Wild-type C. albicans strain SC5314 was provided by Professor Yuanying Jiang, stored at -80°C and activated in liquid yeast extract–peptone–dextrose (YPD) medium (Hope bio-Technology, China). Resuscitated SC5314 was inoculated to the solid YPD medium (Hope bio-Technology, China) by the plate scribing separation method and cultured at 37°C. Then, a selected single colony was amplified to the logarithmic phase of growth at 37°C, and 200 rpm overnight for subsequent experiments.

The RAW264.7 mouse macrophages acquired from the National Collection of Authenticated Cell Cultures were grown in Dulbecco’s Modified Eagle Medium (DMEM) medium (SparkJade, China) at 5% CO₂ and at 37 °C. The medium contained 10% fetal bovine serum (FBS) (Anhui Kangyuan Biotechnology, China) and was supplied with 1% penicillin and streptomycin (PS) (Biosharp, China).

Minimum inhibitory concentration (MIC) values of baicalin or fluconazole (both from Yuanye Biotechnology, China) against C. albicans were tested in microdilution plates at 37°C and examined visually after 24 h according to the recommendations in the Clinical and Laboratory Standards Institute (CLSI) M27-A3 document (Wang et al., 2015; Montoya et al., 2020). Both Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were used as quality control (QC) strains to confirm the accuracy and reproducibility in antifungal susceptibility testing (Guirao-Abad et al., 2018; Shields et al., 2018). Baicalin was serially diluted in 1.953–1000 μg/mL by Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, USA) and co-incubated with C. albicans SC5314 at a final concentration of 1 × 10³ CFU/mL. Fluconazole was tested at concentrations of 0.0625–8 μg/mL. The MIC endpoint was defined as the lowest concentration of the drug that results in a 50% reduction in fungal growth compared to the growth control (Holtappels et al., 2017; Shields et al., 2018; Brennan-Krohn et al., 2021).

Using the Cell Counting Kit-8 (CCK-8), the dehydrogenase activity within the mitochondria of C. albicans SC5314 was determined to characterize cell viability (Jin et al., 2021). Briefly, 5 × 10⁴ CFU/mL of C. albicans SC5314 cells were dispensed into 96-well plates at 100 μL/well. Subsequently, these cells were exposed to 100 μL of different baicalin concentrations at 37°C. This enables the investigation of the concentration-dependent effects of baicalin on the cells. Afterward, 20 μL of CCK-8 reagent (Biosharp, China) was introduced into each well, followed by a 30-min incubation at 37°C. Thereafter, a multi-well spectrophotometric plate reader was deployed to quantitatively determine the optical density (OD) at 450 nm.

To study the drug effect on of C. albicans growth, a slight modification was made according to a previously outlined protocol (Wang et al., 2018). Fungal cells in the logarithmic phase were resuspended by RPMI-1640 medium and 10-fold diluted to create five final concentrations of 2 × 10¹ – 2 × 10⁵ CFU/mL. The above concentrations of C. albicans SC5314 were co-incubated with multiple concentrations of baicalin or fluconazole in a ratio of 1:1 at 37°C. Fluconazole-treated group was used as the positive drug control group. After spotting 5 μL of the mixed culture solution onto YPD solid plates, the growth differences and clonogenic potential were observed following 24-h incubation at 37°C.

A time-kill curve was used to examine the killing kinetics of the baicalin or fluconazole against C. albicans. The experimental procedures were performed based on earlier described methodologies (Cantón et al., 2004; Elgammal et al., 2024), with appropriate modifications to suit our objectives. Logarithmic phase C. albicans cells were subjected to dilution in RPMI 1640 medium until reaching a final concentration of 2 × 10³ CFU/mL and then incubated with either baicalin or fluconazole at 37°C. Equal volumes of samples were extracted at the pre-determined time intervals, diluted, and seeded onto YPD agar plates to determine the number of viable cells.

The growth of C. albicans SC5314 at a final concentration of 2.5 × 10⁴ CFU/mL was investigated in RPMI 1640 medium, both in the presence and absence of baicalin or fluconazole, for 24 h at 37°C. The transmission electron microscopy (TEM) experiments were conducted based on the methodologies outlined in previous literature (Grigor’eva et al., 2020; Xie et al., 2022; Bing et al., 2024) with the incorporation of modest optimizations. The C. albicans samples were fixed with 2.5% glutaraldehyde and then by 1% osmium tetraoxide. Afterward, the samples were exposed to dehydration through an ethanol series and then embedded with an epon-araldite mixture. The embedded blocks were sliced into ultrathin sections of 70 nm in thickness, and then the slices were captured and supported on copper grids. A sequential staining process was then performed as follows, initially with 1% uranyl acetate, followed by Reynolds’ lead citrate (a mixture of lead nitrate and sodium citrate) for several minutes each. The TEM (Hitachi HT7700, Japan) was employed to capture images of stained sections.

The drug treatment procedure for C. albicans is outlined in Section 2.6. The cell surface staining was based on previous literature (Guirao-Abad et al., 2018; Wagner et al., 2023) and followed the manufacturer’s instructions. Both fluconazole- and caspofungin-treated groups were used as the positive drug control groups. For total chitin detection, the fungal cells collected by centrifugation were stained by 10 μg/mL of calcofluor white (Sigma-Aldrich, the United States) for 30 min with gentle shaking. The β-1,3-glucan monoclonal antibody (Biosupplies, Australia) and its corresponding Cyanine3-labeled secondary antibody (Abbkine, the United States) were utilized to evaluate exposed β-1,3-glucan. A confocal laser scanning microscope (Olympus FV3000, Japan) or flow cytometer (BD FACSCelesta™, the United States) was employed to examine the stained cells. Image J and FlowJo were used to analyze microscope photographs and flow cytometry data.

A 2.5 × 10⁴ CFU/mL of C. albicans strain SC5314 was co-cultured with 500 μg/mL baicalin in RPMI-1640 medium that contained 10% FBS. The control group was not supplemented with baicalin. There were three biological replicates in both the baicalin-treated and the control groups. Total RNA extraction was conducted through an RNA extraction kit (Majorbio, China) and the 5300 Bioanalyser (Agilent, the United States) was used for analyzing its quality. Only high-quality RNA samples meeting the criteria (amount > 1 μg, RIN ≥ 6.5, OD260/230 ≥ 2.0, OD260/280 = 1.8–2.2, and 28S: 18S ≥ 1.0) were utilized for RNA sequencing (RNA-seq) library construction. The library was generated following the Illumina^® Stranded mRNA Prep Ligation kit (Illumina, the United States). Using the Illumina NovaSeq 6000 high-throughput sequencer, the paired-end RNA-seq library sequencing was performed (PE library, 2 × 150 bp read length). The procedure for RNA extraction and sequencing is illustrated in Supplementary Figure S1 .

The fastp tool (Chen et al., 2018) was employed to conduct statistical evaluation and quality control on the raw paired-end reads. Then, the resulting clean reads were subjected to an individual mapping to the reference genome of C. albicans SC5314 (GCF_000182965.3) by HISAT2 (Kim et al., 2015) software to obtain mapped reads for subsequent analysis. The StringTie (Pertea et al., 2015) was applied to assemble the mapped reads for each sample.

Quantification and calculation of gene expression abundances were performed using RSEM (Li and Dewey, 2011) based on the approach of transcripts per million (TPM) reads. Using DESeq2 (Love et al., 2014) with parameters |log₂foldchange| > 1 alongside adjusted P < 0.05, differentially expressed genes (DEGs) were identified between the control and baicalin groups. Furthermore, utilizing the Database for Annotation, Visualization and Integrated Discovery (DAVID), DEGs can be categorized through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) annotations on the basis of the biological processes (BP) they participate in, the cellular components (CC) they constitute, and the molecular functions (MF) they perform. A workflow of transcriptomic analysis is shown in Supplementary Figure S1 .

Raw and processed data for transcriptome analysis was uploaded to the GEO database with accession number GSE249820.

The treatment procedures for C. albicans were the same as those outlined in Section 2.8, “RNA extraction and sequencing”, while the handling of RAW264.7 macrophages was detailed in Section 2.11, “Macrophage-mediated phagocytosis of C. albicans”. Following total RNA isolation from either C. albicans or RAW264.7 by yeast RNA kit (SparkJade, China) or FastPure cell/tissue total RNA isolation kit (Vazyme, China), respectively, we assessed RNA concentration and its quality through the NanoDrop™ One microvolume spectrophotometer (Thermo Fisher Scientific, the United States). Next, the extracted RNA was employed to synthesize complementary DNA (cDNA) via a cDNA synthesis kit (YEASEN, China). Tables 1 , 2 list sequence-specific primers designed for quantitative real-time PCR (qRT-PCR). The qRT-PCR amplification system was prepared according to the qPCR SYBR green master mix kit (YEASEN, China) and performed on the LightCycler^® 96 instrument (Roche, Switzerland). The qPCR cycling parameters were set as follows: denaturation at 95°C for 2 min, annealing at 60°C for 10 s, and extension at 60°C for 30 s, with a total of 40 cycles. The internal reference genes ACT1 (for C. albicans SC5314) and Actb (for Mus musculus) were detected for data normalization. Here, the 2^-ΔΔCt method was employed as a relative quantification strategy for data analysis. All the operations were performed in an RNase-free environment.

The RAW264.7 macrophages were seeded at 6 × 10⁵ cells/mL into 35 mm confocal dishes (NEST, China) and cultured overnight. A 2.5 × 10⁴ CFU/mL of C. albicans SC5314 was pretreated with or without 1000 μg/mL of baicalin at 37°C for 24 h. Subsequently, harvested C. albicans cells were co-incubated with RAW264.7 cells at a 3:1 multiplicity of infection (MOI; C. albicans: macrophage) in DMEM for 1 or 3 h. After incubation, the culture medium was eliminated, and fungus-macrophage co-cultures were gently rinsed with PBS three times. Nonphagocytosed C. albicans SC5314 cells were labeled with 10 μg/mL of calcofluor white (CFW), while phagocytosed C. albicans cells remained unstained. Afterward, excess CFW dye was washed away with PBS, followed by fixing the cells with 4% paraformaldehyde. The sample image was acquired by the Olympus FV3000 microscope imaging system. The formula described before was applied to calculate the phagocytic index (Tripathi et al., 2020). Total RNA was extracted from fungus-macrophage co-cultures, and the relative IL-1β/10 and TNF-α mRNA expression from macrophages was detected and calculated.

Statistical significance was conducted utilizing Student’s t-test (two-tailed), with each experiment being performed with three biological replicates. The data were statistically analyzed, and the corresponding figures were generated via GraphPad Prism Version 9.5.0.

The MIC of baicalin on C. albicans SC5314 yeast cell strain and fluconazole were 500 and 0.25 μg/mL, respectively. Figure 1A depicts that C. albicans SC5314 cell viability was 15% when treated with 500 μg/mL (MIC) and 21% when treated with 250 μg/mL (1/2 MIC). When the initial inoculation concentration of C. albicans SC5314 was less than or equal to 10² CFU/mL, the number of colonies formed on plates after baicalin treatment exhibited a marked decrease in contrast to the control group ( Figure 1B ). In the time-dependent killing curve, the killing activity of baicalin in the first 12 h was similar to that of the control. However, after 12 h, C. albicans SC5314 growth was significantly impeded by baicalin above 250 μg/mL ( Figure 1C ). The maintenance of CW integrity is a critical virulence determinant in C. albicans SC5314 pathogenicity (Lima et al., 2019). Upon exposing C. albicans SC5314 cells to baicalin, alterations in the cellular ultrastructure were imaged with TEM. The structural outline of the CW appeared blurred, with partial thinning observed, and in some instances, exhibited signs of breakage and disintegration ( Figure 1D ). Collectively, baicalin can suppress C. albicans SC5314 virulence by interfering with its CW integrity.

The mechanism underlying the baicalin-induced CW remodeling remains unclear at present. This study thoroughly examined the antifungal mechanism of baicalin by focusing on β-glucan exposure, which is a main PAMP in the CW. The microscopy examination revealed that the CW surface exhibited more staining for β-1,3-glucan and chitin in the baicalin-treated group compared to the control group ( Figure 2A ). Cell surface fluorescence was quantitatively assessed through flow cytometry by determining the mean fluorescence intensity (MFI). The results confirmed that the MFI of β-1,3-glucan was enhanced upon baicalin treatment ( Figure 2B ). The same as β-1,3-glucan, the MFI of CFW-labeled total chitin was increased with baicalin treatment ( Figure 2C ). This indicated that baicalin induces β-glucan unmasking and chitin deposition in C. albicans SC5314.

Triplicate samples from each of the baicalin-treated and control groups were subjected to RNA-seq using the Illumina NovaSeq 6000 high-throughput transcriptome sequencing platform. A total of 5768 transcripts were annotated in the sequencing samples from both groups, of which 5656 transcripts were shared between the two groups ( Figure 3A ). Out of the 842 identified DEGs, 496 were up-regulated and 346 were down-regulated in the baicalin-treated groups compared to the control group ( Figure 3B ). The GO annotation and functional enrichment analysis results showcased that DEGs were primarily enriched in CC, such as fungal CW and plasma membrane, as well as BP, including translation, cell adhesion, and iron ion transport ( Figure 3C ; Supplementary Table S1 ). The KEGG pathway enrichment analysis results manifested that DEGs exhibited a main enrichment in carbon metabolism, glycolysis, and peroxisome pathways ( Figure 3D ; Supplementary Table S2 ). To investigate the mechanism behind β-1,3-glucan exposure, GO annotation was employed to screen out 50 genes related to the CW organization or biogenesis in C. albicans ( Figure 3E ; Supplementary Table S3 ). Additionally, the 15 genes associated with signal transduction pathways (Cek1, Hog1, and Mkc1 pathways) within C. albicans that regulate β-1,3-glucan unmasking (Chen et al., 2022) were of particular interest ( Figure 3E ; Supplementary Table S3 ). The products encoded by genes, including MNN12, CHS3/4, PHR1/2, BGL2, GSC1, GSL1/2, ENG1, and GWT1, were involved in CW organization and might directly or indirectly regulate β-1,3-glucan unmasking ( Figure 3E ; Supplementary Table S3 ).

The mRNA expression of 15 genes in the three MAPK pathways governing β-1,3-glucan exposure was confirmed by qRT-PCR. After treatment with baicalin, the expression of most genes within these pathways was up-regulated ( Figures 4A–C ). Moreover, α-1,3-mannosyltransferase (encoded by MNN12) was down-regulated after baicalin treatment ( Figure 4D ). The loss of the mannan outer layer of the CW may cause the inner layer β-1,3-glucan to unmask. Chitin synthases, particularly those encoded by CHS3/4, exhibited increased expression levels ( Figure 4D ), which was consistent with the elevated expression of total chitin observed by laser scanning confocal microscopy ( Figure 2A ) and flow cytometry ( Figure 2C ).

In C. albicans SC5314, the genes GSC1 and GSL1/2 encode for β-1,3-glucan synthases. An evident GSC1 and GSL1 overexpression was detected, whereas GSL2 expression displayed a significant decrease ( Figure 4E ). This observed distinct gene expression pattern indicated a possible β-1,3-glucan exposure induction through corresponding synthesis pathways. Relative expressions of glucan 1,3-β-glucosidase (encoded by BGL2) and glucan endo-1,3-β-D-glucosidase 1 (encoded by ENG1) was down-regulated, which may increase exposure by reducing the degradation of β-1,3-glucan ( Figure 4E ). Inhibition of GPI biosynthesis caused the unmasking of C. albicans β-glucan layer (Huang et al., 2020; Chen et al., 2022). GPI-anchored wall transfer protein 1 (encoded by GWT1) was down-regulated ( Figure 4E ). The pH-responsive proteins 1/2 (encoded by PHR1/2, respectively) have 1,3-β-glucanosyltransferase activity, in which the PHR1 expression level decreased, whereas the PHR2 expression level increased ( Figure 4E ).

Aiming to investigate the host immune response against C. albicans pretreated with baicalin, RAW264.7 macrophages were exposed to C. albicans pretreated with baicalein or not. The phagocytosis of macrophages towards C. albicans cells, particularly those exhibiting increased β-1,3-glucan unmasking post-baicalin treatment ( Figure 2A ), exhibited a significant enhancement. This enhancement was quantified as a 6.8-fold increase after 1 h ( Figures 5A, B ) and an 8.3-fold increase after 3 h ( Figures 5C, D ) in the phagocytic index. Furthermore, infection of C. albicans SC5314 caused macrophages to secrete more cytokines. The C. albicans SC5314 pretreated with baicalin stimulated significantly higher pro-inflammatory cytokine (IL-1β and TNF-α) and anti-inflammatory cytokine (IL-10) levels from RAW264.7 in comparison to C. albicans SC5314 unpretreated with baicalin ( Figures 5E–G ).