Preparation and analysis of quinoa active protein (QAP) and its mechanism of inhibiting Candida albicans from a transcriptome perspective

Plant materials

The quinoa seeds (Cheng Li No.1) utilized in this study were supplied by the Key Laboratory of Coarse Cereal Processing, Ministry of Agriculture and Rural Affairs, Chengdu University (Fig. 1). This quinoa cultivar, meticulously developed by Chengdu University, is characterized by a high protein content. Specifically, the protein content of this variety is approximately 147.38 g/kg, while the starch content is approximately 563.36 mg/g.

Strains and culture conditions

C. albicans (SIIA 2284) was provided by the Department of Urology at the Affiliated Hospital of Chengdu University. The strain was initially cultured in Sabouraud Dextrose Broth (SDB) and subsequently activated by incubation for 18 h at 180 rpm and 37 °C.

Preparation of QAP

Quinoa seeds were initially screened to eliminate impurities, followed by drying and grinding into a fine powder. The resultant powder was then passed through a 100-mesh sieve and subsequently immersed in petroleum ether for defatting. Post-defatting, the quinoa flour was subjected to extraction at a 1:2 ratio using a 7 mM phosphate extraction buffer (pH 7.0, containing 10 mM NaCl and 1 mM EDTA) at 4 °C for 2 h. The extraction mixture was centrifuged at 4 °C and 10,000 rpm for 20 min, and the supernatant was collected as the crude QAP extract. The protein content of the crude QAP extract was quantified using the protein quantification assay kit according to the instructions provided by the Nanjing Jiancheng Bioengineering Institute. Solid ammonium sulfate was finely ground, and the crude extract of QAP was subjected to stepwise salting out using a 10% gradient. Following incubation at 4 °C for 40 min, the precipitates obtained from each fraction were dissolved in a minimal volume of phosphate extraction buffer (pH 7.0) containing 20 mmol/L. These solutions were subsequently dialyzed against a phosphate-buffered saline (PBS) buffer solution (20 mmol/L). Finally, the QAP solution was concentrated to a uniform volume, and the protein concentration was measured and adjusted as necessary. The inhibitory effects of QAP on C. albicans were evaluated using the Oxford cup-hole method. Under aseptic conditions, Oxford cups were placed into Petri dishes. Once the Sabouraud dextrose agar medium had cooled to approximately 50 °C, a suspension of C. albicans at a concentration of 3 × 10⁸ CFU/mL was added at a volume ratio of 1,000:1 and thoroughly mixed before the medium was poured into the Petri dishes. Following the solidification of the medium, the Oxford cups were meticulously removed using sterilized tweezers. Subsequently, 200 µL of QAP extracts, which were prepared under different ammonium sulfate saturation conditions, were added into the cavities left by the Oxford cups. The Petri dishes were then incubated at 37 °C for 24 h. Physiological saline served as the control group, while the clinical drug fluconazole (FLC) functioned as the positive control group. The criterion for assessing the antifungal efficacy was predicated on the external diameter of the Oxford cup. Specifically, the diameter of the inhibition zone was quantified using the cross-streaking technique, with the center of the indentation created by the Oxford cup serving as the reference point for measurement. A diameter exceeding 7.8 mm was considered indicative of a significant antifungal effect. Conversely, diameters below this threshold were deemed to demonstrate no antifungal activity, as the growth and reproduction of C. albicans were not effectively inhibited. In accordance with clinical pharmacological guidelines, the concentration of FLC was maintained at 160 µg/mL.

Quinoa bioactive proteins are separated and purified using gel chromatography

The preparation of the QAP involved utilizing Sephadex G-75 and Sephadex G-50 as packing materials within a chromatographic column with dimensions of 2.0 cm × 40 cm. An appropriate quantity of Sephadex G-75 powder was accurately weighed and transferred into a 500 mL beaker. Subsequently, deionized water or elution buffer, in a volume 7–10 times that of the Sephadex G-75, was added. The resulting (wet) gel was subjected to a digital constant temperature water bath set near boiling for 30 min. After this thermal treatment and cooling, the upper layer containing gel debris and impurities was carefully removed. Additional deionized water or elution buffer was added, and the mixture was equilibrated at ambient temperature for a duration of 2 h to ensure complete swelling. Subsequently, the supernatant was decanted from the swollen Sephadex G-75 gel, and a volume of phosphate extraction solution equivalent to 1–2 times the gel volume was added to form a suspension. This suspension was then gently agitated and transferred into the column using a glass rod to facilitate slurry packing. Initially, a small portion of the gel was permitted to settle within the column prior to opening the outlet valve. The slurry packing process continued was carried out by incrementally adding and uniformly distributing the gel until a settling height of approximately 35 cm was attained, at which point the outlet valve was closed. Following the formation of the gel column, the extraction solution was added into the elution bottle. The gel column was equilibrated by passing three column volumes of the extraction solution through the column at a flow rate of 1.3 mL/min. The procedure for handling Sephadex G-50 filler was identical to that employed for Sephadex G-75.

Protein mass spectrometry

The supernatants containing quinoa proteins, precipitated at varying levels of ammonium sulfate saturation, were analyzed using SDS-PAGE electrophoresis followed by silver staining. For protein identification, the QAP extracted under different ammonium sulfate saturation conditions, along with those purified via Sephadex G-75 and Sephadex G-50 gel filtration chromatography, were subjected to LC-MS/MS analysis (Triple ToF 5600+AB-SCIEX). The resulting peptide sequences were identified using ProteinPilot software. Subsequently, the proteins were classified via the InterProScan (IPR)-based domain annotation method.

Quantification of QAP’s minimum inhibitory concentration (MIC)

The QAP solution (with concentrations ranging from 728 µg/mL to 2.8 µg/mL) and the FLC solution (with concentrations from 256 µg/mL to 0.5 µg/mL) were prepared by serial two-fold dilution. According to the method of Xie et al. (2024), the antifungal activity of the QAP solution was determined using the Oxford cup punching method, consistent with the procedure detailed in the “Preparation of QAP” section. Using a sterile pipette, 200 µL of each concentration of the QAP and FLC solutions was aspirated and then added into the holes created by the Oxford cups to establish test wells. An equivalent volume of sterile physiological saline served as a blank control. The culture dishes were then sealed and incubated at a constant temperature of 37 °C for 24 h. Following incubation, observations were made regarding the areas surrounding the test wells in the culture dishes. The minimum inhibitory concentration (MIC) for the QAP solution was identified as the lowest concentration that resulted in an inhibition zone exceeding 7.8 mm in diameter. Similarly, the MIC for the FLC solution was determined as the lowest concentration that produced an inhibition zone with a diameter greater than 7.8 mm.

Growth kinetics of C. albicans under QAP

Under sterile conditions, 10 µL of C. albicans solution (SIIA 2284, 3 × 10⁸ CFU/mL) was extracted from the blank control group and transferred to a sterilized 96-well plate. Subsequently, 190 µL of RPMI-1640 complete medium was added and thoroughly mixed. For the QAP treatment group, 10 µL of C. albicans solution was obtained from a sterilized 96-well plate and combined with 100 µL of QAP solution (0.78 mg/mL) and 90 µL of RPMI-1640 complete medium. The mixture was meticulously blended before being incubated at 37 °C with agitation at 80 rpm for growth monitoring. The growth curve was constructed by measuring A600 every two hours using a Multifunctional Microplate Reader (Synergy HTX; BioTek, Winooski, VT, USA).

Determination of alkaline phosphatase (AKP) activity

To explore the inhibitory mechanism of QAP against C. albicans, we assessed the activity of alkaline phosphatase (AKP), a commonly utilized indicator for evaluating the cell wall damage or integrity (Song et al., 2018). The activated C. albicans suspension was centrifuged at 4,000 rpm for 2 min and the supernatant was removed. The resultant pellet was subsequently re-suspended in sterile phosphate-buffered saline (PBS) to attain a concentration of 3 × 10⁸ CFU/mL. Subsequently, 200 µL of the C. albicans suspension was added to a sterile conical flask containing 20 mL of SDB solution, followed by the addition of 20 mL of QAP for treatment. In the blank control group, an equal volume of SDB solution was added. The cultures were incubated at 37 °C and 180 rpm for 8 h. Post-incubation, the supernatant was collected by centrifugation at 10,000 rpm for 10 min. The extracellular AKP activity was then determined according to the instructions provided with the AKP assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Activity analysis of Succinate dehydrogenase (SDH), Ca²⁺-Mg²⁺- ATPase and Catalase (CAT)

The conditions for sample processing were maintained in accordance with AKP activity determination, whereas the control group was replaced with PBS. The activity of SDH, CAT and Ca²⁺-Mg²⁺-ATPase was determined by an Enzyme-linked Immunoassay Kit (Jiangsu Meimian industrial Co., Ltd.) according to the manufacturer’s protocol. The specific techniques employed for measurement are provided in Text S1.

C. albicans hyphal morphology assay

The nutrient-rich Spider medium, which utilizes mannitol as its carbon source, is commonly employed to facilitate morphogenesis (Véronique et al., 2024). For the current study, the Spider agar medium was utilized to evaluate the impact of QAP on the yeast-to-hyphal transition in C. albicans (Chu et al., 2025; Sun et al., 2023). C. albicans was cultured on the Spider agar medium with varying treatments: a negative control (0 mg/mL), a test group with 0.78 mg/mL QAP, and a positive control group with 160 µg/mL FLC. The cultures were incubated at 37 °C for 96 h. Following incubation, the morphological characteristics of the C. albicans colonies were documented.

Observation of cell morphology by scanning electron microscope (SEM)

Under aseptic conditions, activated C. albicans cells were centrifuged, and the C. albicans suspension was adjusted to a concentration of 3 × 10⁸ CFU/mL. Two sterilized 150 mL Erlenmeyer flasks were utilized: Flask 1 contained 40 mL of the C. albicans suspension and 20 mL of the QAP component solution at a concentration of 0.78 mg/mL; Flask 2 contained 40 mL of the C. albicans suspension and 20 mL of phosphate extraction liquid.

Following an 8-h incubation period at 37 °C with agitation at 180 rpm, the culture was aseptically transferred to sterilized centrifuge tubes and subjected to centrifugation at 4,000 rpm for 1 min. The resulting supernatant was carefully removed, and the fungal organisms were harvested. Subsequently, the fungal cells underwent three washes with pre-sterilized PBS to eliminate any residual contaminants. The washed cells were subsequently fixed in a 2.5% glutaraldehyde solution at 4 °C under light-excluded conditions for 2 h to ensure optimal preservation of cellular structures. Post-fixation, the samples were subjected to a graded ethanol dehydration series (30%, 70%, and 100%) for approximately 10 min each at room temperature. Following dehydration, the samples were air-dried overnight on sample holders secured with double-sided adhesive tape. Subsequently, gold sputter coating was performed utilizing an HVS-GB vacuum evaporator under meticulously controlled conditions. Following this, the samples were subjected to localization and imaging using a Sirion 200 scanning electron microscope.

Isolation of total RNA and preparation of cDNA libraries

Six 150 mL Erlenmeyer flasks with uniform specification, were prepared and labeled sequentially from 1 to 6. Each flask was filled with 50 mL of SDB and subjected to sterilization at 121 °C for 20 min, followed by cooling for subsequent experimental procedures. Under strictly aseptic conditions, a single colony of C. albicans was inoculated into each flask. The inoculated cultures were then incubated at 37 °C with agitation set at 180 rpm for 18 h. Following the initial incubation phase, Flask 1 was supplemented with 50 mL of QAP solution at a concentration of 0.45 mg/mL; Flasks 2 and 3 underwent the same treatment as Flask 1. Concurrently, Flask 4 received an addition of 50 mL of phosphate extraction liquid, with Flasks 5 and 6 being treated similarly to Flask 4. Subsequently, all six flasks were reincubated under consistent conditions (37 °C, 180 rpm) for an additional 8-h duration. Upon the conclusion of this secondary incubation phase, the contents of each flask were meticulously transferred into sterile centrifuge tubes and centrifuged at 4,000 rpm for 2 min to pellet the biomass. The resultant cell pellets were flash-frozen in liquid nitrogen and were promptly stored at −80 °C for subsequent analysis. The RNA was extracted from C. albicans cells following a standardized protocol and subjected to stringent quality control assessment of RNA integrity using the Agilent 2100 Bioanalyzer. The fragmented mRNA was used as a template for the synthesis first-strand cDNA, employing random oligonucleotides in the M-MuLV reverse transcriptase system. Subsequently, RNase H was utilized to degrade the RNA strand, facilitating the synthesis of second-strand cDNA using dNTPs in the DNA polymerase I system. Following terminal repair, adenylation, and the ligation of sequencing adaptors, the purified double-stranded cDNA was subjected to size selection (approximately 250–300 base pairs) using AMPure XP beads. The selected cDNA fragments were then amplified via polymerase chain reaction (PCR) to construct the library. The statistical power of this experimental design, as calculated using RNASeqPower, is 0.83.

Data analysis following illumina sequencing

Following a comprehensive library inspection, various libraries are categorized according to the prerequisites for optimal concentration and target data volume necessary for Illumina sequencing technology. Amplification is facilitated by the introduction of four fluorescently labeled deoxynucleotide triphosphates (dNTPs), DNA polymerase, and adapter primers into the flow cell. Each incorporation of a fluorescently labeled dNTP generates a corresponding fluorescence signal. This signal is subsequently captured and processed by computer software to produce sequencing peaks, thereby yielding the sequence information of the desired fragments. The acquired sequence data is subsequently subjected to a quality assessment protocol, wherein statistical analyses are conducted to identify discrepancies between the raw data and the quality-controlled processed data.

Following the completion of quality control, the high-quality sequences were aligned to the reference genome using HISAT2 software (Mortazavi et al., 2008). Subsequently, the mapped sequences were assembled to the genome utilizing StringTie software (Pertea et al., 2015). A comparative analysis with existing gene annotations was then conducted using GffCompare software to identify unannotated transcript regions and discover novel transcripts or genes within this species. To ensure accurate assessment of gene expression levels (Garber et al., 2011), FPKM values for each gene were calculated using StringTie (Bray et al., 2016). Subsequently, the Euclidean distance metric was employed to assess the correlations among samples for the purpose of hierarchical clustering. Following the clustering process, a heatmap was constructed, wherein the intensity of color denotes the variance in gene expression patterns between samples, with lighter shades indicating greater differences and darker shades representing smaller differences. The resulting dendrogram illustrates the degree of similarity among samples, with proximal branches signifying higher similarity, and samples exhibiting greater similarity tending to cluster more closely together.

Ultimately, the DESeq2 software was utilized to conduct a significance analysis of variations in gene expression. Typically, a gene demonstrating a fold change (FC) greater than two between two sample groups is considered to exhibit significant differential expression.

Quantitative real-time PCR

The expression levels of regulatory genes in C. albicans inhibited by QAP were quantitatively assessed using real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) technology. This procedure encompassed RNA extraction and purification, followed by reverse transcription into complementary DNA (cDNA) utilizing the FastKing RT Kit (With gDNase) FastKing cDNA, in accordance with the manufacturer’s protocol provided by TianGen Biotech (Beijing) Co., Ltd. The specific genes analyzed in this study, along with their corresponding primers, are enumerated in Table S1. Quantitative real-time PCR (qRT-PCR) assays were conducted in triplicate utilizing a qTOWER $\stackrel{ˆ}{}$ 3G Real-Time PCR system (Analytik Jena AG, Jena, Germany). The thermal cycling protocol comprised an initial denaturation step at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 30 s. The actin gene (ACT1) served as the reference gene, and relative gene expression levels were quantified using the $2{\stackrel{ˆ}{}}^{-\Delta \Delta \mathrm{CT}}$ method.

Statistical analysis

To elucidate the functionality of specific genes, an extensive search was conducted within the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases (Table S2). Subsequently, a comprehensive analysis of GO functional enrichment (Young et al., 2010) and KEGG pathway enrichment was performed on the set of DEGs using the clusterProfiler software (Table S3). Following the Z-score normalization of the expression levels of the identified DEGs, clustering analysis was undertaken to group genes exhibiting similar expression patterns. Ultimately, a clustering heatmap was generated using the Wekemo Bioincloud Platform (http://www.bioincloud.tech).

The data collected in this study were systematically organized using Excel 2016 software and subsequently visualized through GraphPad Prism 9 (version 9.5.0) and Cytoscape (version 3.9.1). Statistical comparisons between two independent samples were analyzed using the t-test, while differences among three or more independent samples were assessed via one-way ANOVA, supplemented by Duncan’s multiple range test. The significance threshold was established at P < 0.05. These statistical analyses were performed using the pertinent functions available in SPSS 27 software. The experiment was conducted in triplicate, and the results are presented as x ± n. Asterisks indicate statistically differences between control and treatment groups. The RNA-seq data have been deposited in the NCBI Sequence Read Archive (SRA) database (accession number: PRJNA1166727).

Isolation and purification of antifungal QAP

To elucidate the final saturation range of ammonium sulfate necessary for QAP precipitation to inhibit C. albicans, an investigation was conducted within the 0–30% ammonium sulfate saturation range. Although QAP demonstrated inhibitory effects against C. albicans within this range, there were no statistically significant differences in the diameters of the inhibition zones (p > 0.05). This suggests that the proteins precipitated under these conditions were likely contaminants (Fig. 2). As the ammonium sulfate saturation increased to the range of 30%–70%, the QAP precipitated within this range exhibited significant variations in inhibitory activity against C. albicans (Fig. 3). However, at a saturation level of 80%, the inhibitory effect markedly weakened, suggesting that the majority of QAP had been fully precipitated under these conditions. Proteomic analysis of protein precipitation at each saturation showed that there were 644, 716, 690, 647, 643 and 622 proteins in different ammonium sulfate saturation segments of 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70% and 70%–80%, respectively. The results showed that most of the active proteins of quinoa could be effectively separated with different saturation of ammonium sulfate, and there were about 149 common proteins in the 30%–80% ammonium sulfate salting phase (Fig. 4).

Analyses of QAP by SDS-PAGE at different saturation of ammonium sulfate

The supernatants containing QAP, precipitated at varying ammonium sulfate saturation levels, were analyzed using SDS-PAGE electrophoresis. As illustrated in Fig. 5, the molecular weights of quinoa proteins were observed to range from approximately 14.4 to 116.0 kDa. Notably, as the ammonium sulfate saturation increased, the proteins within the 18.4–25 kDa range in the supernatant showed a significant decrease. Meanwhile, the antifungal activity of the precipitated proteins exhibited a progressive enhancement. This suggests that the antifungal proteins are likely within this molecular weight range, closely aligning with the molecular mass (24 kDa) of the antimicrobial protein discovered by Wang et al. (2011) from the seed of Cynanchum komarovii Al Iljinski. When considered alongside the separation outcomes obtained from Sephadex G-75 chromatography, the relative abundance of proteins within the specified range was observed to increase. This observation further substantiates the hypothesis that the antimicrobial proteins are situated within the 18.4–25 kDa range, corroborating the results of previous proteomic analyses (Vilcacundo, Martínez-Villaluenga & Hernández-Ledesma, 2017). Furthermore, research conducted by Osman, Mahgoub & Sitohy (2013) demonstrated that the subunits of soybean 11S globulin exhibit antimicrobial activity. These findings indicate that ammonium sulfate fractionation serves as an effective technique for isolating QAP with inhibitory properties against C. albicans, while simultaneously distinguishing these compounds from other proteins.

Purification of QAP by dextran gel chromatography

QAP solutions, purified using Sephadex G-75 and Sephadex G-50 columns, were evaluated for their antifungal activity against C. albicans on Sabouraud dextrose agar medium. The QAP solution derived from Sephadex G-75 exhibited a concentration of 0.78 mg/mL, whereas the solution from Sephadex G-50 had a concentration of 0.4 mg/mL. In comparison to the results obtained from purification using a Sephadex G-75 column, the spectrum of active protein fractions in quinoa was further refined when purified with a Sephadex G-50 column (Fig. 6).

Proteomic analysis revealed that 18 proteins shared between the fractions obtained through Sephadex G-75 and Sephadex G-50 purification (Fig. 7). It was observed that, despite a significant decrease in the concentration of QAP, an increase in the diameter of the inhibition zones was observed between the two groups (p < 0.05). Moreover, when compared to the clinical drug FLC, the inhibitory zone diameter of QAP, purified by Sephadex G-50, was larger than that of FLC (Figs. 8A and 8B).

Determination of MIC of QAP

The MIC of QAP against C. albicans was determined to be 182 µg/mL (Table 1), with an associated antifungal zone diameter of 1.10 ± 0.04 cm. Meanwhile, for the positive control group, the MIC was found to be 16 µg/mL, with a corresponding antifungal zone diameter of 1.84 ± 0.16 cm (Table 2). The effect diagrams of antifungal activity are presented in Data S1.

QAP caused cell wall damage in C. albicans

As shown in Fig. 9, the extracellular AKP activity of C. albicans treated with 0.78 mg/mL of QAP was 0.98 ± 0.09 U/mL (p < 0.05), which is higher than that of the control group (0.62 ± 0.09 U/mL). Therefore, we concluded that QAP has the potential to induce cell wall damage in C. albicans.

QAP exhibits inhibitory effects on the growth and reproduction of C. albicans

As illustrated in Fig. 10, the initial five-hour period was characterized by relatively sluggish growth of C. albicans, with no significant difference in optical density (OD) observed between the two groups. Between 5 and 20 h, the OD of the control group increased rapidly, whereas the growth rate of the OD in the QAP group was markedly lower compared to the control group during this interval. By 30 h, both groups reached a stable state, with the OD of the control group recorded at 1.400 ± 0.008, while the OD of the QAP group was 0.621 ± 0.010. Throughout the duration of the study, the OD of the QAP group consistently remained lower than that of the control group.

Activity analysis of succinate dehydrogenase (SDH), Ca²⁺-Mg²⁺- ATPase and catalase (CAT)

When C. albicans was treated with QAP at concentrations of 0.78 mg/mL, 0.52 mg/mL, and 0.26 mg/mL for 8 h, the succinate dehydrogenase (SDH) activities were recorded as 334.4 ± 5.8 U/L, 341.8 ± 8.8 U/L, and 363.0 ± 6.5 U/L, respectively, while the corresponding calcium-magnesium ATPase (Ca²⁺-Mg²⁺-ATPase) activities were 92.84 ± 0.97 IU/L, 92.85 ± 2.08 IU/L, and 87.10 ± 1.20 IU/L. Concurrently, in the positive control group treated with fluconazole (FLC) at concentrations of 320 µg/mL, 160 µg/mL, and 80 µg/mL, the SDH activities were measured at 317.5 ± 8.9 U/L, 319.3 ± 5.0 U/L, and 345.0 ± 5.9 U/L, with Ca²⁺-Mg²⁺-ATPase activities of 75.21 ± 0.30 IU/L, 79.99 ± 0.61 IU/L, and 77.69 ± 1.31 IU/L. Compared to the blank control group, which exhibited an SDH activity of 407.8 ± 8.1 U/L and a Ca²⁺-Mg²⁺-ATPase activity of 98.56 ± 1.59 IU/L, both the QAP-treated and FLC-treated groups demonstrated statistically significant reductions in SDH and Ca²⁺-Mg²⁺-ATPase activities (P < 0.0001). Through comprehensive data analysis, it is evident that QAP affects the Ca²⁺-Mg²⁺-ATPase located on the cell membrane and the intracellular succinate dehydrogenase (SDH) of C. albicans, thereby disrupting intracellular homeostasis and energy metabolism. This disruption is hypothesized to result from modifications in enzymatic activities, which are crucial for maintaining the normal physiological state of the cell (Figs. 11A and 11B).

Moreover, an increase in the CAT activity of C. albicans was observed following treatment with varying concentrations of QAP and FLC. The CAT activity of the negative control group was measured at 14.03 ± 0.36 U/mL, whereas the CAT activities of the QAP-treated group at concentrations of 0.78 mg/mL, 0.52 mg/mL, and 0.26 mg/mL were 19.92 ± 0.44 U/mL, 17.42 ± 0.37 U/mL, and 18.79 ± 0.52 U/mL, respectively. Concurrently, the CAT activities of the FLC-treated group at concentrations of 320 µg/mL, 160 µg/mL, and 80 µg/mL were 17.34 ± 0.18 U/mL, 16.32 ± 0.22 U/mL, and 18.25 ± 0.41 U/mL, respectively, with these differences being statistically significant. This experimental data suggests that QAP has disrupted the normal metabolic activities of C. albicans, potentially affecting energy metabolism and membrane functions, as evidenced by alterations in SDH and Ca²⁺-Mg²⁺-ATPase activities. Consequently, this disruption results in increased intracellular oxidative stress, leading C. albicans to enhance its CAT activity as a compensatory response to mitigate the excessive reactive oxygen species generated due to the disrupted metabolic activities (Fig. 11C).

The effects of QAP on yeast-to-hyphal transition

The ability of C. albicans to transition from the yeast form to the hyphal form is considered one of its most crucial virulence factors. As depicted in Fig. 12A, the negative control group formed dense long hyphae when cultured on the Spider agar medium. However, upon treatment with either QAP (0.78 mg/mL) or FLC (160 µg/mL), the colonies formed by C. albicans cells displayed irregular edges, and produced only a limited number of hyphae (Figs. 12B and 12C). This could be attributed to the diffusion of QAP or FLC in the Spider agar medium (Yang et al., 2024) potentially disrupting the local concentration gradients of nutrients or signaling molecules essential for the normal hyphal growth of C. albicans, leading to the observed irregular colony edges and reduced hyphal production. These findings suggest that QAP effectively inhibits the transition of C. albicans from the yeast form to the hyphal form in Spider agar medium.

QAP affects C. albicans colony morphology

This study employed SEM to investigate the effects of QAP on the morphology of C. albicans. The results indicated that the C. albicans displayed plump and rough cell bodies undergoing division and growth in the control group. While C. albicans displayed smoother cell surfaces characterized by distinct cracks and a marked decrease in budding sites in the treated group with 0.78 mg/mL of QAP (Fig. 13).

Results of illumina sequencing and assembly

Following the construction and quality assessment of the cDNA library, various library combinations were subjected to Illumina sequencing according to the desired effective concentration and target data volume. Subsequently, the raw sequencing data for each sample were then subjected to quality control using the fastp software. Ultimately, 38.09 G clean reads were obtained, with a GC content of 36.77% and a Q30 value of 93.78% (Table 3). These metrics satisfy the requirements for transcriptome analysis.

In this study, the correlation among samples was evaluated utilizing Euclidean distance, and a hierarchical clustering heatmap was generated to analyze the gene expression level correlations across various samples. The clustering of three biological replicates within each experimental and control group revealed significant differences in expression profiles between the groups, while also demonstrating a high degree of correlation among replicates within each group (Fig. 14A). A comprehensive analysis revealed that 6,263 genes were expressed across all samples. Differential expression analysis was performed using the DESeq2 software package, employing stringent screening criteria of |log2(fold change)| > 1 and an adjusted p-value (p. adj) < 0.05. As a result, 1,413 genes exhibited significant differential expression, with 892 genes up-regulated and 521 genes down-regulated (Figs. 14B and 14C).

Unigene function annotation

A comprehensive annotated and classified analysis of differentially expressed genes (DEGs) was performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses to uncover the underlying biological mechanisms and pathways.

GO analysis revealed that, compared to the control group, the QAP-treated group exhibited significant enrichment in GO terms associated with the cell wall, cellular respiration, reactive oxygen species metabolism, ion transport, ergosterol biosynthesis, and transmembrane transport proteins (Fig. 14D). To further elucidate the molecular mechanisms underlying the inhibitory effects of QAP on C. albicans, these DEGs were further subjected to KEGG pathway annotation and enrichment analysis. The results revealed significant enrichment in pathways including DNA replication proteins, oxidative phosphorylation, the tricarboxylic acid (TCA) cycle, and glycosyltransferase (Fig. 14E). Subsequently, a heatmap was generated to illustrate differential gene expression based on the DEGs within the selected pathways (Fig. 14F).

Through transcriptome analysis, we identified DEGs within specific pathways for subsequent investigation of protein–protein interaction (PPI) networks using the STRING online platform (https://string-db.org/). The selected DEGs and their corresponding analysis results are listed in Table S4. Subsequently, Cytoscape software was utilized to perform topological analyses on the resulting PPI network graph, with node size, coloration, and intensity indicating their respective degree values. Our findings revealed that SDH2 (Succinate dehydrogenase 2) and ACO1 (Aconitase 1) are pivotal proteins (Fig. 15). These proteins jointly participate in the TCA cycle and play a crucial role in the energy metabolism of C. albicans. Additionally, qRT-PCR validation of the expression levels of 13 selected differentially expressed genes revealed consistent trends with our initial transcriptome analysis, and all the results exhibited statistically significant differences (P < 0.05). Specifically, the expression levels of genes ZRT1, SIM1, ALS4, and PRA1 showed a significant increase (P < 0.05) following the treatment with quinoa active proteins. Conversely, the expression levels of the remaining 11 genes showed a significant decrease (P < 0.05). Notably, the expression levels of FBA1, CST20, ERG1, and SDH2 showed an extremely significant decrease after being treated with QAP (P < 0.0001), with the expression level of SDH2 decreasing by more than threefold (Fig. 16).