Signature Garlic Phytochemical as a Potential Anti-Candidal Candidate Targeting Virulence Factors in Candida albicans  ^†

Abstract

Resistance to presently available antifungals and their toxicities is a serious concern throughout the world. It is necessary to investigate innovative, more effective molecules especially derived from medicinally active plants with lesser side effects. Allyl methyl sulfide (AMS), an organosulfur derived from garlic oil, was explored for its activity against Candida albicans strains. The minimum Inhibitory Concentration (MIC) values of AMS were found to be 200 µg/mL and 250 µg/mL, and the Minimum Fungicidal Concentration (MFC) values of AMS were 400 µg/mL and 500 µg/mL for the selected strains, respectively. Fungal growth in C. albicans was 90% inhibited at their respective MIC values, as demonstrated by micro broth dilution experiments. After treatment with AMS, C. albicans’ release of extracellular proteinases, phospholipases, and biofilm formation was significantly inhibited. In C. albicans, AMS treatment also reduces attachment to buccal epithelial tissues as measured microscopically. In addition, AMS exhibited significant control over yeast to hypha transitions in C. albicans cells, which constitutes one of the major virulent features of the Candida species. All the findings of this study indicate that AMS may be a potential alternative to commonly used antifungals.

1. Introduction

Candida is a prominent contributor to nosocomial and mucocutaneous infections in immunocompromised persons. Candida albicans is the frequently isolated and studied species of the genus Candida and is responsible for a wide range of ailments, including thrush, diaper rash, and systemic candidiasis [1]. Despite the availability of antifungal drugs, growing levels of resistance to conventional antifungal treatments have been recorded, necessitating the development of novel therapeutic approaches to combat Candida infections [2]. Phytochemicals, or substances produced from plants, have been extensively investigated for their potential use as antifungal agents. Garlic is a good candidate for the treatment of Candida infections due to its broad-spectrum antifungal action [3].

This work aims to examine the antifungal activity of garlic phytochemical against C. albicans and the effects of these compounds on C. albicans virulence factors. Using in vitro assays, the antifungal activity of garlic phytochemical against C. albicans was investigated along with its effect on virulence factors.

Allyl methyl sulfide (AMS) is present in garlic, onions, and other Allium species. Recent research has demonstrated that AMS has antimicrobial characteristics, implying it can suppress the growth of fungi [4]. This feature makes AMS a possible natural alternative for managing fungal infections in a wide range of settings, including health, medicine, agriculture, and food preservation. The efficacy of AMS against several fungi, including Candida albicans, Aspergillus flavus, and Rhizopus stolonifer, has already been investigated by different researchers at the initial level [5,6,7]. To fully understand the mechanism of its antifungal activity and the potential effects of its virulence-causing mechanism, additional research is required.

In this work, the effectiveness of the allyl methyl sulfide (AMS) against C. albicans is investigated in vitro. The purpose of the study was to examine the effect of AMS on virulence factors and its potential as an anti-Candidal.

2. Materials and Methods

2.1. Growth Parameters, Strain, Media and Chemicals Used

The standard laboratory strains of C. albicans ATCC 5314 and 90028 were employed in the current study and were obtained from the National Centre for Cell Sciences (NCCS), Pune, India. On YPD plates (1% yeast extract, 2% peptone, 2% dextrose, 2.5% agar), all the strains were maintained for experiments, being kept at 4 °C. All chemicals used in the study were of analytical grade and were purchased from Merck (Bengaluru, India). MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyl tetrazolium bromide) and other media components, Trypsin, EDTA, N-acetyl glucosamine, and Dulbecco’s modified Eagle’s medium (DMEM), were purchased from Himedia (Thane, India). Allyl methyl sulfide (AMS) and fluconazole were obtained from Sigma Aldrich (St. Louis, MA, USA). Gibco (Grand Island, NY, USA) provided the fetal bovine serum (FBS).

2.2. Determination of Antifungal Susceptibility

Minimum Inhibitory Concentration and Minimum Fungicidal Concentration Evaluation of AMS

The micro broth dilution method was used in accordance with the recommendations found in CLSI reference document M27-A3 [8] to determine the minimum inhibitory concentration (MIC) values of AMS for the Candida strains. MIC was defined as the lowest concentration that caused a 90% decrease in absorbance as compared to that of the control. In total, 20 µL aliquots were obtained from each well, containing different concentrations of AMS and control. In order to determine the minimal fungicidal concentration (MFC), these were subcultured on YPD agar plates for 3–4 days at 35 °C until growth was visible in control samples. The MFC for AMS was defined as the lowest AMS concentration that resulted in no fungal growth on agar plates [9].

2.3. Determination of AMS Impact on Virulence

2.3.1. Proteinase and Phospholipase Assay

The Candida culture was cultivated overnight, and its concentration was standardized using saline. Then, a desired concentration of AMS equivalent to MIC, MIC/2, and MIC/8 was applied to the standardized culture. To determine proteinase secretion, 2 µL aliquots were placed at equal intervals on agar plates, containing 2% agar, 0.2 g of BSA fraction V, yeast nitrogen base, 20 g of glucose, and enough distilled water to reach a final volume of 1000 mL. Similarly, to determine phospholipase secretion, aliquots were placed on agar peptone media comprising 2% agar, 10 g of peptone, 30 g of glucose, 57.3 g of NaCl, 0.55 g of CaCl₂, and distilled water in a total volume of 900 mL, which was subsequently supplemented with 10% egg yolk emulsion. Plates were incubated at 37 °C for 2–4 days. Measuring the degradation or precipitation zones (Pz) allowed for the estimation of proteinase and phospholipase secretion [10].

2.3.2. Buccal Epithelial Cells Adhesion Assay

The overnight-grown Candida cells were washed in sterile PBS (pH 6.8) and resuspended in spider media (pH 7.2). The first author voluntarily donated epithelial cells by scraping the cheek mucosa with sterile cotton swabs, delicately stirring and washing them in PBS, and then donated them. In order to conduct adhesion tests, 1 mL of each suspension was combined in a test tube and incubated for 2 hours at 37 °C with mild stirring in the presence of 200 μg/mL AMS. After incubation, two droplets of a 0.4% trypan blue solution were added to each tube. In total, 10 μL of the stained suspension was transferred to a glass slide and examined at 40× magnification using confocal microscopy [11].

2.3.3. Biofilm Formation Assay

Biofilms of Candida were investigated on the polystyrene surface of 96-well plates. In total, 100 μL of a PBS cell suspension containing 1 × 10⁷ cells/mL was injected into each well for overnight culture. The plates were incubated at 37 °C and 50 rpm for 90 minutes in order to adhere the cells to the surface. To remove non-adherent cells, the wells were rinsed two to three times with PBS. After adding 200 μL of YPD medium and 20 μg/mL of AMS to the plates, they were incubated at 37 °C for 24 hours. Biofilms were treated with 20 μg/mL AMS for 24 hours. The MTT assay was employed to measure biofilms. Each well was provided with a 100 μL aliquot of MTT, which was mixed gently for one minute. The plates were then incubated in a CO2 incubator for 5 hours at 37 °C in the dark. Using a microtiter plate reader (BIO-RAD, iMark, Hercules, CA, USA), the sample was measured at 450 nm, and the results were expressed as a percentage of viability [12].

2.3.4. Yeast to Hypha Virulence Trait

The cells that were developed overnight were collected, and then they were washed twice. After that, the cells were starved for six hours at 37 °C while suspended in PBS. After the cells had been incubated, they were moved to the media for hyphal growth in the presence of AMS, which included 10% (v/v) horse serum and glucosamine. Hyphae were observed under a confocal microscope at magnifications of 40× [13].

2.4. Statistical Analysis

All experiments were carried out at least three times independently. To investigate the statistical difference between the control and treated samples, the statistical significance of the data was determined using the Student’s t-test. The significance level was set at p ≤ 0.05.

3. Results

3.1. Antifungal Activity Determination

Allyl methyl sulfide (AMS) had minimum inhibitory and fungicidal concentrations of 200 µg/mL and 400 µg/mL against C. albicans ATCC 5314 and 250 µg/mL MIC and 500 µg/mL MFC against C. albicans ATCC 90028, respectively (

Table 1. MIC90 and MFC of isolates as determined by broth microdilution assay.

Strains	Allyl Methyl Sulfide MIC90/MFC (µg/mL)	Fluconazole MIC90/MFC (µg/mL)
C. albicans ATCC 5314	200/400	8/16
C. albicans ATCC 90028	250/500	10/18

).

3.2. Effect of AMS against Virulence Traits of C. albicans

3.2.1. Effect on Proteinase and Phospholipase Secretion

Extracellular hydrolytic enzymes, primarily proteinases and phospholipases, are crucial in the development of fungal infection because they promote adhesion, invasion, and tissue damage in the host. Therefore, proteinase and phospholipase’s secretory activities were examined at MIC and sub-MIC concentrations (MIC/2 and MIC/4) of AMS along with untreated cells. As shown in Figure 1A, Candida cells grown in agar plates show different precipitation zones (Pz) in the presence of AMS. However, the reduction at MIC/4 is negligible with inhibition of only 6% in proteinase secretion. However, at MIC/2 and MIC concentrations, it demonstrated 13% and 55% reductions in proteinase secretion (Figure 2A). On the other hand, the inhibition of phospholipase secretion is less prominent at MIC and sub-MIC concentrations of AMS. It accounts for only 3%, 6%, and 10% of MIC/4, MIC/2, and MIC of AMS, respectively (Figure 1B and Figure 2B). The inhibition was more pronounced in the case of proteinase rather than phospholipase secretion.

3.2.2. Effect of AMS on Adherence to Host Epithelial Tissue

To infect the host, adherence to the mucosal and outer epithelial tissues is the prerequisite for the C. albicans to exert its pathogenic potential. Thus, a buccal epithelial cells (BECs) adhesion assay plays a crucial role in testing the efficacy of antifungals targeting virulent factors. At MIC concentration, AMS can detach the C. albicans cells from the BEC surface and also cause a significant reduction in cell population as compared to the regular pattern of attached cells in the case of the control sample (Figure 3).

3.2.3. Effect of AMS on Biofilm Formation

In comparison to untreated controls, AMS at various inhibitory and sub-inhibitory concentrations was found to reduce biofilm formation in a concentration-dependent manner. The biofilm formation was estimated in terms of cell viability percentage, which corresponded to the value of absorbance at 490 nm. As Figure 4 suggests, untreated control cells show 100% cell viability in terms of biofilm formation, and the rest of the concentrations are taken in reference to it. The results show that biofilm formation was inhibited by 53%, 68%, and 81% at MIC/4, MIC/2, and MIC concentrations of AMS, respectively.

3.2.4. Effect of AMS on Morphological Switching

To avoid a hostile host environment and stress situation, C. albicans cells undergo yeast to hypha morphological switching. In the presence of 10% serum as well as glucosamine-containing hypha-inducing media at 37 °C, the induction of a germ tube or hyphae was significantly reduced at the MIC value of AMS (Figure 5). Furthermore, a significant reduction in the number of cells along with the disappearance of hypha was observed under confocal microscopy. Therefore, it is clear that AMS blocked C. albicans filamentation at MIC, a mechanism essential to fungal pathogenesis.

4. Discussion

The most prevalent human fungal pathogen, Candida albicans, can cause systemic infections as well as mucosal infections. C. albicans’ pathogenicity is linked to a number of virulence factors, such as adhesion, phenotypic and morphological alterations, the production of numerous hydrolases (secreted proteinase and phospholipases), and the presence of efflux pumps [14]. Additionally, cells are covered in adherent biofilms that make it more difficult for conventional antifungals to work and build host defenses that are resistant, which raises the risk of multidrug resistance [15]. Therefore, a different approach to treating Candida infections that are linked to virulence attributes and biofilms is required.

Garlic is said to have medicinal, insecticidal, antibacterial, and antifungal effects. Additionally, there has recently been a surge in interest in garlic as an anti-fungal agent. It has been shown that garlic extract is fungicidal to pathogenic yeasts, particularly C. albicans. Allicin was one of the components of garlic that was shown to prevent the proliferation of fungi by blocking succinate dehydrogenase [5]. According to reports, the main active metabolite component of allicin is AMS [5,16]. It has been demonstrated that AMS, a key constituent of volatile garlic metabolites, possesses antibacterial, antioxidant, and anticancer effects [3,6,17]. However, no concrete study is available highlighting its effect on virulence factors in C. albicans.

We showed that AMS had a MIC of 200 µg/mL against C. albicans ATCC 5314 and 250 µg/mL against C. albicans ATCC 90028. In both the strains, the values of MFC were found to be double of MIC, which were 400 µg/mL and 500 µg/mL, respectively. We also put the negative control of standard antifungal fluconazole, and it showed a similar ratio to AMS between MIC and MFC. However, the concentrations of MIC and MFC for AMS were quite high compared to fluconazole, but its natural origin and negligible toxicity in that range should not limit its efficiency.

C. albicans secretes virulence agents, including proteases and phospholipases that increase its pathogenicity. These hydrolases effectively break down host membranes and surface membrane proteins, which promotes the invasion of C. albicans into host tissues [15]. AMS was successful in preventing C. albicans from producing these virulent enzymes. This finding inspired us to hypothesize that AMS might be able to prevent candidemia from developing. The adherence of C. albicans to the host epithelial surface is considered to be an important factor in the development of infection [18]. Also, the ability of C. albicans to produce biofilms is linked to the majority of infections caused by this organism [13]. Our study reported that AMS was able to reduce the adherence of Candida cells to host epithelial tissues and inhibit biofilm formation at the abiotic surface at its MIC concentration. In addition to the above-mentioned factors, the yeast-to-hyphal transition was thought to be critical for C. albicans’ pathogenicity. The hyphal cells colonized endothelium and epithelial tissues and promoted systemic infection [19]. Our results showed that AMS-treated cells had less hyphal development, whereas control cells had a higher filamentous structure. Overall, the results of this study showed that AMS had the potential to be employed as an antifungal agent, particularly against C. albicans.

5. Conclusions

The study suggested that allyl methyl sulfide (AMS) had the potential as an antifungal candidate against Candida albicans. The effectiveness of AMS against C. albicans originated from the inhibition of proteinase and phospholipase secretion. In addition, it also inhibited biofilm formations, yeast-to-hyphal transitions, and adhesions of Candida cells to epithelium.