Quercetin Inhibits the Proliferation and Aflatoxins Biosynthesis of Aspergillus flavus

In this work of quercetin’s anti-proliferation action on A. flavus, we revealed that quercetin can effectively hamper the proliferation of A. flavus in dose-effect and time-effect relationships. We tested whether quercetin induced apoptosis in A. flavus via various detection methods, such as phosphatidylserine externalization and Hoechst 33342 staining. The results showed that quercetin had no effect on phosphatidylserine externalization and cell nucleus in A. flavus. Simultaneously, quercetin reduced the levels of reactive oxygen species (ROS). For a better understanding of the molecular mechanism of the A. flavus response to quercetin, the RNA-Seq was used to explore the transcriptomic profiles of A. flavus. According to transcriptome sequencing data, quercetin inhibits the proliferation and aflatoxin biosynthesis by regulating the expression of development-related genes and aflatoxin production-related genes. These results will provide some theoretical basis for quercetin as an anti-mildew agent resource.


Introduction
Aspergillus flavus is a saprophytic filamentous fungus that produces aflatoxins (AF), which are mutagenic, teratogenic and carcinogenic toxins for humans and animals [1][2][3][4]. Currently, there is a large amount of natural products, synthetic compounds, and extracts from diverse organisms for inhibitors of A. flavus growth, and aflatoxin biosynthesis that were investigated for application in food and feed preservation due to their low impact on the environment and human health [5][6][7][8][9][10].
The addition of anti-mildew agent is one of the important measures to prevent mildew pollution. Natural anti-mildew agent is a more ideal choice. Quercetin (3,3 ,4 ,5,7-pentahydroxy-flavone) is a natural resource found in many plants, fruits and vegetables [11]. Due to its anti-oxidant [12], anti-inflammatory [13], anti-cancer [5], antiviral, antibacterial [11], and anti-proliferative activity [5,11] and so on, it has been chemically synthesized and commercially sold. Previous studies revealed that sucked out of the 96-well plate, centrifuged at 8000 rpm for 5 min, washed with quercetin, and then suspended the A. flavus with 0.9% normal saline. The washed A. flavus was then coated onto potato dextrose agar (PDA) plates, and the single colony was cultured for counting. All data were expressed as mean ± standard deviation (n = 3).

Morphological Changes of A. flavus
Spore (10 7 CFU/mL) were treated with quercetin (200 μg/mL). After 24 h, A. flavus cells were harvested. The morphological changes of A. flavus were observed with the aid of a microscope with a 100-fold oil mirror. The result is shown in Figure 2. Compared with the control group, the mycelia of A. flavus were significantly degraded in the quercetin treated group. sucked out of the 96-well plate, centrifuged at 8000 rpm for 5 min, washed with quercetin, and then suspended the A. flavus with 0.9% normal saline. The washed A. flavus was then coated onto potato dextrose agar (PDA) plates, and the single colony was cultured for counting. All data were expressed as mean ± standard deviation (n = 3).

Morphological Changes of A. flavus
Spore (10 7 CFU/mL) were treated with quercetin (200 µg/mL). After 24 h, A. flavus cells were harvested. The morphological changes of A. flavus were observed with the aid of a microscope with a 100-fold oil mirror. The result is shown in Figure 2. Compared with the control group, the mycelia of A. flavus were significantly degraded in the quercetin treated group.

Cell Apoptosis
We used annexin-V-FITC/propidium iodide (PI) double staining to differentiate intact cells from non-apoptotic cells (annexin-V negative and PI negative), early apoptotic cells (annexin-V positive and PI negative), late apoptotic cells (annexin-V positive and PI positive), and dead (necrotic) cells (PI positive) and to examine apoptosis more deeply [14]. As shown in Figure 3A, only the quercetintreated group produced dead (necrotic) cells. The observations suggested that A. flavus cells have died via necrosis but not through the apoptotic pathway. In addition, compared with the control group, quercetin did not cause changes in the nuclear integrity of A. flavus ( Figure 3B). Generation of ROS happens at the onset of apoptosis [5,15]. However, in our work, quercetin did not cause reactive oxygen species to rise, but caused reactive oxygen species to decrease ( Figure 3C). This further indicates that quercetin does not induce the death of A. flavus through apoptotic pathway. In conclusion, these results demonstrated that quercetin does not induce apoptosis in A. flavus.

RNA-Seq Data
The transcriptome of A. flavus was put together from scratch with paired-end raw reads brought forth by the Illumina HiSeq2500 instrument. After redundancy and short reads had been weeded out, the clean reads in the QT group and CK group were 50561156 and 51441686, respectively (Table S1). The Illumina guidelines were used to sequence data for every sample found to have Q30 as its quality score. The GC counts for the QT group and the CK group were 52.39% and 52.33%, respectively (Table  S1). Also, 45577031 (90.14%) and 46843066 (89.88%) clean reads that we got from the two groups effectively matched the value for the A. flavus genome. 89.42% of the reads were individually mapped to the genome for the QT group and 89.68% for CK group (Table S1) according to the statistics. Moreover, 0.46% and 0.46% of the reads were multiply mapped to the genome for the CK group and the QT group, respectively (Table S1). These results showed that the sequencing quality was suitable for the unigenes of subsequent annotation analysis.

Cell Apoptosis
We used annexin-V-FITC/propidium iodide (PI) double staining to differentiate intact cells from non-apoptotic cells (annexin-V negative and PI negative), early apoptotic cells (annexin-V positive and PI negative), late apoptotic cells (annexin-V positive and PI positive), and dead (necrotic) cells (PI positive) and to examine apoptosis more deeply [14]. As shown in Figure 3A, only the quercetin-treated group produced dead (necrotic) cells. The observations suggested that A. flavus cells have died via necrosis but not through the apoptotic pathway. In addition, compared with the control group, quercetin did not cause changes in the nuclear integrity of A. flavus ( Figure 3B). Generation of ROS happens at the onset of apoptosis [5,15]. However, in our work, quercetin did not cause reactive oxygen species to rise, but caused reactive oxygen species to decrease ( Figure 3C). This further indicates that quercetin does not induce the death of A. flavus through apoptotic pathway.
In conclusion, these results demonstrated that quercetin does not induce apoptosis in A. flavus.

RNA-Seq Data
The transcriptome of A. flavus was put together from scratch with paired-end raw reads brought forth by the Illumina HiSeq2500 instrument. After redundancy and short reads had been weeded out, the clean reads in the QT group and CK group were 50561156 and 51441686, respectively (Table  S1). The Illumina guidelines were used to sequence data for every sample found to have Q30 as its quality score. The GC counts for the QT group and the CK group were 52.39% and 52.33%, respectively (Table S1). Also, 45577031 (90.14%) and 46843066 (89.88%) clean reads that we got from the two groups effectively matched the value for the A. flavus genome. 89.42% of the reads were individually mapped to the genome for the QT group and 89.68% for CK group (Table S1) according to the statistics. Moreover, 0.46% and 0.46% of the reads were multiply mapped to the genome for the CK group and the QT group, respectively (Table S1). These results showed that the sequencing quality was suitable for the unigenes of subsequent annotation analysis.

Identification and Functional Annotation
From the FPKM (Reads Per Kilobase of exon model per Million mapped reads) values, we identified 665 differentially expressed genes (log2[fold change] = log2[QT/CK] > 1, Probability > 0.8) between the QT and CK groups. Of these, 340 genes up-regulated and 325 genes down-regulated following exposure to quercetin (Table S2). We carried out a GO functional enrichment analysis of these differently expressed genes. The results demonstrated that these genes played a role in structural constituent of ribosome, structural molecule activity, electron carrier activity, rRNA binding, cis-trans isomerase activity, translation, cellular protein metabolic process, protein metabolic process, cellular biosynthetic process, biosynthetic process, organic substance biosynthetic process, cellular metabolic process, cellular macromolecule biosynthetic process, gene expression, macromolecule biosynthetic process, organic substance metabolic process, primary metabolic process, cellular macromolecule metabolic process, cellular process, organonitrogen compound biosynthetic process, purine nucleoside triphosphate biosynthetic process, purine ribonucleoside triphosphate biosynthetic process, ribosome, ribonucleoprotein complex, non-membrane-bounded organelle, intracellular non-membrane-bounded organelle, cytoplasmic part, macromolecular complex, cytoplasm, cell, cell part, intracellular part, intracellular, organelle, intracellular organelle, ribosomal subunit, small ribosomal subunit, or proton-transporting ATP synthase complex (Table S3). KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic pathway enrichment analysis shown that these genes were primarily involved in the ribosome, Oxidative phosphorylation, Huntington's disease, Parkinson's disease and Alzheimmer's disease (Table S3). Our analysis of KEGG metabolic pathway enrichment showed that these genes played a role in the ribosome, Huntington's disease, Oxidative phosphorylation, Parkinson's disease and Alzheimer's disease (Table S4).

Expression Analysis of Conidial Development-and A. flavus Growth-Related Genes in Response to Quercetin
To elucidate the effects of quercetin on the regulation of conidia and mycelia, based on the differentially expression genes (Table S2) of A. flavus in the CK and QT groups, we found that some genes that played a role in conidial and mycelial development were down-regulated when quercetin was used (Table 1), including sexual development transcription factor NsdD (AFLA_020210), sexual development transcription factor SteA (AFLA_048650), G protein complex alpha subunit GpaB (AFLA_018540), APSES transcription factor StuA (AFLA_046990), conidiation-specific protein Con-10 (AFLA_083110) and conidiation-specific family protein (AFLA_044790).

Expression Analysis of A. flavus AF Biosynthesis-Related Genes in Response to Quercetin
To elucidate the effects of quercetin on the regulation of aflatoxin biosynthesis, based on the analysis of differentially expressed genes data of A. flavus in the CK and QT groups (Table S2), the transcription regulator gene aflS (AFLA_139340) was significantly down-regulated. Further, aflS gene was validated by real-time RT-PCR analysis. The data confirmed the significant down-regulated of gene aflS (Figure 4), which was consistent with transcriptome data (Table S2).

Expression Analysis of A. flavus AF Biosynthesis-Related Genes in Response to Quercetin
To elucidate the effects of quercetin on the regulation of aflatoxin biosynthesis, based on the analysis of differentially expressed genes data of A. flavus in the CK and QT groups (Table S2), the transcription regulator gene aflS (AFLA_139340) was significantly down-regulated. Further, aflS gene was validated by real-time RT-PCR analysis. The data confirmed the significant down-regulated of gene aflS (Figure 4), which was consistent with transcriptome data (Table S2).

Figure 4.
Relative lives of aflS mRNA from A. flavus exposed to quercetin for 24 h. The expression of aflS was quantified by SYBR quantitative polymerase chain reaction (qPCR) assay. A. flavus cells untreated with quercetin were used as the control. Data are presented with mean ± standard deviation (n = 5). ** p < 0.01, compared with the control group.

Discussion
Quercetin is one of the natural flavonoids that play a crucial role in antibacterial activity [11]. Studies of flavonoid molecules of structure activity relationship have demonstrated that the oxygen atoms at position 4 in the C ring and the hydroxyl at positions 5 and 7 in the A ring constitute the primary group of antibacterial activity; next to them is the hydroxyl at position 3 in the C ring for antibacterial activity of such compounds. The hydroxyl at positions 3′ and 4′ in the B ring also shows some antibacterial activity [11]. Quercetin includes the oxygen atoms at position 4 in the C ring and the hydroxyl at position 5, 7, 3, 3′ and 4′. Previous studies have shown that quercetin can inhibit the proliferation of A. flavus [6]. However, in our work, we found that quercetin not only inhibited the growth of A. flavus, but also killed A. flavus, with a minimum inhibitory concentration of 505 μg/mL, and a minimum fungicidal concentration of 505 μg/mL ( Figure 1). In addition, quercetin inhibits the growth of A. flavus in a dose-effect and time-effect relationship ( Figure 1C).
Apoptosis is a kind of physiological programmed cell death and is different from necrosis [18]. One important mechanism referred to the function of antifungal drugs is the activation of the apoptotic pathway [7,19,20]. Antifungal agents trigger morphological features characteristic of apoptosis including PS externalization, nuclear condensation and ROS generation and so on, when they induce apoptosis on fungi [7,[18][19][20]. However, in our works, the results of Annexin V-FITC/PI staining shown only the quercetin-treated group produced dead (necrotic) cells ( Figure 3A). Subsequently, the morphological features characteristics of nuclear condensation was observed, we found that compared with the control group, quercetin did not cause changes in the nuclear integrity of A. flavus ( Figure 3B). In addition, generation of ROS happens at the onset of apoptosis [5,15], in our work, quercetin did not cause reactive oxygen species to rise, but caused reactive oxygen species to decrease ( Figure 3C). This result is contrary to the PS externalization morphological features characteristics of apoptosis. From the above, we concluded that quercetin might not induce apoptosis in A. flavus. How does quercetin inhibit the growth of A. flavus? We used transcriptome sequencing to reveal its possible mechanism. In our work, the mechanism by which quercetin inhibits A. flavus proliferation and aflatoxin biosynthesis was investigated adopting an RNA-seq analysis.
Based on our transcriptome data, we found that some genes that played a role in conidial and mycelial development were down-regulated when quercetin was used (Table 1), including sexual

Discussion
Quercetin is one of the natural flavonoids that play a crucial role in antibacterial activity [11]. Studies of flavonoid molecules of structure activity relationship have demonstrated that the oxygen atoms at position 4 in the C ring and the hydroxyl at positions 5 and 7 in the A ring constitute the primary group of antibacterial activity; next to them is the hydroxyl at position 3 in the C ring for antibacterial activity of such compounds. The hydroxyl at positions 3 and 4 in the B ring also shows some antibacterial activity [11]. Quercetin includes the oxygen atoms at position 4 in the C ring and the hydroxyl at position 5, 7, 3, 3 and 4 . Previous studies have shown that quercetin can inhibit the proliferation of A. flavus [6]. However, in our work, we found that quercetin not only inhibited the growth of A. flavus, but also killed A. flavus, with a minimum inhibitory concentration of 505 µg/mL, and a minimum fungicidal concentration of 505 µg/mL (Figure 1). In addition, quercetin inhibits the growth of A. flavus in a dose-effect and time-effect relationship ( Figure 1C).
Apoptosis is a kind of physiological programmed cell death and is different from necrosis [18]. One important mechanism referred to the function of antifungal drugs is the activation of the apoptotic pathway [7,19,20]. Antifungal agents trigger morphological features characteristic of apoptosis including PS externalization, nuclear condensation and ROS generation and so on, when they induce apoptosis on fungi [7,[18][19][20]. However, in our works, the results of Annexin V-FITC/PI staining shown only the quercetin-treated group produced dead (necrotic) cells ( Figure 3A). Subsequently, the morphological features characteristics of nuclear condensation was observed, we found that compared with the control group, quercetin did not cause changes in the nuclear integrity of A. flavus ( Figure 3B). In addition, generation of ROS happens at the onset of apoptosis [5,15], in our work, quercetin did not cause reactive oxygen species to rise, but caused reactive oxygen species to decrease ( Figure 3C). This result is contrary to the PS externalization morphological features characteristics of apoptosis. From the above, we concluded that quercetin might not induce apoptosis in A. flavus. How does quercetin inhibit the growth of A. flavus? We used transcriptome sequencing to reveal its possible mechanism. In our work, the mechanism by which quercetin inhibits A. flavus proliferation and aflatoxin biosynthesis was investigated adopting an RNA-seq analysis.
Based on our transcriptome data, we found that some genes that played a role in conidial and mycelial development were down-regulated when quercetin was used (Table 1), including sexual development transcription factor NsdD (AFLA_020210), sexual development transcription factor SteA (AFLA_048650), G protein complex alpha subunit GpaB (AFLA_018540), APSES transcription factor StuA (AFLA_046990), conidiation-specific protein Con-10 (AFLA_083110) and conidiation-specific family protein (AFLA_044790). When the development of A. flavus is inhibited, the sexual development transcription factor NsdD (AFLA_020210) [9] and SteA (AFLA_048650) were significantly down-regulated. Concurrently, transcriptions of conidia-specific genes, such as conidiation-specific family protein (AFLA_044790) and Con-10 (AFLA_083110) were significantly down-regulated [21]. The APSES transcription factor StuA that affects the orderly differentiation and spatial organization of cell types of the conidiospore [8] is encoded by transcription of the stuA gene (AFLA_046990), and the G protein complex alpha subunit GpaB (AFLA_018540) was significantly decreased. During aflatoxin biosynthesis, AflR is essential for expression of most of the genes in the aflatoxin genes cluster [6], which AflS (AFLA_139340) was reported to interact with activating AflR to give play to its regulatory effect [22]. As is known to all that fungal growth was closely related to biosynthesis of secondary metabolism [2,23]. APSES transcription factor StuA to be required for fungal conidial and mycelium growth [8,24]. Down-regulation of APSES transcription factor StuA inhibited the aflatoxin biosynthesis [24]. In our works, the transcription regulator genes aflS were significantly down-regulated (Table S2 and Figure 4). In addition, the redox state in the mycelia of A. flavus has been proved to be closely related to aflatoxin production [6]. Quercetin reduced the ROS level in the A. flavus ( Figure 3C). So, quercetin may reduce the production of aflatoxin by lowering levels of ROS.

Reagents
The quercetin (purity > 98.0%) was bought from the National Institutes for Food and Drug Control (Beijing, China). Muse®Oxidative Stress Assay Kit was bought from Merckmillipore (Billerica, MA, USA). Hoechst 33342 and Annexin V-FITC Kit were bought from Beyotime (Shanghai, China).

Fungus Strain and Cultivation
A. flavus (CGMCC3.6434) was bought from the China General Microbiological Culture Collection Center (CGMCC, Beijing, China). The A. flavus was cultured at 28 • C in a potato dextrose agar (PDA) and preserved in a refrigerator at 4 • C.

Measurement of Reactive Oxygen Species
Spores (10 7 CFU/mL) were treated with quercetin (200 µg/mL). After 24 h, A. flavus cells were harvested. The A. flavus cell walls were digested with 1.5% nailase (Solarbio, Beijing, China) and 1.5% Lyticase (Sigma, St Louis, MO, USA) and 1.5% cellulase (Onozuka, Tokyo, Japan) at 30 • C on a rotary shaker (80 rpm) for 3 h. They were washed twice in PBS and filtered through five layers of sterile lens paper to eliminate mycelial debris; then the protoplasts were obtained. According to the recommendations of the manufacturer, prepare cell samples in 1× assay buffer at 1 × 10 6 CFU/mL, and then 10 µL of prepared cells were add to 190 µL of oxidative stress working solution. Incubate at 37 • C for 30 min. the cells were analyzed using Muse®Cell Analyzer (Merck, MA, USA).

cDNA Preparation and Illumina Sequencing
Construction of library and RNA-Seq were performed at Realbio Technology (Shanghai, China). Total RNA from quercetin-untreated (CK) and quercetin-treated (QT) groups was isolated with TRIzol Reagent (Invitrogen, Shanghai, China) following the recommendations of the manufacturer. The integrity and total concentration of RNA were assessed with a NanoDrop (Implen, Westlake Village, CA, USA), a Qubit®Fluorometer 2.0, and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) instruments. The mRNA was separated with the use of oligo (dT)-attached magnetic beads. The separated mRNA and the fragmentation buffer were mixed and cut into tiny fragments using divalent cations under high temperatures. The cDNA was synthesized with these cleaved RNA fragments as templates. Afterward, the short fragments and the adapters were connected. The fragments found suitable were picked as templates for the amplification of PCR. During the QC steps, Agilent 2100 Bioanaylzer (Agilent Technologies, Santa Clara, CA, USA) and ABI StepOnePlus Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) were exploited for the qualification and quantification of the sample library. Lastly, the library was carried out with an Illumina HiSeq 2500 (Illumina, San Diego, CA, USA).

RNA-Seq and Enrichment Analysis of Differentially Expressed Genes
Raw data (raw reads) based on fastq format were initially processed with the use of in-house perl scripts. Clean data (clean reads) were procured by eliminating reads containing adapter and poly-N as well as reads of low quality from the raw data. The Q20, Q30, GC content, as well as level of sequence duplication of the clean data, were calculated. Analysis of downstream used clean data with high quality. Sequenced clean reads were mapped against predicted transcripts of the A. flavus NRRL 3357 genome1 (http://www.ncbi.nlm.nih.gov/genome/?term=aspergil-lus+flavus) using TopHat V2.1.1 and Bowtie v2.2.5 [40], and only unique matches were allowed. The FPKM (Fragments Per Kb of exon per Million reads) method was used to calculate and normalize the expression levels of the gene [41]. The genes expressed differentially were analyzed with the R edge R package V3.6.2 [35], and both a twofold change cut-off and an adjusted p-value of ≤0.05 were put in place as thresholds. Enrichment analysis of differential expression was carried out with the use of the GO-TermFinder v0.86 [42]. GO terms (including molecular function, cellular component, and biological process) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were recognized as well enriched among genes expressed differentially when their p-values were ≤0.05.

Validation of RNA-Seq Analysis by Quantitative Real-Time PCR (qRT-PCR)
The totality of RNA was then separated by use of Trizol reagent (Invitrogen, Carlsbad, CA, USA). Briefly, the qRT-PCR conditions were thus: 95 • C for 10 min and 40 cycles of 95 • C for 15 s and 60 • C for 60 s. The fold or percentage of change in the relative expression of the mRNA of the target gene was assessed by the 2 −∆∆Ct approach. The gene-specific primers are listed in Table S5.

Statistical Analysis
Data were expressed as mean ± standard deviation. Statistical analysis was carried with a one-way analysis of variance test for multiple comparisons. Differences between comparisons were deemed statistically significant at p < 0.05. SPSS software version 17.0 (SPSS Inc., Chicago, IL, USA) was deployed for analysis of data.