health Organization in 2022 with the release of its first-ever fungal priority list. In this context, antimicrobial peptides present promising candidates for the development of alternative antimicrobial agents. In this study, we evaluated the antifungal activity of the Proteus mirabilis urease β subunit (PmUreβ; 12.2 kDa) against Candida species. PmUreβ reduced the viability of the tested Candida species by over 50 % at concentrations ranging from 2.25 to 9 µM, with the extend of the effect varying according to species and incubation temperature. It also decreased Candida albicans biofilm formation by 30 % at a higher concentration (18 µM). The mechanism of action of PmUreβ involves disruption of the cell wall integrity, as C. albicans cells treated with the recombinant peptide were protected by sorbitol, exhibited increased deposition of chitin in the cell wall, formed cell agglomerates, and downregulated genes associated with cell wall biosynthesis. Additionally, PmUreβ did not appear to cause cell membrane damage, as evidenced by the absence of propidium iodide permeation in treated cells. This peptide also demonstrated a synergistic and predominantly additive effect with fluconazole against the emergent Candida auris. Importantly, no harmful effects were observed in mammalian cells. Our findings suggest that PmUreβ is a fungitoxic peptide with significant biotechnological potential for treating infections caused by antifungal-resistant pathogens.

Graphical abstract

Image, graphical abstract

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1. Introduction

The incidence of systemic mycoses has increased over the past three decades, particularly in immunocompromised individuals (Momtazmanesh et al., 2023; Thomaz et al., 2021; WHO Fungal Priority Pathogens List to Guide Research, Development and Public Health Action, 2022). Risk factors for invasive fungal infections include human immunodeficiency virus (HIV) infection, immunosuppressive therapy, cancer chemotherapy, and the use of long-term devices such as intravenous catheters, especially when combined with broad-spectrum antibiotics (Aranda-Audelo et al., 2018; Hosseini et al., 2021). Rising Candida infections, particularly invasive candidiasis, contribute significantly to morbidity and mortality in elderly and neonatal populations (Biernasiuk et al., 2021; CDC, 2024; Dermitzaki et al., 2024; Kilpatrick et al., 2022). It is estimated that invasive candidiasis occurs in over 626,000 individuals annually, leading to a total mortality of 254,000 (Denning, 2024). In addition to Candida albicans, non-albicans species have also emerged (Billamboz et al., 2021; Kumar et al., 2022; Lee et al., 2021). The World Health Organization (WHO) listed C. albicans and Candida auris as critical pathogens, while Candida parapsilosis, Candida glabrata, and Candida tropicalis are considered high-priority pathogens (WHO Fungal Priority Pathogens List to Guide Research, Development and Public Health Action, 2022). Some Candida species are commensals, but can cause infections when host-microbe balance is disrupted (Jacobsen, 2023). Given the increasing prevalence of fungal infections and antifungal resistance, there is a need for new therapies (Agbadamashi and Price, 2025; Croitoru et al., 2024; Mobeen et al., 2022).

Therapeutic peptides are promising due to their high target affinity, low immunogenicity, and adequate tissue absorption (Purohit et al., 2024; Rossino et al., 2023). In addition, antimicrobial peptides (AMPs) can act efficiently on various fungal structures, such as the plasma membrane, cell wall, or cytoplasm (Brady et al., 2019; Kumar et al., 2024; Ul Haq et al., 2024). Antifungal peptides from natural sources, with potent and broad-spectrum activity against fungal pathogens, are a promising alternative for controlling fungal infections (Roque‐Borda et al., 2025).

Ureases catalyze the hydrolysis of urea into carbon dioxide and ammonia and are widespread in plants, fungi, and bacteria, but absent in animals (Mobley and Hausinger, 1989). Beyond catalysis, ureases have other biological roles, including platelet activation, glycoconjugate interaction, insecticidal, and antifungal activities (Carlini et al., 1985; Follmer et al., 2004; Postal et al., 2012). Jaburetox, from Canavalia ensiformis urease, and soyuretox, from soybean urease, demonstrated antifungal effects against filamentous fungi and yeasts (Mulinari et al., 2007; Kappaun et al., 2019; Postal et al., 2012).

Proteus mirabilis, a bacterial pathogen causing urinary tract infections, colonizes the urethra and catheters, leading to complications such as obstruction, stones, catheter blockage, and bacteremia (Yuan et al., 2021). P. mirabilis urease (PMU) consists of a trimer of trimers (ABC)3, encoded by an operon composed of the structural genes ureA, B and C, which encode the γ (UreA), β (UreB) and α domains (UreC) of the enzyme, with molecular masses of 11, 12.2 and 61 kDa, respectively. Like other ureases, PMU exerts non-enzymatic effects, including platelet activation, and antifungal and entomotoxic activities, primarily attributed to its β domain (PmUreβ) (Broll et al., 2021).

Given the potential biotechnological applicability of PmUreβ as an antimicrobial peptide, the present study investigated the effects and mechanism of action on different Candida species. The recombinant peptide exhibits fungitoxic activity against C. albicans, C. glabrata, C. krusei, C. parapsilosis, and Candida guilliermondii. In C. albicans, PmUreβ acts causing damage to the cell wall, resulting in chitin accumulation, and promoting cell aggregation. Additionally, PmUreβ modulates the expression of genes involved in cell wall biosynthesis and demonstrates a synergistic effect with fluconazole against C. auris resistant cells. Notably, the recombinant peptide does not exhibit cytotoxicity to human cells.

2. Material and methods

2.1. Fungal strains and growth conditions

The following Candida species were used in this study: C. albicans (ATCC 24433), C. glabrata (ATCC MYA 2950), C. krusei (ATCC 6258), C. parapsilosis (ATCC 22019), C. auris (Ca 446) and C. guilliermondii (CE022). All strains were grown in Sabouraud broth (1 % peptone [w/v], 2 % glucose [w/v]), incubated at 30 °C with rotatory shaking (180 rpm) overnight and grown to the exponential phase for further experiments. The strains were stored on Sabouraud plates (1.5 % [w/v] agar) at 4 °C, and for long-term storage the strains were kept in glycerol stock at −80 °C.

2.2. PmUreβ production

The recombinant peptide PmUreβ was expressed and purified as described (Broll et al., 2021). This subunit contains a His-tag in the C-terminal portion of the protein. Escherichia coli (Lemo21 (DE3)) cell cultures were carried out using Luria Bertani (LB) medium with 100 µg/mL of ampicillin and 40 µg/mL of chloramphenicol (Sigma-Aldrich, St. Louis, MO, USA). Overnight culture of single colonies of E. coli transformed with pET15b-PmUreβ was performed at 37 °C, under constant agitation. Protein expression was induced by the addition of 0.3 mM Isopropyl-ßβ-d-thiogalactopyranoside (IPTG) and 100 µM rhamnose, when cellular growth reached an OD600nm of 0.7, and kept overnight at 18 °C. Recombinant E. coli culture was centrifuged at 6000 x g for 10 min at 4 °C. The pellet was suspended in a buffer containing 50 mM Tris–HCl pH 7.5, 500 mM NaCl and 20 mM imidazole. Cells were disrupted by sonication (15 cycles - amplitude: 70 %, sonication pulse rate: 10 s ON, 30 s OFF), and cellular debris were centrifuged at 14,000 x g for 30 min. PmUreβ was found in the culture supernatant, and was subjected to affinity chromatography purification using Chelating Sepharose resin (GE Healthcare, Little Chalfont, UK) equilibrated in 50 mM Tris–HCl pH 7.5 buffer, 500 mM NaCl and 20 mM imidazole. The column was washed with the same buffer containing 70 mM imidazole and then the peptide was eluted with 500 mM imidazole. Before each assay, a dialysis was conducted to change the buffer to 10 mM Tris–HCl pH 7.5 buffer and 1 mM DTT. Solutions of PmUreβ were sterilized by filtration through 0.22 µm syringe filters to perform the biological assays. Protein contents were determined by the Bradford method (Bradford, 1976) using bovine serum albumin as a standard.

2.3. Broth microdilution assay

Five Candida species were evaluated in this assay: C. albicans, C. glabrata, C. krusei, C. parapsilosis, and C. guilliermondii. All strains were grown in Sabouraud broth at 30 °C for 24 h, and the cells were then harvested, transferred to fresh Sabouraud broth, and cell density determined in a Neubauer chamber. A 100 µL suspension of 5 × 105 cells/mL was plated in each well in 96-well plates. The recombinant protein was then added to the wells in concentrations of 2.25, 4.5, and 9 µM, along with a buffer control, resulting in a final assay volume of 200 µL. The plates were incubated at 30 °C or 37 °C for 24 hours. To determine the cell viability, colony-forming units (CFUs) were counted after further incubation at 30 °C for 24 hours on Sabouraud agar plates using the drop plate method (Postal et al., 2012).

2.4. Biofilm assay

C. albicans were grown for 24 h on Sabouraud broth at 30 °C with agitation. After growth, cells were harvested and transferred to RPMI-1640 (Invitrogen, Carlslad, CA, United States) supplemented with 15 % fetal bovine serum (FBS) (Sigma-Aldrich). The cells were counted using a Neubauer chamber, and 100 µL of the cell suspension (5 × 105 cells/mL) were added to 96-well plates containing RPMI broth. The recombinant protein was then added to the wells at concentrations of 2.25, 4.5, 9, and 18 µM, for a final assay volume of 200 µL. Then, the yeasts were incubated for 24 h at 37 °C. The XTT (2,3-bis-(2‑methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reduction assay was then performed as previously described (Jin et al., 2004). Briefly, the cells were washed with PBS to remove loosely adherent cells. Subsequently, the XTT mixture was added to the cells and incubated in the dark for 4 hours at 37 °C. After the incubation, the solution was transferred to a new well and the colorimetric change in the solution was measured using a SpectraMax® M3 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) at 492 nm. For the Scanning Electron Microscope (SEM) analysis, the cells were washed with PBS buffer after the incubation period. Then, it was fixed with 2.0 % glutaraldehyde for 7 days, incubated with PBS for 30 min to remove the glutaraldehyde, and dried at room temperature. The control consisted of yeast samples with no recombinant peptide. The samples were dehydrated in acetone solutions of increasing concentration (50 %, 70 %, 90 %, and 100 %) for 10 min each, dried, metallized and stored in a desiccator for observation by scanning electron microscopy (SEM) (Zeiss Evo MA10 - German) at Microscopy and Microanalysis Center of the Federal University of Rio Grande do Sul (UFRGS), as described (Trentin et al., 2011).

2.5. Checkerboard assay

The synergistic action of fluconazole in combination with the PmUreβ was tested by the checkerboard method based on EUCAST guidelines (Bellio et al., 2021). After overnight culture on YPD agar plates, colonies of C. auris were suspended in sterile water, counted in a Neubauer chamber and a suspension of 5 × 105 cells/mL was prepared. A gradient of PmUreβ was established along the horizontal axis and the fluconazole along the vertical axis. One hundred microliters of the cell suspension were inoculated into each well of the plate, and incubated for 24 h at 30 °C. The Minimum Inhibitory Concentration (MIC) values of the compounds alone or in combination were determined using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific, Grand Island, NY, USA) at 530 nm. In vitro interactions between PmUreβ and fluconazole were analyzed by determining the fractional inhibitory concentration index (FICI): FICI = FICPmUreβ + FICfluconazole = (MIC of PmUreβ in combination/MIC of PmUreβ alone) + (MIC of fluconazole in combination/MIC of fluconazole alone). FICI ≤ 0.5 indicated synergy, FICI > 4 denoted antagonism, FICI between 0.5 and < 1 was considered addition, and between 1 and < 4 was considered indifferent (Carton et al., 2025).

2.6. Cytotoxic evaluation assays

The HEK293 cell line was cultivated in DMEM (Thermo Fisher Scientific, Grand Island, NY, USA) supplemented with 10 % heat-inactivated FBS and 0.1 % Penicillin-Streptomycin and maintained at 37 °C with 5 % CO2. HEK293 cells were incubated for 24 h with 2.25, 4.5 or 9 µM of PmUreβ, buffer, or 0.01 % Triton X-100 as positive and negative controls, respectively. The supernatants were removed, and cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 5 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA) for 4 h at 37 °C with 5 % CO2. Plates were centrifuged (1000 x g, 10 min at room temperature), and the precipitates were resuspended with 100 µL 100 % DMSO. Plates were read at 570 nm in a M2 spectrofluorometer (Molecular Devices, San Jose, CA, USA), as described (Olivera-Severo et al., 2017). Viability was also determined using the trypan blue dye exclusion test. After incubation, 20 μL of cell culture was mixed with 5 μL trypan blue (0.4 %, Gibco-BRL). The total number of cells, including those that had excluded the dye, was counted in a Neubauer chamber, and cell viability was calculated as the percentage of viable cells in the samples relative to the untreated cells. The experiments were conducted in triplicate.

To determine intracellular ROS production in HEK293 cells, carboxy-H2DCFDA (5-(-6)-carboxy2′,7′-difluorodihydrofluorescein diacetate, Thermo-Scientific, Waltham, MA, USA) was used following the protocol described previously (Uberti et al., 2013). Cells (106 cells/well) were seeded in 96-well plates and pre-incubated with the dye (2 mM) for 30 min at 37 °C in the dark. Then, cells were treated with 2.25, 4.5 and 9 µM of PmUreβ or buffer (control), and 0.1 % of H2O2 was used as a positive control. ROS production was measured 24 h after treatment. Fluorescence (λex 495 nm/λem 527 nm) was measured using a SpectraMax®M2 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Experiments were performed in triplicate. The time dynamics in fluorescence were normalized against the control group df/F0 (%), where F0 represents the fluorescence expressed by the control and df represents the change in fluorescence over time during the cell stimulus.

2.7. RNA sequencing

C. albicans cells were grown in Sabouraud medium for 24 hours at 30 °C with agitation, washed with PBS, and 5 × 105 cells/mL were plated in Sabouraud with or without 4.5 µM of PmUreβ in 24-well plates for 24 h at 30 °C in three biological replicates. Total RNA was then isolated and sequenced as previously described (Maier et al., 2015). Briefly, the cultures were treated with an ice-cold stop solution (5 % Tris-saturated phenol in ethanol) to prevent RNA degradation, and then the cultures were collected by centrifugation. Cells were suspended in TRIzol reagent (Life Technologies) and lysed by mechanical bead-beating at 4 °C with 0.5-mm silica-zirconia beads (4 cycles of 3 min of beating followed by 2 min on ice). Following lysis, total RNA was extracted according to the manufacturer’s instructions. Residual DNA was removed with the TURBO DNA-free kit (Life Technologies). cDNA libraries were prepared and sequenced using the Illumina NextSeq platform for paired-end 2  × 75 bp reads at LaCTAD (Laboratório Central de Tecnologias de Alto Desempenho, Universidade Estadual de Campinas, Unicamp, São Paulo, Brazil). The reads quality was evaluated by FastQC (Babraham Bioinformatics, 2023). Fastq files were aligned to the C. albicans genome (GCF_000182965.3) (Skrzypek et al., 2017) using STAR (Dobin et al., 2013) with the –quantMode GeneCounts flag to generate a count table, and differential gene expression was analyzed with DESeq2 (Love et al., 2014). Genes with absolute values of Log2 fold change > 1.0 and p-adjusted < 0.05 were considered differentially expressed.

2.8. Sorbitol protection assay

The effect of PmUreβ on the integrity of the fungal cell wall was evaluated by the sorbitol protection assay. C. albicans were grown for 24 h on Sabouraud broth at 30 °C with agitation. After growth, cells were harvested, transferred to a new Sabouraud broth, and counted using a Neubauer chamber. A suspension of 100 µL of 5 × 105 cells/mL was plated in 96-well plates containing Sabouraud broth with and without 0.8 M sorbitol (Sigma Aldrich, St. Louis, MO, USA), as a fungal cell wall osmoprotectant. The peptide was added at concentrations of 4.5 or 9 µM, and the cells were incubated for 24 h at 30 °C. The cell viability was then assessed by CFU after further incubation at 30 °C for 24 h on Sabouraud agar plates.

2.9. Fluorescence microscopy and flow cytometry

C. albicans cells were cultivated in Sabouraud broth at 30 °C for 24 h. Following growth, cells were collected and transferred to a fresh Sabouraud broth, counted, and 100 µL of a suspension of 5 × 105 cells/mL was plated in 96-well plates. The recombinant peptide was then added to the wells in concentrations of 2.25, 4.5, and 9 µM, along with a buffer control, resulting in a final assay volume of 200 µL. The yeast cells were incubated for 24 hours at 30 °C. To evaluate the chitin of the cell wall, the culture medium was centrifuged (4000 x g, 5 min), washed with PBS, and the cells were stained with 5 µg/mL CalcoFluor White (CFW) for 30 min at 37 °C. The samples were washed and resuspended in 10 µl PBS for fluorescence microscopy. Cells were visualized by confocal microscopy (Olympus FV1000) at Microscopy and Microanalysis Center, Universidade Federal do Rio Grande do Sul. For flow cytometry, the samples were washed with PBS 1x and resuspended in 1 mL of PBS. A total of 20,000 events were recorded for each sample, and the CFW mean fluorescence intensity (MFI) was measured using a violet laser and 450/40 nm filter. For the cell aggregation analysis, 10,000 events were recorded. Cells were separated into four quadrants: Q1 (FSC-A-; SSC-A-), Q2 (FSC-A-; SSC-A+), Q3 (FSC-A+; SSC-A-), and Q4 (FSC-A+; SSC-A+). Increased cell agglomerates are demonstrated in Q4, as highlighted by the images. The assays were performed using the flow cytometer Attune Cytpix, Thermo Fisher Scientific.

2.10. Propidium iodide (PI) uptake assay

Membrane permeability of Candida cells after PmUreβ treatment was evaluated using a PI uptake assay with some modifications (Sun et al., 2022). In brief, an overnight culture of C. albicans in Sabouraud broth was harvested by centrifugation, resuspended, and counted. PmUreβ (2.25, 4.5, and 9 µM) was added to 500 µL of cell suspension (1 × 107 cells/mL) and then incubated for 6 h at 30 °C. Cells were washed with PBS, resuspended, and incubated at 30 °C with 5 μg/mL PI (Merck Life Science - Darmstadt, Germany) in PBS in the dark for 20 min, under agitation. Then, cells were washed in PBS twice and resuspended. Membrane permeability was assessed by fluorescence intensity of PI (λex 525 nm/λem 590 nm), determined in a SpectraMax®M3 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA), and the OD600nm was also measured for normalization. Amphotericin B (128 µM) was used as positive control. Three replicates were performed, and values were reported as mean ± SD. PI uptake was expressed as the fluorescence/ OD600nm ratio, as arbitrary units.

2.11. Statistical analysis

The results from independent replicate experiments are expressed as means ± SD. The Shapiro-Wilk test was performed to evaluate the normality distribution of the data. Statistical analysis was performed using a one-way ANOVA with Dunnett’s post-hoc test. When the results displayed unequal variance, the Kruskal–Wallis non-parametric or Dunnett’s T3 test was applied. Significance was determined as p < 0.05.

2.12. Data availability

RNA-Seq data files are available at NCBI under the accession number PRJNA1249100.

3. Results

3.1. PmUreβ presents antifungal activity against different Candida species

Antimicrobial peptides represent a promising source of new antifungal therapies. Our results demonstrated that PmUreβ exhibits antifungal activity against a variety of Candida species, including C. albicans, C. glabrata, C. krusei, C. parapsilosis, and C. guilliermondii. The antifungal activity of PmUreβ was determined at 30 °C (Fig. 1) and 37 °C (Fig. 2). Inhibition exceeding 50 % of cell viability was observed at concentrations ranging from 2.25 to 9 µM across the different tested Candida species. At 30 °C, C. albicans and C. krusei exhibited a reduction in cell viability greater than 50 % at 4.5 µM, whereas C. glabrata and C. guilliermondii required 2.25 µM to achieve a comparable effect. C. parapsilosis, in contrast, required the highest tested concentration (9 µM) to reach a similar level of inhibition (Fig. 1). At 37 °C, C. albicans, C. glabrata, and C. guilliermondii demonstrated over 50 % reduction in cell viability only at 9 µM. Conversely, C. krusei and C. parapsilosis reached this threshold at lower concentrations, 4.5 µM and 2.25 µM, respectively (Fig. 2).

Fig. 1.

Fig 1

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PmUreβ exhibits antifungal activity against a variety of Candida species at 30 °C. Evaluation of the fungitoxic effect of PmUreβ in Candida spp. Yeast cells were incubated with PmUreβ for 24 h at 30 °C, and CFU was determined by the drop plate method. CFU percentage was calculated as the ratio of CFU in the presence of the recombinant protein by the control. Results are means ± standard deviations (SD). * represents p ≤ 0.05; ⁎⁎ ≤ 0.01; ⁎⁎⁎p ≤ 0.001; ⁎⁎⁎⁎p ≤ 0.0001.

Fig. 2.

Fig 2

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PmUreβ exhibits antifungal activity against a variety of Candida species at 37 °C. Evaluation of the fungitoxic effect of PmUreβ in Candida spp. Yeasts were incubated with PmUreβ for 24 h at 37 °C. Cell proliferation was determined by the drop plate method. CFU percentage was calculated as the ratio of CFU in the presence of the recombinant protein by the control. Results are means ± SD. * represents p ≤ 0.05; ⁎⁎ ≤ 0.01; ⁎⁎⁎p ≤ 0.001; ⁎⁎⁎⁎p ≤ 0.0001.

In addition, the ability of the PmUreβ peptide to inhibit biofilm formation was evaluated using the XTT reduction assay. The results revealed that PmUreβ significantly reduced the C. albicans biofilm by 30 % compared to the control group at the highest tested concentration (18 µM) (Fig. 3A). This inhibitory effect on biofilm formation was further corroborated by scanning electron microscopy images (Fig. 3B), which showed a reduction in pseudo-hyphae formations and the overall biofilm layer.

Fig. 3.

Fig 3

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PmUreβ reduces C. albicans biofilm formation. Evaluation of biofilm formation of C. albicans. (A) Yeasts were incubated with PmUreβ (2.25, 4.5, 9, and 18 µM) for 24 h at 37 °C. The adherent biofilms were quantified by XTT. (B) Scanning electron microscopy of C. albicans (biofilm forming) incubated for 24 h with or without PmUreβ. (I-II) control; (III-IV) PmUreβ 18 µM. Images (II) and (IV) (bar = 1 µm) are the magnification of (I) and (III) (bar = 2 µm) respectively. Results are means ± SD. * represents p ≤ 0.05.

3.2. PmUreβ exhibits additive and synergistic activity with fluconazole

Considering the fungitoxic activity of PmUreβ, we aimed to evaluate whether the recombinant peptide could effectively target the emerging pathogen C. auris. Employing a checkerboard assay, we observed that PmUreβ displayed predominantly additive interactions with fluconazole, along with synergistic effects that significantly enhanced antifungal efficacy (Table 1). Notably, the recombinant peptide reduces fluconazole’s minimum inhibitory concentration (MIC) from 8 to 4 µg/mL, even at low concentrations of 0.008 and 0.017 µM (Table 1). This MIC reduction suggests that PmUreβ enhances fluconazole activity.

Table 1.

In vitro activity of PmUreβ in combination with FLC against C. albicans.

MIC alone MIC combination
FLC (µg/mL) PmUreβ (µM) FLC (µg/mL) PmUreβ (µM) FICIa Interpretation of interaction
8 2.25 4 0.008 0.50 Synergic
8 2.25 4 0.017 0.50 Synergic
8 2.25 4 0.035 0.51 Addition
8 2.25 4 0.070 0.53 Addition
8 2.25 4 0.140 0.56 Addition
8 2.25 4 0.281 0.62 Addition
8 2.25 4 0.525 0.75 Addition
8 2.25 4 1.125 1 Addition
8 2.25 2 1.125 0.75 Addition
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a

FICI ≤ 0.5: synergism; FICI > 4.0: antagonism; 0.5 < FICI ≤ 4.0: addition.

FLC: fluconazole

3.3. PmUreβ does not induce toxicity in mammalian cells

To evaluate the cytotoxic effect of PmUreβ on mammalian cells, we incubated HEK293 cells with different concentrations of PmUreβ for 24 h, followed by viability assays and ROS production tests. These results demonstrated that PmUreβ did not harm cell viability, either evaluated by alterations in cell membrane permeability (Fig. 4A), or metabolic alterations (Fig. 4B). Furthermore, PmUreβ did not lead to increased intracellular ROS production (Fig. 4C).

Fig. 4.

Fig 4

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PmUreβ does not present a harmful effect on mammalian cells. HEK293 cells were incubated with different concentrations of PmUreβ for 24 h. (A) Trypan blue exclusion; (B) MTT viability assay; (C) ROS production. Results are means ± SD. Groups identified by different superscripts are significantly different (p < 0.05) and *p < 0.05 compared to the control group.

3.4. PmUreβ affects fungal cell wall synthesis and integrity

To gain insights into the mechanism of action of PmUreβ, we performed total RNA sequencing of C. albicans cells in the presence of 4.5 µM PmUreβ for 24 h at 30 °C. The expression of PGA6, ECM331, and RBR1 genes, all involved in cell wall formation (De Groot et al., 2003), was decreased in C. albicans incubated with the recombinant peptide (Table 2). Other differentially expressed coding genes were related to transmembrane transport (potassium ions), ergosterol synthesis (ERG6), and tRNA synthesis (Table 2). To test if PmUreβ acts at the fungal cell wall, we performed the sorbitol assay, since sorbitol is an osmotic protector that stabilizes protoplasts and protects their cell wall from environmental stresses (Frost et al., 1995). When added to the medium, the MIC of the compounds that damage the cell wall is expected to increase in the presence of osmotic support (Frost et al., 1995). The results of our assay showed that the concentration of PmUreβ required to inhibit >50 % of C. albicans cell viability significantly increased in medium supplemented with sorbitol after 24 h of incubation, compared to the medium without sorbitol (Fig. 5).

Table 2.

Genes differentially expressed upon PmUreβ exposure.

Biological process Organism Gene name Log2FC* Padj⁎⁎ Description
Cell wall formation C. albicans
ATCC 24433 		RBR1 -2.33 0.027617619 Glycosylphosphatidylinositol (GPI)-anchored cell wall protein
Cell wall formation C. albicans
ATCC 24433 		ECM331 -1.77 9.16E-05 Glycosylphosphatidylinositol (GPI)-anchored protein
Cell wall formation C. albicans
ATCC 24433		 PGA6 -1.41 1.23E-05 GPI-anchored cell wall adhesin-like protein
Ion transport C. albicans
ATCC 24433		 HAK1 -1.42 0.015801762 Potassium ion transmembrane transport
Ergosterol biosynthesis C. albicans
ATCC 24433		 ERG6 1.60 0.000476815 Sterol 24-C-methyltransferase
C. albicans
ATCC 24433		 tR(UCU)4 -2.53 0.012792535 tRNA-Arg
C. albicans
ATCC 24433		 tE(UUC)1 -2.52 6.85E-12 tRNA-Glu
C. albicans
ATCC 24433		 uncharacterized protein -1.74 0.029599279
C. albicans
ATCC 24433		 uncharacterized protein -1.57 0.001021735
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⁎

Log2 fold change for treatment compared to control.

⁎⁎

Padj: FDR-adjusted p value.

Fig. 5.

Fig 5

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PmUreβ affects the fungal cell wall integrity. The concentration of PmUreβ that inhibited 50 % of C. albicans viability was tested in the presence of sorbitol (0.8 M). After 24 h of incubation at 30 °C, the CFU was determined and compared to the control (without sorbitol). The values are expressed as a mean of three independent experiments ± SD. Groups identified by different superscripts are significantly different (p < 0.05).

To further investigate the impact of PmUreβ in impairing cell wall integrity, we evaluated the chitin content of the C. albicans cell wall in the presence of the recombinant peptide. An increased deposition of chitin in cells incubated with PmUreβ was observed compared to the control group, as determined by calcofluor white fluorescence quantified either by confocal microscopy (Fig. 6A and B) and flow cytometry (Fig. 6C and D). Additionally, further evidence of cell wall disruption by the peptide is the increased formation of cell aggregates, which are more abundant in peptide-treated cells. Flow cytometry of C. albicans cells treated with distinct concentrations of PmUreβ for 24 hours at 30 °C revealed impaired bud detachment, as evidenced by the increased formation of cell aggregates (Fig. 7). While some yeast cells successfully underwent budding, the process was not uniformly effective across the entire population.

Fig. 6.

Fig 6

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PmUreβ affects chitin production in C. albicans. (A)C. albicans upon 24h-treatment at 30 °C with different concentrations of PmUreβ and stained with calcofluor white (CFW) (bar = 10 µm). (B) The graph represents the quantification of the calcofluor fluorescence of the cells in figure A. (C) The graph represents the mean fluorescence intensity of the C. albicans stained with CFW that were measured with flow cytometry. (D) Represents of the overlay of the control and treatment histograms of the flow cytometry assay. Results are means ± SD. ⁎⁎⁎⁎ represents p ≤ 0.0001.

Fig. 7.

Fig 7

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PmUreβ treatment induces cell aggregates in C. albicans. Flow cytometry of C. albicans cells treated for 24 hours at 30 °C (A) with buffer, (B) 2.25 µM, (C) 4.5 µM, and (D) 9 µM PmUreβ. Left panel: dot plot of 10,000 events; Q4 represents cell aggregates (FSC-Ahigh and SSC-Ahigh). The percentage of cells in each quadrant is represented. Right panel: representative image of one event of each panel. Q4 image is highlighted in red. Flow cytometer objective of 20x.

3.5. PmUreβ does not induce membrane perturbation in C. albicans

To determine whether PmUreβ also affects the integrity of the fungal cell membrane, we conducted a propidium iodide (PI) permeability assay. Amphotericin B was used as a positive control, as it binds to ergosterol in the fungal cell membrane, leading to ion leakage and membrane disruption (Stone et al., 2016). The results revealed that 6 hours of PmUreβ treatment did not increase PI uptake in C. albicans cells, indicating that the peptide does not induce cell membrane perturbation, in contrast to amphotericin B (Fig. 8).

Fig. 8.

Fig 8

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PmUreβ did not induce membrane perturbation in C. albicans. After 6 h treatment with PmUreβ at 30 °C, propidium iodide (PI) uptake was detected by spectrofluoro-photometer. Error bars represent the standard deviation of three independent experiments. Results are means ± SD. *p < 0.05 compared to the control group.

4. Discussion

Candida-related fungal infections are an emerging public health problem that is increasing mortality and cost per hospitalized patient (Cui et al., 2022). The growing threat of drug-resistant fungi is heightened by the limited number of antifungal agents in the market, challenging the recent task of dealing with fungal infections (Song et al., 2024). Resistance arises through drug target alteration or overexpression, efflux pump activity, or activation of stress response pathways (Lee et al., 2023). The emergence of inherently drug-resistant fungi, such as C. auris and C. glabrata, further exacerbates this issue (Pfaller et al., 2019). Furthermore, the emergence of multidrug-resistant isolates towards conventional antifungals and their inherent toxicity to humans, further intensifies the challenge (Cui et al., 2022; Rakhshan et al., 2023; Teng et al., 2023). In light of this scenario, antifungal peptides (AFPs), such as Novexatin® (NP213), Omiganan, PAC 113, and CZEN-002, currently in the clinical phase, hold great promise as effective molecules against a wide range of fungal infections (Freitas and Felipe, 2023).

In the present study, we investigated the antifungal activity of the β subunit of P. mirabilis urease. A previous study demonstrated that this urease, composed of three subunits, exhibited antifungal activity (Broll et al., 2021). Here, we show that the recombinant PmUreβ has a broad-spectrum antifungal activity against various Candida species, both at 30 °C and 37 °C, at low doses (Figs. 1 and 2). Notably, PmUreβ also displayed antifungal activity against resistant C. auris (Table 1) (Muñoz et al., 2020) and is not toxic to human cells (Fig. 4).

Our findings indicate that at 37 °C, although most of the tested Candida species showed increased resistance to PmUreβ, the peptide still significantly reduced yeast growth (Fig. 2). This may be due to the fact that at this temperature, Candida species are more likely to develop pseudohyphal structures, which enhance their infectivity and resistance to antifungal drugs (Malinovská et al., 2023; Talapko et al., 2021). On the other hand, the peptide significantly reduced C. albicans biofilm formation at the highest tested concentration (18 µM) (Fig. 3). Co-cultivation of P. mirabilis and C. albicans suggests that secretory products from the bacteria inhibit the transition of C. albicans from the yeast form to the hyphal form (Lee et al., 2017). This is a significant finding, as biofilm formation contributes to treatment inefficiency, high morbidity and mortality rates, and is a major virulence factor in candidiasis pathogenesis (Pereira et al., 2021; Sharma et al., 2023; Malinovská et al., 2023). A limitation of this study is the low yield of the recombinant peptide, restricting the use of higher concentrations in experimental procedures. The choice of the CFU counting method instead of the standardized microbroth dilution protocols recommended by CLSI or EUCAST represents another limitation, but, at the same time, CFU determination provides a direct and accurate measure of cell viability.

Our RNA-seq analysis revealed that PGA6, ECM33, and RBR1 genes involved in cell wall formation were downregulated upon exposure to PmUreβ (Table 2). These genes play critical roles in cell wall restructuring and reinforcement. Specifically, RBR1 null mutants in C. albicans exhibit filamentation defects. At the same time, both PGA6 and ECM33 are associated with cell wall reinforcement and restructuring, suggesting that the peptide disrupts these pathways, resulting in the cell wall impairment (Degani et al., 2016; Gil-Bona et al., 2016; Lotz et al., 2004). This response is further supported by the observation that C. albicans treated with PmUreβ for 24 h resulted in increased calcofluor staining (Fig. 6), indicative of elevated chitin deposition in the cell wall. A similar phenomenon has also been observed in C. albicans treated with caspofungin (CSF), where CSF-induced cell wall stress triggers a compensatory increase in chitin and beta-1,3-glucan exposure (Mora-Montes et al., 2011; Walker et al., 2008; Wheeler et al., 2008).

Furthermore, our flow cytometry analysis revealed an increase in incomplete daughter cell release in PmUreβ-treated cells, resulting in cell aggregates that had not fully completed the budding process. This defect in bud release was previously linked to disruptions in the C. albicans cell wall integrity (Fiołka et al., 2020). Notably, the PGA6 ortholog in S. cerevisiae is also associated with cell wall reinforcement in emerging buds (Ragni et al., 2011). Additionally, the cell wall impairment induced by PmUreβ was restored by the osmotic protector sorbitol (Fig. 5). Together, these findings suggest that PmUreβ treatment induces cell wall damage, prompting the downregulation of genes involved in cell wall maintenance and restructuring, increases chitin deposition as a protective response, and induces defects in budding due to impaired cell wall integrity.

The RNA-seq analysis also revealed that the ERG6 gene, which is involved in ergosterol biosynthesis, was upregulated in C. albicans treated with PmUreβ (Table 2). The ERG6 is essential to the maintenance of cellular membrane integrity and fluidity (Elias et al., 2022; Sharma, 2006). The increased expression may act as a compensatory response to enhance the stability and functionality of the plasma membrane, given the cell wall impairment caused by the recombinant peptide. Additionally, the expression of HAK1 gene, a potassium membrane transporter, was also affected (Table 2). Despite these transcriptional changes in cell membrane-related genes, cell membrane permeability, assessed by PI uptake, did not show significant difference between PmUreβ-treated and untreated cells (Fig. 8). This indicates that PmUreβ does not directly disrupt the cell membrane integrity. Although ERG6 is a member of the ergosterol biosynthesis pathway, its overexpression leads to cell wall sensitivity in S. cerevisiae, increasing the yeast doubling time (Bhattacharya et al., 2018). In C. albicans, the ergosterol biosynthetic pathway is regulated by Pkc1, which also plays a central role in the cell wall damage response, further highlighting the interplay between cell membrane and cell wall homeostasis (LaFayette et al., 2010). These findings suggest that the upregulation of ERG6 in response to PmUreβ treatment may indirectly contribute to cell wall stress responses rather than directly affecting the membrane integrity.

The observed additive and synergistic effects between PmUreβ and fluconazole likely arises from their complementary multi-target mechanisms: fluconazole disrupts ergosterol biosynthesis, compromising membrane integrity, while PmUreβ targets the cell wall. This synergy is particularly advantageous given fluconazole's dose-dependent hepatotoxicity (Gupta et al., 2020; Rakhshan et al., 2023). Through this synergistic interaction, combination therapy may not only reduce the required fluconazole dosage, minimizing liver damage in critically ill patients while preserving therapeutic efficacy, but also potentially restore fluconazole sensitivity in clinically resistant strains (Toepfer et al., 2024). This finding highlights PmUreβ potential as a valuable adjuvant in antifungal therapy and offers a promising strategy to combat drug resistant C. auris infections.

The cell wall plays a crucial role in host-fungus interactions, mediating essential processes such as adhesion and phagocytosis, which are critical during infection (Garcia-Rubio et al., 2020; Karkowska-Kuleta and Maddi, 2022). Disruptions in cell wall dynamics can inhibit fungal growth and impair host-fungal interactions required for pathogenesis (Lima et al., 2019). In addition to playing a crucial role in fungal biology, the fungal cell wall is primarily composed of molecules absent in the human body, making it an ideal target for antifungal development and immunotherapy.

5. Conclusion

The PmUreβ peptide demonstrates promising antifungal activity against various Candida species, including clinically significant strains such as C. albicans, C. glabrata, and C. auris. It also disrupts C. albicans biofilm formation at higher concentrations and acts additively and synergistically with fluconazole to lower the MIC against C. auris. Importantly, PmUreβ does not induce toxicity in mammalian cells, suggesting its safety for further research. Mechanistically, PmUreβ disrupts fungal cell wall integrity by downregulating genes involved in cell wall formation and increasing chitin deposition, leading to cell aggregation in C. albicans. Furthermore, it does not affect the fungal cell membrane permeability, distinguishing its action from amphotericin B. Overall, PmUreβ is a promising candidate for further development as an antifungal agent, particularly as a potential adjuvant in combination with existing therapies to combat fungal infections.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work the authors used OpenAI ChatGPT to check English grammar, with caution. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

CRediT authorship contribution statement

Ana Paula A. Perin: Writing – original draft, Writing – review & editing, Methodology, Investigation, Conceptualization, Formal analysis, Data curation. Julia C.V. Reuwsaat: Writing – review & editing, Methodology, Investigation. Heryk Motta: Writing – review & editing, Methodology, Visualization. Fernanda Cortez Lopes: Writing – review & editing, Project administration, Conceptualization. Matheus V.C. Grahl: Investigation, Methodology, Visualization. Andrea G. Tavanti: Investigation, Data curation, Software. Marilene H. Vainstein: Resources, Writing – review & editing, Funding acquisition. Charley C. Staats: Investigation, Data curation, Software. Célia R. Carlini: Resources, Writing – review & editing, Supervision, Conceptualization, Funding acquisition. Rodrigo Ligabue-Braun: Writing – review & editing, Supervision, Conceptualization. Lívia Kmetzsch: Resources, Writing – review & editing, Supervision, Project administration, Conceptualization, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

L.K., C.R.C., C.C.S., F.C.L, M.H.V., and R.L.B. were supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS, Brazil). A.P.A.P., M.V.C.G., and H.M. were recipients of post-doctoral fellowships from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil). J.C.V.R. was recipiente of post-doctoral fellowship from Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS). A.G.T was recipiente of posgraduat fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil). This work was supported by the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Grant numbers 405810/2023-7, 312797/2021-4 and 408717/2022-0), The National Institute of Science and Technology INCT FUNVIR (Grant number 405934/2022-0) and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS, Grant number 17/2551-0000516-3).

We thank Dra. Valdirene Gomes, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, Brazil for generously providing us the C. guilliermondii strain and Dr. Douglas Sato, Pontifícia Universidade Católica do Rio Grande do Sul, Brazil, for generously providing us the HEK293 cell line. We thank the Microscopy and Microanalysis Center (CMM) of the Federal University of Rio Grande do Sul (UFRGS) for the confocal and scanning electron microscopy analysis, as well as Henrique Biehl and Francis Almeida for technical assistance.

Data availability

Data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

RNA-Seq data files are available at NCBI under the accession number PRJNA1249100.

Data will be made available on request.

Articles from Current Research in Microbial Sciences are provided here courtesy of Elsevier

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