The Inhibitory Effect of Validamycin A on Aspergillus flavus

Aspergillus flavus is one of the most common isolates from patients with fungal infections. Aspergillus infection is usually treated with antifungal agents, but side effects of these agents are common. Trehalase is an essential enzyme involved in fungal metabolism, and the trehalase inhibitor, validamycin A, has been used to prevent fungal infections in agricultural products. In this study, we observed that validamycin A significantly increased trehalose levels in A. flavus conidia and delayed germination, including decreased fungal adherence. In addition, validamycin A and amphotericin B showed a combinatorial effect on A. flavus ATCC204304 and clinical isolates with high minimum inhibitory concentrations (MICs) of amphotericin B using checkerboard assays. We observed that validamycin A and amphotericin B had a synergistic effect on A. flavus strains resistant to amphotericin B. The MICs in the combination of validamycin A and amphotericin B were at 0.125 μg/mL and 2 μg/mL, respectively. The FICI of validamycin A and amphotericin B of these clinical isolates was about 0.25–0.28 with synergistic effects. No drug cytotoxicity was observed in human bronchial epithelial cells treated with validamycin A using LDH-cytotoxicity assays. In conclusion, this study demonstrated that validamycin A inhibited the growth of A. flavus and delayed conidial germination. Furthermore, the combined effect of validamycin A with amphotericin B increased A. flavus killing, without significant cytotoxicity to human bronchial epithelial cells. We propose that validamycin A could potentially be used in vivo as an alternative treatment for A. flavus infections.


Introduction
Aspergillus flavus is a fungus commonly found in the environment, and when it contaminates food, it produces aflatoxins, which are associated with increased risk of developing liver cancer in humans [1,2]. Moreover, A. flavus is an infectious fungus and can colonize organs leading to conditions such as keratitis, cutaneous infections, sinusitis, and invasive pulmonary aspergillosis [3][4][5]. Knowledge and understanding of the epidemiology and pathogenesis of A. flavus infection in humans are still very limited as there are only a few reports on A. flavus in comparison to other effects (e.g., nephrotoxicity) [9]. Owing to socioeconomic status of patients and availability of this agent, the use of amphotericin B as a treatment against aspergillosis is very common in developing countries, including ailand [10][11][12]. Unfortunately, recent studies have demonstrated increasing incidence of A. flavus clinical isolates with resistance to amphotericin B [13,14].
Although patients with aspergillosis are treated with standard antifungal therapy as mentioned, evidence shows that the morbidity and mortality rates in patients with these infections are still high (up to 80%) [15]. erefore, the discovery of novel antifungal agents with fewer side effects is crucial for treatment of aspergillosis. Many studies have reported virulence factors and metabolic pathways that are specific to this fungus, and these could potentially be new targets for the development of antifungal agents [16,17]. For example, trehalose is a disaccharide that is only found in bacteria, plants, insects, and invertebrates. It is composed of two glucose molecules conjugated with α, α-1, 1-glycosidic linkage, and serves as an energy source, particularly when fungi are exposed to environmental stresses such as cold, heat, and desiccation [18][19][20].
In previous studies, it was demonstrated that Rhizoctonia solani, a rice fungal pathogen, was inhibited by the trehalase inhibitor, validamycin A [33][34][35]. Validamycin A was originally isolated from Streptomyces hygroscopicus var. limoneus [33,36,37], and it was shown that it inhibited branching of R. solani [33,38]. Another study found that validamycin A delayed conidial production of Fusarium culmorum [38]. However, the effectiveness of validamycin A against human fungal pathogens and its toxicity on human cells are unknown. Here, we investigated the effects of validamycin A alone and in combination with amphotericin B on the growth of A. flavus, including the cytotoxicity of validamycin A to a human cell line. Oxoid, ermo Fisher Scientific) Petri-dish plates at 37°C for three days before harvesting A. flavus conidia using sterile distilled water with 0.01% Tween 80. In brief, 5 mL of sterile distilled water with 0.01% Tween 80 was utilized to harvest A. flavus conidia on SDA Petri-dish plates using cell scrapers. e mixture between distilled water with Tween 80 and A. flavus conidia was filtered using Miracloth. A number of conidia were counted from the filtrate using a hemocytometer. en, 10 3 conidia were inoculated into culture media [39], i.e., glucose peptone agar (peptone 10 g, glucose 20 g, agar 20 g, distilled water 1000 ml, and pH 6.8-7.0), trehalose peptone agar (peptone 10 g, trehalose 10 g, agar 20 g, distilled water 1000 ml, and pH 6.8-7.0), and peptone agar (peptone 10 g, agar 20 g, distilled water 1000 ml, and pH 6.8-7.0), incubated at 37°C for 2-5 days. e radial fungal growth was measured in three biological replicates.

Materials and Methods
A. flavus clinical isolates were obtained from the Mycology Laboratory, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, and King Chulalongkorn Memorial Hospital during 2019. Patient characteristics were collected from medical records/charts. Patients with invasive aspergillosis (IA) were classified as proven, probable, and possible invasive aspergillosis according to EORTC/MSG criteria [40,41].

Trehalose Measurements.
Conidia of A. flavus ATCC 204304 from SDA treated with or without 1 μg/mL validamycin A were collected at day 5 after incubation at 37°C. Trehalose levels of A. flavus conidia were measured, as previously described [42]. In brief, 2 × 10 8 conidia in 500 uL distilled water with Tween 80 were boiled at 100 o C for 20 min and centrifuged at 11,000 ×g for 10 min. e supernatant was collected for trehalose measurement (with biological triplicates) using the glucose oxidase assay protocol (Sigma; GAGO20). e reaction was measured at 490 nm using a spectrophotometer (Lambda 1050+ UV/Vis/ NIR, PerkinElmer, USA).

Germination Assay.
Conidia of A. flavus ATCC 204304 at 1 × 10 8 cells were incubated in 10 mL Sabouraud dextrose broth at 37°C in an orbital shaker at 200 rpm. e cultured broth (500 μL) was used for counting percentage of germlings. e germinated conidia are counted using a microscope. At each time point, 100 conidia were counted, and the number of germinated conidia was calculated as a percentage out of total 100 conidia [43]. Each strain was cultured up to 24 h at 37°C in three biological replicates [44].

XTT Assay.
XTT assays (sodium 2,3-bis (2-methoxy-4nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium) were performed as described previously [45,46]. In brief, 10 3 conidia of A. flavus ATCC 204304 were incubated with different culture media with or without validamycin A in a 96-well plate at 37°C for 18 h. XTT solution (0.5 mg/mL in PBS) was added into each well and incubated at 37°C for 15 min. e plate was centrifuged, and the supernatant was collected to measure the OD at 490 nm using a  [48]. e additive/synergistic effect of validamycin A and amphotericin B was identified using the checkerboard assays [49]. Fractional inhibitory concentration index (FICI) was calculated for each antifungal drug, in each combination used, with the following formula [49]: FICI results were determined as follows: synergy: <0.5; additivity: 0.5-1; indifference: >1-4; and antagonism: >4. precoated 96-well plate, and then validamycin A was added at different concentrations (1 μg/mL-1 mg/mL, final concentration). LDH reaction mixture was added, and the cells were incubated at 37°C for 30 min. LDH released from the cells was measured at 450 nm using a spectrophotometer. e percentage of cytotoxicity was calculated using the following formula: High control is cells with lysis buffer, while low control is cells alone as a background.

Statistical Analysis.
All statistical analyses were conducted with Prism 8 software (GraphPad Software, Inc., San Diego, CA). Comparison between groups was performed with unpaired two-tailed Student's t-tests for two data groups and one-way ANOVA tests with post hoc Bonferroni's multiple comparison tests for more than two data groups. Error bars represent standard errors of the means. Significant differences were considered when P value < 0.05.

Ethical Statement.
is study was approved by the Institutional Review Board (IRB no. 546/60), Faculty of Medicine, Chulalongkorn University, Bangkok, ailand. e results showed that AFLA_090490 protein, containing one signal peptide at positions 1-18 and two O-glycosyl hydrolase domains (EC 3.2.1) at positions 70-339 and 407-638, was similar to acid trehalase of S. cerevisiae and A. fumigatus (Figure 1(a)). AFLA_052430 protein, containing a neutral trehalase calcium-binding domain at positions 105-134 and an O-glycosyl hydrolase domain (EC 3.2.1) at positions 162-725, was similar to neutral trehalase of S. cerevisiae and A. fumigatus (Figure 1(b)). Our findings suggest that A. flavus has both acid and neutral trehalases, as seen in S. cerevisiae and A. fumigatus.

Trehalase Homologs in
Next, we investigated the ability of A. flavus to utilize trehalose as a sole carbon source. e result showed that growth and viability of A. flavus on glucose peptone media and trehalose peptone media were similar (Figures 2(a) and 2(b)). is finding supports the idea that A. flavus utilizes trehalose as a sole carbon source and implies that it degrades extracellular trehalose into glucose for its growth.  Figure 2: Aspergillus flavus utilizes trehalose as a sole carbon source similar to glucose. (a) Aspergillus flavus ATCC 204304 was incubated at 37°C on glucose peptone, trehalose peptone, and peptone alone media. e radial growth of these fungal growths was measured on the second day of incubation. Data are presented as means ± SE from three biological replicates. * P value < 0.05; * * P value < 0.01 (one-way ANOVA with post hoc Bonferroni's test). (b) Aspergillus flavus ATCC 204304 was incubated at 37°C on glucose peptone, trehalose peptone, and peptone alone liquid media for 24 hours, and viability tests using XTT assays were performed. Data are presented as means ± SE from three biological replicates. * P value < 0.05 (one-way ANOVA with post hoc Bonferroni's test).

Growth Inhibition and Decreased Fungal Adherence of Aspergillus flavus by Validamycin A.
To observe the inhibitory effect of validamycin A on A. flavus ATCC204304, broth microdilution and XTT assays were performed. e results showed that the minimal inhibition concentration (MIC) of validamycin A against A. flavus was 1 μg/mL (Table 1), and the viability of A. flavus ATCC204304 after validamycin A treatment at this concentration was significantly decreased when compared with 0.5 μg/mL of validamycin A, 0.25 μg/mL of amphotericin B, and the control group ( Figure 3).
Next, A. flavus ATCC204304 was cultured and treated with or without 0.5 and 1 μg/mL of validamycin A, and trehalose levels in the conidia were measured. e results demonstrated that conidia collected from A. flavus treated with validamycin A showed significantly higher levels of trehalose than the control (untreated) group, suggesting that validamycin A inhibited trehalase enzymes in the conidia of A. flavus (Figure 4(a)). In addition, the rate of conidial germination was investigated in A. flavus conidia treated with 1 μg/mL of validamycin A. e results showed that validamycin A significantly delayed conidial germination of A. flavus ATCC204304 particularly at 10 and 12 h (Figure 4(b)). ese data suggest that validamycin A delays conidial germination of A. flavus via inhibition of trehalase enzymes.
To observe the effect of validamycin A on exopolysaccharides of A. flavus, the crystal violet adherence assays were performed. We observed that 1 μg/mL of validamycin A decreased the adherence property of A. flavus ATCC204304 (Figure 4(c)).
ese data suggest that validamycin A affects the fungal adherence of A. flavus. e results demonstrated that 0.125, 0.5, and 1 μg/mL of validamycin A, 1 and 2 μg/mL of amphotericin B, and a combination of these two drug concentrations of 0.125 μg/mL of validamycin A and 2 μg/mL of amphotericin B showed no significant cytotoxicity to human bronchial epithelial cells ( Figure 5).

Discussion
e trehalose pathway is a major mechanism for growth and metabolism of many fungi; however, the presence of trehalase enzymes in many of these fungi is still unknown [19,[21][22][23][30][31][32]. Validamycin A is a trehalase enzyme inhibitor produced by Streptomyces hygroscopicus and is used for fungal inhibition in plants and insects [33,36,37,51,52]. From many previous reports, in plants and insects, the effect of validamycin A is to inhibit trehalase activity in their cells [53][54][55][56]. In a rice fungal pathogen, Rhizoctonia solani, validamycin A was shown to inhibit trehalase activity but not cellulase, pectinase, chitinase, amylase, or glucosidases [57]. Additionally, validamycin A also inhibited the growth of Rhizoctonia solani and Fusarium culmorum [33,38]. However, there are only few studies demonstrating the effects of validamycin A on human fungal pathogens [58]. From our study, we observed that a human fungal pathogen, A. flavus, had two trehalase enzymes that shared similar conserved domains and possessed high similarity and identity to Saccharomyces cerevisiae and Aspergillus fumigatus (Figures 1(a) and 1(b)), including Rhizoctonia solani and Candida albicans (Figures S1(a)and S1(b)). erefore, we hypothesize that validamycin A may inhibit trehalase enzyme activity in A. flavus similar to previous reports [33,38,57].
In this study, we investigated the presence of trehalase enzymes and the effect of the trehalase inhibitor, validamycin A, on the growth of a common pathogenic fungus in humans, A. flavus. e results showed that A. flavus possesses trehalase homologs and grows on trehalose peptone media, similar to growth on glucose peptone media (Figures 2(a) and 2(b)). ese findings imply that A. flavus utilizes trehalase enzymes to degrade trehalose for use as a carbon source and energy. In addition, we observed inhibitory effects of validamycin A on the growth of A. flavus (Figure 3).
is finding suggests that trehalase activity is required for A. flavus growth. However, direct evidence, such as genetic approaches (e.g., generating trehalase genedeletion mutants) to support the importance of trehalase, is needed to confirm this observation.
In a previous study, it was found that validamycin A increased trehalose levels in a pathogenic fungus, C. albicans [58]. is result is similar to our findings that showed an increase in trehalose levels of A. flavus conidia after validamycin A treatment (Figure 4(a)). However, further trehalase activity assay using high-performance liquid chromatography (HPLC) is also necessary to confirm the effect of validamycin A against trehalase enzymes in A. flavus. As the trehalose pathway is crucial in the early stages of conidial germination [18,19,47,59], we further investigated the effect of validamycin A on conidial germination of A. flavus. Expectedly, validamycin A significantly delayed International Journal of Microbiology  (Figure 4(b)). erefore, these observations suggest that the inhibition of trehalase enzymes depletes the source of energy and the growth for A. flavus. Nonetheless, we observed that conidial germination, in the presence of validamycin A, was not different from the untreated group at 24-hour incubation. is result suggests that A. flavus could probably increase conidial germination by alternative pathways following trehalase inhibition (e.g., mannitol pathway) [60,61]. A wide variety of different media is still necessary to further investigate the trehalose phenotypes in A. flavus. In addition, this study further investigated the combinative effect between validamycin A and amphotericin B on A. flavus ATCC204304, which is a standard strain for the antifungal susceptibility test. e result demonstrated that these two drugs showed an additive effect on growth inhibition of A. flavus. Interestingly, the combination of these drugs had a synergistic effect on A. flavus clinical isolates with high MICs of amphotericin B. Although the cutoff value of MIC for amphotericin B resistance in A. flavus was unknown, Barchiesi et al. suggested that MIC of amphotericin B ≥ 2 μg/ mL should be considered as a resistant strain [48,62].
Trehalose pathway is clearly associated with cell wall components, including chitin and beta-glucan, as shown in many previous reports [18,19,42,47]. Disturbance in substrates of trehalose or enzymes or proteins associated with the trehalose pathway in Aspergillus fumigatus would lead to changes in the cell wall components and structure [18,19,42,47]. Furthermore, trehalose level and proteins associated with the trehalose pathway may affect exopolysaccharide galactosaminogalactans (GAGs), which are important for fungal adherence and biofilm formation, as shown in A. fumigatus previous reports [42,47]. In this study, we also observed that validamycin A decreased fungal adherence (Figure 4(c)). ese data imply that the structure or components of exopolysaccharide GAGs may be affected by validamycin A.
Besides, trehalase enzymes in many eukaryotic organisms may play important roles in carbon metabolism, chitin biosynthesis, and stress tolerance, i.e., sucrose and trehalose homeostasis in Arabidopsis thaliana and Phaseolus vulgaris, regulation of chitin biosynthesis in insects, and carbon partitioning in many plants [63][64][65][66][67][68][69][70]. erefore, we hypothesize that inhibition of the trehalase enzyme via validamycin A may change the structure and components of the fungal cell wall and exopolysaccharide through changes in the carbon metabolism of A. flavus leading to increased permeability and synergistic effects of amphotericin B against A. flavus in the presence of validamycin A. However, further studies of cell wall/GAG structures via the electron microscope and cell wall/GAG components through HPLC, including RNA sequencing and metabolomic analyses, are necessary to decipher the effect of validamycin A on A. flavus [18,47].
Additionally, MICs of validamycin A in each A. flavus clinical isolate were varied. is variation of MICS of validamycin A in these clinical isolates is probably due to the difference in the cell wall/GAG structure and components of each strain (e.g., glucan or chitin), as a previous study showed that amphotericin B-resistant A. flavus contained higher (1,3)-β-D-glucan in their cell wall than the sensitive strains [71]. Furthermore, previous studies suggest that some clinical isolates of A. fumigatus had different phenotypes including cell wall components and virulence [72,73].
We further characterized these clinical isolates and observed that the growth rate and conidial trehalose levels International Journal of Microbiology 7 showed no difference from A. flavus ATCC204304 (Figures S2(a) and S2(b)). However, these isolates possessed different fungal adherence properties ( Figure S2(c)). Different exopolysaccharide components and/or structure of these isolates may lead to decreased permeability of amphotericin B and validamycin A into the fungal cell membrane and cytoplasm affecting MICs in each clinical isolate. Nonetheless, the cell wall/GAG structure and components of these clinical isolates need to be further studied. Moreover, more clinical isolates and animal models are also necessary to confirm synergistic effects between validamycin A and amphotericin B.
Cytotoxicity of validamycin A was tested in our study, and the result demonstrated that validamycin A at concentrations showing synergistic effects on A. flavus had no cytotoxicity on human bronchial epithelial cells ( Figure 5). Nevertheless, different human cell lines together with different concentrations of validamycin A and amphotericin B are still needed to be further investigated for the cytotoxicity. In addition, in vivo studies are required as acute toxicity was

Data Availability
All data used to support the findings of this study are included within the article, and the raw data for each figure are available from the corresponding author upon request.   cells. e cytotoxicity test was performed to observe the toxicity of validamycin A and amphotericin B on BEAS-2B cells using Lactate Dehydrogenase (LDH) Cytotoxicity Colorimetric Assay Kit II. Cell cultures were incubated at 37°C in a humidified environment containing 95% air and 5% CO 2 . After 24 hours, LDH reaction mixture was added (25 μl) and incubated at 37°C for 30 minutes. en, ODs were measured at 450 nm using a spectrophotometer. Data are presented as means ± SE from three biological replicates. No significant difference was observed (one-way ANOVA with post hoc Bonferroni's test). similarity and 55% identity, 71% similarity, respectively. AFLA, Aspergillus flavus; Trehalase_Ca-bi, neutral trehalase calcium-binding domain; trehalase: trehalose hydrolysis domain (adapted from SMART analyses). Figure S2: different Aspergillus flavus isolates show no difference in the radial growth rate and conidial trehalose levels but possess different fungal adherence properties. (a) Aspergillus flavus ATCC 204304 and three clinical isolates were incubated at 37°C on glucose media. e radial growth of these fungal growths was measured on the third day of incubation. Data are presented as means ± SE from three biological replicates. No significant difference was observed (one-way ANOVA with post hoc Bonferroni's test). (b) Aspergillus flavus ATCC-204304 and three clinical isolates were cultured at 37°C on Sabouraud dextrose agar for five days with or without 1 μg/mL validamycin A. Trehalose assays were performed to measure trehalose levels in the conidia using glucose oxidase assays. Data are presented as means ± SE from three biological replicates. No significant difference was observed (one-way ANOVA with post hoc Bonferroni's test). (c) Aspergillus flavus ATCC 204304 and three clinical isolates were cultured at 37°C in Sabouraud dextrose broth with or without 1 μg/mL validamycin A in 96-well plates for 24 hours, and crystal violet adherence assays were performed. Data are presented as means ± SE from three biological replicates. * P value < 0.05; * * P value < 0.01 (one-way ANOVA with post hoc Bonferroni's test compared to the ATCC-204304 strain). (Supplementary Materials)