Effect of Allium cepa on LAC1 gene expression and physiological activities in Cryptococcus neoformans

Background and Purpose: This study aimed to investigate the effects of Allium cepa ethanolic extract (EAC) on Cryptococcus neoformans biological activities and LAC1 gene expression. Materials and Methods: The minimum inhibitory concentration (MIC) of EAC was determined based on the Clinical and Laboratory Standards Institute M27-A4 method at a concentration range of 125-4000 µg/ml. The EAC synergism activity was determined in combination with fluconazole (FCZ) as an antifungal azole. Laccase activity, melanin production, and cell membrane ergosterol content of C. neoformans were assessed at the 0.5× MIC concentration of EAC (1000 μg/ml) and FCZ (64 μg/ml) by approved methods. The expression of the LAC1 gene was studied in the fungus exposed to 0.5× MIC concentration of EAC and FCZ using the real-time polymerase chain reaction. Results: Based on obtained results, MIC of EAC and FCZ were 2000 and 128 μg/ml, respectively. A combinatory effect was reported for FCZ and EAC by a fractional inhibitory concentration index of 0.25. The cell membrane ergosterol content was inhibited in EAC- and FCZ-treated C. neoformans by 58.25% and 49.85%, respectively. The laccase activity and melanin production were reduced in EAC-treated C. neoformans by 45.37% and 51.57%, and in FCZ-treated fungus by 54.64% and 53.68%, respectively. The expression of fungal LAC1 at messenger RNA (mRNA) level was measured 0.46 and 0.58 folds and significantly decreased in both EAC- and FCZ-treated C. neoformans at the 0.5×MIC concentration, respectively (P<0.05). Conclusion: The findings revealed that EAC contains inhibitory compounds which interact with biological activities in C. neoformans and thereby, it could be considered as a potential source for the development of novel antifungal drugs.


Introduction
ryptococcosis caused by Cryptococcus spp. is considered a life-threatening fungal infection in immunocompromised patients [1,2]. The Cryptococcus neoformans is responsible for the majority of cases of cryptococcosis and possesses virulence factors, including polysaccharide capsule, melanin, and secretory enzymes production, and rapid growth at 37˚C which provide the fungus with advantages that help it reside in host cells [3,4]. Two enzymes, namely phenoloxidase and laccase, are involved in melanin production by the fungus which is accumulated in the fungal cell wall and protects it from oxidative stress [4,5]. The genes LAC1 and LAC2 encode two laccases, and LAC1 is essential for melanin production [3,5].
Amphotericin B and azoles, effective drugs for clinical cryptococcosis, are reported to have considerable side effects and lead to the emergence of resistant strains [6]. The concerns about the toxicity and limited availability of these drugs have stimulated the search for natural therapeutic alternatives alone or in combination which provide a broader spectrum of action, greater potency, and reduction in the number of resistant organisms [7][8][9][10][11]. The virulenceassociated enzymes are introduced as potential targets for the discovery of antifungal drugs in medicinal plants [12][13][14][15].
Allium cepa (A. cepa; Onion) belongs to the Liliaceae family and is known as a traditional plant with important medicinal properties. These properties have been mainly attributed to organosulfur compounds as well as a wide array of other chemically different ingredients [16][17][18]. According to the World Health Organization, this traditional plant has been widely used for the treatment of various disorders, such as bruises, colic, high blood pressure, jaundice, and even varicose veins [19][20][21][22]. Although the plant has been shown to have antimicrobial activity against a wide array of microorganisms, including bacteria, parasites, viruses, and fungi [23][24][25][26][27][28][29], little has been documented about its mode of antimicrobial action. The present study aimed to investigate the effect of ethanolic extract of A. cepa (EAC) on major physiological factors of C. neoformans, including growth, cell membrane ergosterol (CME), laccase activity, melanin production, and LAC1 gene expression as an important fungal virulence factor.

Fungal strain
This research was approved by the Ethics Committee of Tarbiat

Preparation of aqueous and alcoholic extracts of Allium cepa
For the purposes of the study, 1,000 g of fresh onion white bulbs were mixed by blender and dried by a freeze-dryer (Christ, Germany). To prepare A. cepa extracts, 50 g of the dried powder were mixed with 250 ml of each ethanol, methanol, and sterilized distilled water, separately and sonicated at 120 Htz for one h by a sonicator (Hielscher UP400S Ultrasonic, Germany). After incubation at room temperature for 48 h on a shaker at 120 rpm, the extracts were filtered through Whatman No. 1 filter paper (Merck, Germany) to remove the debris. Moreover, ethanol and methanol extracts were dried by a rotary evaporator at 40°C [28]. The aqueous extract was concentrated to dryness using a freeze drier. The yield percentage was calculated using the following formula: Extract yield (%)=R (the dry weight of the extracted plant)/S (the raw plant sample)×100

Screening of antifungal activity of Allium cepa extracts
Agar well diffusion technique was used to screen and determine the antifungal activities of A. cepa extracts. The final concentration of each extract was prepared in sterile distilled water at 4000 μg/ml. The fungal suspension (2.5×10 3 colony-forming unit [CFU]/ml) was cultured on SDA medium and 100 μl of each extract that contained 400 μg dry material was inoculated in wells created on the plates and stored at 35°C for 48 h. The EAC that showed the strongest inhibitory effect against the fungal growth was selected for further steps.

Evaluation of Allium cepa ethanolic extract antifungal activity
Antifungal activity was measured according to the guidelines of the National Committee for Clinical Laboratory Standards CLSI M27-A4 method [29]. A 100 µl cell suspension of C. neoformans (0.5-2.5 × 10 3 CFU/ml) prepared in RPMI-1640 (Sigma-Aldrich, USA) plus MOPS (3-(N-morpholino) propanesulfonic acid) medium were added to each well of a 96-wells microplate. The EAC was prepared in dimethylsulfoxide (DMSO, Sigma-Aldrich) and two-fold serial dilutions were prepared in RPMI in a microplate to obtain the final concentrations of 125 to 4000 μg/ml. For fluconazole (FCZ), serial two-fold concentrations of 0.5-256 µg/ml were prepared from a stock solution of the drug (Sigma-Aldrich, USA) in methanol (Merck, Germany). Microplates were incubated at 35°C for 72 h.
The minimum inhibitory concentrations (MIC) of EAC and FCZ were defined based on the inhibition of fungal growth in 96 well microplates by visual assay. For determining the minimum fungicidal concentration (MFC) of EAC, 50 µl of the contents of wells with no visual fungal growth was cultured on SDA plates and incubated at 37°C for 48 h. The MIC was defined as the lowest concentration of the EAC capable of interrupting any visible fungal growth. The MFC of EAC was the lowest concentration of the extract in which no fungal colony appeared on SDA plates. The MICs of FCZ were defined as the concentrations of the drug that constantly reduced the turbidity, compared to the control group.

Effect of Allium cepa ethanolic extract on the ergosterol content
The ergosterol content was measured according to the instructions of Arthington-Skaggs et al. [32]. The resultant heptane extract was scanned from 200 to 300 nm using a UV-visible spectrophotometer (Shimadzu, Japan) and the absorbance of the samples was read at two wavelengths of 281.5 nm (A281.5) and 230 nm (A230). The ergosterol content was calculated as a percentage of the wet weight of the cell using the

Effect of Allium cepa ethanolic extract on laccase activity of Cryptococcus neoformans
Laccase activity was measured in C. neoformans (2.5×10 6 cells/ml) exposed to EAC (1000 μg/ml) and FCZ (64 μg/ml) in 250 ml flasks containing YPD broth according to the instructions given by Albino et al. [34]. The enzyme unit was defined as the amount of enzyme which oxidized one µmol of substrate per min in the assay conditions.
It should be mentioned that amplification products were analyzed by agarose (1%) gel electrophoresis. To calibrate the real-time quantitative reverse transcription PCR system, a standard curve from serial dilutions (10 −1 to 10 −4 ) of the cDNA template of C. neoformans was used. Real-time PCR was carried out using SYBR green master mix (Applied Biosystems, USA) using the ẞ-actin as a reference gene. The PCR was performed with an initial incubation at 95°C for 10 min, followed by 40 cycles of denaturation (95°C for 15 sec), annealing (60°C for 1 min) and extension (72°C for 15 sec) and a final extension step at 72°C for 1 min by the ABI PRISM 7500 thermal cycler (Applied Biosystems, USA). To determine the mRNA level of LAC1 expression, the differences between the threshold cycle (CT) of samples and calibrator [ΔCT=CT (target) -CT (reference)] were calculated using the following formula: (2 −ΔΔCT ) and analyzed by REST© software (version 2.0.13).

Statistical analysis
All data were analyzed in GraphPad Prism software (version 6.0, Sandiego, CA). A p-value of less than 0.05 was considered statistically significant in all the analyses.

Antifungal activity of Allium cepa
Screening results on agar well diffusion assay showed that EAC (15 mm) was more effective than the aqueous (2 mm) and methanolic (10 mm) extracts which were used for all the next steps in this study. The MIC and MFC values of EAC were determined in comparison with FCZ against the C. neoformans (Table 1). Results indicated that MIC and MFC of EAC against C. neoformans were 2000 and 4000 μg/ml, respectively. For FCZ, these values were reported at 128 and 256 μg/ml, respectively. The FICIs for EAC combined with FCZ were calculated as well. Results of the checkerboard microtiter assay indicated significant synergistic interaction between EAC and FCZ against C. neoformans ( Table 1). The synergistic interaction between EAC and FCZ (0.5>FICI=0.25) against C. neoformans was observed after 72 h of incubation at 35°C.

Effect of Allium cepa ethanolic extract on cell membrane ergosterol content
The CME contents of C. neoformans in EAC-and FCZ-treated cultures were measured at 0.42 and 0.50 µg/g, respectively ( Table 2). The CME was inhibited

Effect of Allium cepa ethanolic extract on melanin production and laccase activity
Melanin production decreased in C. neoformans treated with 0.5× MICs of both EAC (0.092) and FCZ (0.088) as summarized in Table 3. The melanin production was significantly inhibited by 51.57% and 53.68% in EAC-and FCZ-treated cultures, compared to the control group (P<0.05) (Figure 1). The laccase activity was measured at 0.053 and 0.044 in C. neoformans exposed to EAC and FCZ, respectively (Table 3). Furthermore, the enzyme activity was inhibited by 45.37% in EAC-treated and 54.64% in FCZ-treated cultures. The results showed that both EAC and FCZ were able to significantly reduce laccase activity in C. neoformans, compared to the control group (P<0.05) (Figure 2).

Effect of Allium cepa ethanolic extract on LAC1 gene expression
The relative quantification of the LAC1 expression ratio normalized to β-actin indicated that there were significant differences between the gene expressions of EAC-and FCZ-treated fungal cells and the untreated control group. Real-time PCR demonstrated that the expression levels of LAC1 in all the treated samples with 0.5× MIC concentration of EAC (1,000 μg/ml) and FCZ (64 μg/ml) were 0.43 and 0.53 folds lower than the control samples, respectively ( Figure 3). The mRNA transcripts level of the LAC1 gene underwent a significant decrease and down-regulation in comparison with non-treated control samples (P<0.05).

Discussion
To clarify the exact mode of antifungal action of A. cepa against C. neoformans, we evaluated the potential targets of EAC in vitro. For this purpose, the fungal plasma membrane ergosterol synthesis, laccase activity, melanin production, and LAC1 gene expression were studied in C. neoformans exposed to A. cepa.
Several studies have confirmed the antifungal activity of white onion against various pathogenic fungi. Lanzotti  Aspergillus niger, and Trichophyton rubrum [28]. Based on the results of another study, the organic ethanol extracts of onion (A. cepa) (MFC: 275 mg/ml) inhibited the growth of Aspergillus flavus, A. niger, and Cladosporium herbarum [37]. Shams-Ghahfarokhi et al. [38] also found ultrastructural cell damage in T. rubrum and Trichophyton mentagrophytes after exposure to fresh A. cepa aqueous extract in a concentration of 200 mg/ml. They indicated different changes in the fungal cytoplasmic membranes and other membranous structures of organelles, such as nuclei and mitochondria, cell wall, complete or partial destruction of organelles, degeneration of the cytoplasm, and formation of electron-dense material in hyphae cells which led to cell death. In another study, the zones of inhibitions are shown by gel of red onion at different concentrations (5, 7.5, and 12.5%) against T. rubrum [39]. The gel with a concentration of 12.5% showed significant antifungal activity against T. rubrum (10-20 mm) (P<0.05).
In this study, the screening results showed that EAC was more effective than the aqueous and methanolic extracts against C. neoformans. The EAC inhibited the fungal growth by 50% at 1,000 μg/ml while the fungal growth was completely inhibited at 4,000 μg/ml. The EAC at a concentration of 125 μg/ml increased the sensitivity of C. neoformans to FCZ (MIC: 0.5 μg/ml). The FICI values of EAC in combination with FCZ was reported at 0.25 which shows a combinatory effect for these compound. Moreover, it was found that EAC in the concentration of 1,000 µg/ml decreased the cell membrane ergosterol content to 0.42 µg/g fungal dry weight in the treated C. neoformans. The EAC probably increases the susceptibility of C. neoformans to FCZ by changing the cell membrane ergosterol content. Reduced ergosterol content interferes with the permeability of fungal cells and causes the release of various important components from inside of the fungal cell.
Sulfhydryl organic compounds have been reported to be laccase inhibitors. Nickavar and Yousefian found that ethanol extracts of Allium spp. were able to inhibit α-amylase activity with IC50 values around 15 mg/ml [40]. In this study, the laccase activity and consequently, melanin production was potently reduced in C. neoformans exposed to both EAC and FCZ. This inhibitory effect may be attributed to the direct effect of EAC on laccase activity or may be the consequence of the release of important fungal cell components essential for the biosynthesis of laccase after changing fungal cell membrane permeability by EAC.
To our knowledge, this is the first report on the effect of EAC on LAC1 gene expression in C. neoformans. The LAC1 gene has been shown to be important as one of the crucial steps of melanin biosynthesis in C. neoformans. Results of this study showed that LAC1 expression underwent a significant reduction in C. neoformans exposed to EAC (1000 μg/ml) and FCZ (64 μg/ml) by 0.46 and 0.58 folds, respectively.

Conclusion
In conclusion, the findings of this study indicated the effectiveness of EAC against C. neoformans through affecting the cell membrane ergosterol content, laccase activity, melanin production, and LAC1 gene expression. The EAC could be considered a potential source for novel antifungal drugs which may be effective in the treatment of invasive cryptococcosis. Since biological activities of EAC against C. neoformans may be attributed to flavonoids and organosulphur compounds as two major classes of phytochemicals of A. cepa, further studies are recommended to isolate and characterize antifungal components of EAC.