In vitro influence of certain essential oils on germ tube formation, cell surface hydrophobicity, and production of proteinase and hemolysin in Candida albicans
Correspondence Address: DOI: 10.4103/2229-5119.102755
Introduction: Plant essential oils especially Cinnamomum verum J. Presl. (Lauraceae), Cymbopogon citratus Spreng. (Poaceae), Cymbopogon martini Roxb .(Poaceae), Syzygium aromaticum Linn. (Myrtaceae) are used in Indian traditional medicine for various ailments including fungal infections. We investigated in vitro antivirulence activity of the above-mentioned essential oils and some of their major active compounds namely cinnamaldehyde, citral, geraniol, and eugenol against the strains of Candida albicans at their sub-MICs. Materials and Methods: Broth macrodilution method was used for the determination of MIC of oils. Specific spectrophotometric assays were performed for detection and inhibition of virulence factors production namely germ tube formation (GTF), cell surface hydrophobicity (CSH), proteinase, and hemolysin in test strains. The concentrations used to assess inhibition of these virulence factors were below the MIC values of each oil. Results: Among the oils tested, C. martini, citral, and cinnamaldehyde exhibited strong antifungal activity with the MIC ranging from 45 to 200 μg/ml against the test strains. At sub-MIC (50 μg/ml), cinnamaldehyde reduced the % GTF from 89.33 to 10.0, whereas % CSH was reduced from 61.75 to 19.26 in C. albicans 04. Oils of C. martini, citral, and geraniol reduced the production of proteinase (>80%) in C. albicans 04 at 45 μg/ml. A maximum of 85.20% reduction in the production of hemolysin was exhibited by cinnamaldehyde at 25 μg/ml in C. albicans 16. Conclusions: Our study has highlighted the promising and broad spectrum in vitro antivirulence activity of oils of S. aromaticum, C. verum, eugenol, and cinnamaldehyde against C. albicans. Keywords: Antivirulence activity, Candida albicans, virulence factors
Infections with C. albicans, called candidiasis, predominantly occur in the immunosuppressed patients. [1] C. albicans causes oral, vaginal, and systemic diseases, with the highest morbidity rate (30-50%) occurring in systemic Candida infections in neutropenic transplant patients. [2] C. albicans is also associated with the 78% of fungal nosocomial infections and over 10% of all the nosocomial infections documented. [3] Conventional antifungal drugs available for treatment of fungal infections have limitations, such as host toxicity and emergence of resistant strains among Candida spp. Polyene agents are highly nephrotoxic and hepatotoxic, [4] whereas azole resistant strains isolated from vulvo-vaginal and oral candidiasis are continued to be a common problem in immunocompromised individuals. [5] An alternative approach to combat such pathogenic fungi is to develop antipathogenic drugs that target virulence factors that are required for establishment of infections by causing host damage and evasion from the immune cells. In the recent years, virulence is considered as a promising anti-infective drug target and has been attempted by many researchers to obtain effective antipathogenic drugs against fungal infections. [6] Pathogenicity is a multifactorial phenomenon where a number of virulence factors contribute. Various virulence factors have been characterized in C. albicans such as germ tube formation, adherence, production of proteinases, phospholipases, hemolysin, and formation of biofilms, which have been shown to be involved in the pathogenesis of C. albicans. [1],[6] Several antifungal drugs such as fluconazole, ketoconazole, nystatin, amphotericin B, and 5-fluorocytosine are known to interfere with certain virulence factors. [7] In the past few decades interest in natural products has increased, and medicinal plants have been investigated for various biological activities and therapeutic potentials. [8],[9] Recent scientific literatures have documented essential oils for potential antifungal activities [10] including activity against drug-resistant fungi. [11] However, little information is available on antivirulence activity from plant essential oils against C. albicans. Therefore, considering the need of exploring novel antivirulence activity from plant products, the present study was planned to investigate essential oils of Cinnamomum verum J.Presl. (Lauraceae), Cymbopogon citratus Spreng. (Poaceae), Cymbopogon martini Roxb. (Poaceae), Syzygium aromaticum Linn. (Myrtaceae) and their major active compounds namely cinnamaldehyde, citral, geraniol, and eugenol, respectively, for their ability to inhibit production of virulence factors such as germ tube formation (GTF), cell surface hydrophobicity (CSH), proteinase and hemolysin in the strains of C. albicans.
Organisms and media In this study, 18 clinical (C. albicans 01-18) and two reference strains (C. albicans NRRLY12983 and C. albicans MTCC183) were included. The clinical strains were isolated from patients of known candidemia, urinary infections, and vaginitis attending the Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh, India. The strains were identified and characterized on the basis of morphological and physiological methods such as use of HiCrome Candida differential agar (HiMedia Laboratories, Mumbai, India), ability to form germ tubes and biochemical tests such as sugar and nitrogen assimilation, nitrate reduction and urease tests (data not shown). Strain C. albicans NRRLY12983 was kindly provided from the fungal culture collection of the Agricultural Research Service, USDA, Peoria, USA and C. albicans MTCC183 was purchased from the Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India. The test strains were maintained on Sabouraud dextrose agar (SDA) slants at 4°C and subcultured in the Sabouraud dextrose broth (SDB) prior to use in assays. Plant essential oils A total of eight essential oils/active compounds used in this study were obtained from Himalaya Drug Co., Dehradun, India (Cinnamomum verum, cinnamon); Himedia Laboratories, Mumbai, India (eugenol and cinnamaldehyde), Aroma Sales Corporation, New Delhi, India (Cymbopogon citratus, lemongrass; Cymbopogon martini, palmrosa; citral and geraniol) and Dabur India Ltd., New Delhi, India (Syzygium aromaticum, clove). All test oils were checked for quality assessment using physico-chemical tests such as specific gravity, refractive index and solubility in alcohol at Fragrance and Flavour Development Centre, Kannauj, India and chemical composition by gas chromatography-mass spectrometry at Sophisticated Analytical Instrument Facility of Indian Institute of Technology, Mumbai, India (data not shown). Essential oils were diluted 10 times in 1% dimethyl sulfoxide before use in assays. Macrobroth dilution method The minimum inhibitory concentrations (MICs) of essential oils and active compounds were determined against the test strains by the broth macrodilution method as adapted by Sokovic and Griensven [12] with some modifications. Essential oils were serially diluted in sugar tubes containing 1 ml SDB to achieve a range of concentrations from 45 to 800 μg/ml. Next, 10 μl of yeast suspension (0.5 McFarland) was added to the tubes and incubated at 37°C for 24 hours. MIC was defined as the lowest concentration that inhibited visible growth. Inhibition of growth was confirmed by spot inoculating the treated broth culture on to SDA plates and incubating at 37°C for 24 hours. Each experiment was repeated three times and mean values were calculated for MICs. Determination of viability of candida cells at Sub-MICs of Oils A 10 μl of yeast suspension (1.0×10 6 cfu/ml) was inoculated into tubes containing 1 ml of SDB amended with sub-MICs (0.5× and 0.25× MICs) of oils. The test tubes were incubated at 37°C for 24 hours. An untreated control without test agents but containing yeast inoculum was also run. Viable counts were obtained from 10-fold serial dilutions of test and control solutions by plating 100 μl of 10-fold dilution onto SDA plates and incubating at 30°C for 24 hours. Each experiment was performed three times with three replicates per experiment and the mean colony count for each experiment was recorded. Detection and inhibition of virulence factors in C. Albicans Germ tube formation The germ tube formation in test strains was evaluated by the method of Taschdjian et al.[13] with some modifications. Briefly, Candida cells were allowed to grow in SDB in the presence of 0.5× and 0.25× MICs of oils at 37°C for 24 hours. Agent free SDB was used as untreated control. Next, 0.5 ml of yeast suspension (1.0×10 6 cfu/ ml) was mixed with 0.5 ml of serum and incubated at 37°C for 3 hours. A random 100 yeast cells from each sample were selected to count the number of germ tubes using hemocytometer (Axiva Co., New Delhi, India). Each experiment was performed three times with three replicates per experiment and the mean values were considered for calculation of % formation of germ tubes in untreated and treated samples. Cell surface hydrophobicity The method of microbial adhesion to hydrocarbons (MATH) as described by Rosenberg et al.[14] was used with some modifications to determine the % CSH of Candida cells. Briefly, Candida cells were grown on SDA plates amended with 0.5× and 0.25× MICs of test agents at 25°C for 48 hours. Candida cells grown on SDA plates without amendment of test agents were considered as untreated control. Yeast suspensions were made in PBS (A 520 of 0.400) and allowed to contact with xylene in 5:1 ratio. The mixtures were vortexed for 1 minute at room temperature and then allowed to separate the two phases for 10 minutes. The absorbance of aqueous phase was read at 520 nm. The % CSH was calculated as follows: % CSH = [(A i -A f )/A i ] ×100, where A i and A f are the absorbance values of aqueous yeast suspension at 520 nm before and after contacting yeast suspension to the organic phase. Each experiment was conducted three times with three replicates per experiment and the mean values were considered for calculation of % CSH in untreated and treated samples. Proteinase assay The proteinase production and its inhibition by oils in test strains were determined using the modified method of Kuriyama et al.[15] Briefly, 50 ml Erlenmeyer flasks containing 10 ml of induction medium as described by MacDonald and Odds, [16] with and without sub-MICs (0.5× and 0.25× MICs) of test agents were inoculated with 100 μl of Candida cells (1.0×10 6 cfu/ml) and incubated at 26°C, 160 rpm for 7 days. The broth culture was centrifuged at 5000 rpm for 30 minutes to obtain supernatant. Furthermore, 200 μl of supernatant was mixed with 800 μl of substrate [1% (w/v) bovine serum albumin (BSA) in 0.025 M sodium citrate buffer, pH 3.2] and incubated at 37°C for 3 hours. The reaction was halted by the addition of 2.0 ml of 5% trichloroacetic acid (TCA) resulting in precipitation of undigested BSA. Tubes were kept at ice for 30 minutes and then centrifuged at 2000 rpm for 20 minutes. Proteolysis was determined by measuring the absorbance of soluble peptides at 280 nm. A control was run by mixing substrate to supernatant and reaction was immediately stopped by adding TCA. The absorbance value of control was subtracted from absorbance values of treated samples to obtain values for enzyme activity in test strains. Each experiment was conducted three times with three replicates per experiment and the mean absorbance values were considered for calculation of % reduction in the production of proteinase in treated samples over untreated control. Hemolysin assay The hemolysin production and its inhibition by oils in test strains were detected by using the method of Manns et al.[17] with some modifications. Briefly, Candida cells were grown in SDB [8% (w/v) glucose] containing 0.5× and 0.25× MICs of test agents at 37°C for 4 days. The SDB without test agents was considered as untreated control. The broth cultures were centrifuged at 5000 rpm for 30 minutes to obtain supernatant. Furthermore, the culture supernatant and RBC suspension were mixed in 1:1 ratio and incubated at 37°C for 4 hours. Triton X-100 [0.1% (v/v) in Phosphate Buffer Saline (PBS)] was used as a positive control whereas 1% DMSO and PBS were used as negative controls. Tubes were centrifuged at 2000 rpm for 10 minutes and the absorbance of supernatant was read at 540 nm. The % hemolysis was calculated as [{(A-B)/(C-B)}×100], where A and B are the absorbance values of supernatant from the test sample and PBS (solvent control), respectively and C is the absorbance value of supernatant from the sample after 100% lysis. Each experiment was conducted three times with three replicates per experiment. Mean absorbance values were used to calculate % reduction in the production of hemolysin in treated samples over untreated controls. Statistical analysis All the experiments were performed three times with three replicates per experiment and data are expressed as Mean±Standard deviation. Statistical significance of the differences was determined by the one-way ANOVA test using Minitab (V.11.0 for Windows). Reduction in % CSH in test strains in the presence of oils was compared to untreated control by one-way ANOVA using Duncan's method. Similarly, log cfu/ml of treated samples (0.5× and 0.25× MICs) was compared to untreated control. P-values of ≤0.05 were considered as statistically significant.
Production of virulence factors in C. albicans Production of virulence factors such as GTF, CSH, proteinase and hemolysin in the test C. albicans strains is depicted in [Table 1]. All the tested strains showed GTF in the range of 52.33-89.33%, whereas 75% of the test strains were positive for CSH (24.76-66.21%CSH) as well as proteinase production (A 260 nm 0.45-1.61). Seven clinical strains form different sites of infection and two reference strains exhibited higher proteolytic activity with absorbance values >1.0. Hemolysin production was recorded in 50% of the test strains. Four strains C. albicans 01, 03, 06 and 09 exhibited % CSH >50 and also produced significant amount of proteinase; however, C. albicans 01 in addition could also produce significant amount of hemolysin.
MIC of oils and viability of candida cells at Sub-MICs MIC of test oils and viability of cells at their sub-MICs were evaluated against Candida strains that were used in the inhibition assays. The Candida strains exhibiting a significant amount of production of one or other tested virulence factors were selected for determining the potential of oils to inhibit virulence factors production. As presented in [Table 2], oils C. martini, citral, and cinnamaldehyde were strongly active with MIC range of 45-200 μg/ml. There was no significant decrease observed in the viability in terms of log cfu count of strains treated with 0.5× and 0.25× MICs of oils compared to untreated control [Table 3].
Effect of essential oils on germ tube formation Oils S. aromaticum, C. verum and their respective major active compounds eugenol and cinnamaldehyde were highly effective at both the 0.5× and 0.25× MICs in inhibiting the GTF in C. albicans 04 exhibiting highest amount of GTF (89.33%) in untreated control. In this strain, GTF was reduced to 10.0, 11.20, 15.60, and 7.8% when treated with cinnamaldehyde (50 μg/ml), S. aromaticum (100 μg/ml), C. verum (200 μg/ml) and eugenol (200 μg/ml), respectively [Figure 1]. Other test oils were least effective in inhibiting GTF at tested sub-MICs.
Effect of essential oils on cell surface hydrophobicity As evident from [Table 4], CSH in test strains was affected in varying capacity when treated with sub-MICs of oils. Cinnamaldehyde, eugenol, and citral at both the 0.5× and 0.25× MICs were significantly ( P0≤0.05) effective in reducing the CSH in one or other strains. % CSH of C. albicans 04 was reduced from 61.75 to 28.89 and 19.26 when treated with cinnamaldehyde at 25 and 50 μg/ml, respectively. Test oils at 0.5× MIC significantly reduced the %CSH in C. albicans 07 from 62.74 to in the range of 11.56-24.54 and the order of efficacy of oils in terms of effective concentration required (12.5 and 22.5 μg/ml) was cinnamaldehyde >S. aromaticum >C. citratus >C. verum >eugenol >citral > geraniol. Oil C. martini was ineffective in reducing the CSH in test strains.
Effect of essential oils on the production of proteinase A ≥ 70% reduction in the production of proteinase was recorded in the test strains treated with both the sub-MICs of test oils [Table 5]. On the basis of effective concentration (45-100 μg/ml), reduction of proteinase production in C. albicans 04 by oils was recorded in the order of citral > geraniol > C. martini > S. aromaticum > cinnamaldehyde > C. citratus > C. verum > eugenol.
Effect of essential oils on the production of hemolysin The oils C. verum, S. aromaticum, cinnamaldehyde, eugenol and geraniol at tested sub-MICs showed ≥50% reduction in the production of hemolysin in one or other test strains. Cinnamaldehyde was most inhibitory and showed reduction of 85.20%, 67.18%, and 77.52% in the hemolysin production by test strains at effective concentrations of 25, 50, and 100 μg/ml, respectively [Table 5].
Development of new and effective antifungal agents remains a challenge for researchers and has encouraged the search for new alternative strategies to combat Candida infections. One approach is to target production of virulence factors whose absence or inhibition, although not lethal for fungi but may results in attenuation/loss of virulence potential. Therefore, the present study was carried out to determine expression of four virulence factors namely germ tube formation, cell surface hydrophobicity, and extracellular production of proteinase and hemolysin in C. albicans. Further, certain essential oils and some of their major active compounds were studied for their efficacy to inhibit production of these virulence factors. In our study, a varying level of phenotypic expression of these virulence factors was observed among 20 strains of C. albicans tested. Expression and quantity of virulence factors produced by microorganisms are attributed to the specificity of strains, sites of infection it was isolated and their own genetic variations. For example blood isolates generally produce much higher levels of virulence factors than do isolates from wounds and urine. [18] Since strains under study were isolated from different clinical conditions such as patients of known vaginitis, candidemia, and urinary tract infections, so variation in amount and expression of virulence factors was obvious. Furthermore, on the basis of higher levels of production of virulence factors certain strains of C. albicans were selected to determine antivirulence potential of test oils/active compounds at sub-MICs (0.5× and 0.25× MIC) in terms of reduction in the amount of virulence factors produced. Test oils especially oils of S. aromaticum, C. verum, eugenol and cinnamaldehyde significantly inhibited the GTF in test strain. Our study has first time reported the anti-GTF activity by these oils in pathogenic strains of C. albicans. Other workers have reported oils of tea tree and other plants to inhibit GTF in C. albicans. [19],[20] The dimorphic transition from yeast to hyphae form, i.e., germ tube formation plays a pivotal role in the pathogenesis of C. albicans. GTF has been shown to contribute in increased adherence and invasiveness in models of skin, mucosal, and disseminated candidiasis. [21] Therefore, anti-GTF activites by these test oils have highlighted their promising antivirulence property. It is considered that hydrophobic interaction plays an important role in the adherence of opportunistic pathogen yeasts to eukaryotic cells and is an important event in the initiation of pathogenic process. [22] Considering this the test oils were also evaluated for their anti-CSH activity. Except C. martini, all the oils at tested sub-MICs significantly reduced the CSH in test strains. Similar to our findings, concentration-dependent inhibition of CSH in C. albicans by tannins from Stryphnodendron adstringens is reported by Ishida et al.[4] Hydrophobic cells are more adherent and resistant to phagocytosis, and therefore, more virulent than hydrophilic cells. In this regard, lowering of CSH by test oils suggests their importance in arresting the candidal colonization in pathogenesis. Production of proteinase is a highly regulated process and is transcriptionally coregulated with other virulence attributes in C. albicans. Our findings have revealed strong inhibition of proteinase production in C. albicans by all the test oils at both the sub-MICs. These observations are in agreement with the report of Hofling et al., [23] who have reported inhibition of proteinase production in C. albicans by six plant extracts. Proteinase activity helps Candida cells in vivo in obtaining nutrients, adherence, invasiveness, dimorphism, phenotypic switching, biofilm formation, and degradation of immune components such as antibodies, complements, and cytokines. [11],[15],[24] Therefore, test oils exhibiting strong antiproteinase activity could be considered as potential antipathogenic agents against Candida infections. Moreover, these test oils especially C. verum, S. aromaticum, cinnamaldehyde, and eugenol at 0.5×MIC significantly reduced the production of hemolysin in test strains. In our knowledge, there is no report on inhibition of hemolysin production by essential oils in Candida spp. Production of hemolysin in Candida cells is considered as an important factor to acquire iron from blood for the growth of pathogen during the systemic infection. Adherence of cells is also shown to be reduced in the iron limiting environment. [25] Therefore, antihemolysin activity of these oils may further aid to their potential in reducing the pathogenicity of Candida spp. These drug targets have been attempted by many researchers to obtain effective antipathogenic drugs against fungal infections. [6] In general, test oils or compounds inhibited production of one or more virulence factors tested in C. albicans strains in the order of proteinase > CSH > GTF > hemolysin. Oils S. aromaticum, C. verum, eugenol, and cinnamaldehyde were highly effective at 0.5× as well as 0.25× MICs in reducing the production of all the four virulence factors tested. Furthermore, since different virulence factors are expressed in varying amount in a strain, the efficacy of oils to inhibit these virulence factors is also varying. However, it is to be considered that not all the virulence factors are expressed in a pathogen during infection [26] and inhibition of even a single virulence factors by an agent could be of practical significance. Moreover, eugenol constitutes 74.32% in oil of S. aromaticum and cinnamaldehyde shares 79.10% of C. verum as a major active ingredient as reported elsewhere. [27] Activity of these ingredients being at par with their respective oils indicates that mechanistic role of these oils is attributed to their major active ingredients and other minor compounds are of less importance. Therefore, oils exhibiting broad spectrum antivirulence activity may be potential candidates for development as antipathogenic drugs. Essential oils interrupt cell walls, cell membranes, nuclear and mitochondrial membranes, and respiration in fungal cells. [4],[26],[27] It has been suggested that where membrane integrity is adversely affected, membrane associated functions may also be compromised. [28] Membrane associated enzymes such as chitin, mannan, 1,3-β-d-glucan synthase and adenosine triphosphatase (ATPase) are involved in GTF [29] in C. albicans. Therefore, loss of these enzymes together with the lack of energy production in Candida cells treated with oils may prevent the morphological transition from blastoconidia to hyphae, a form that is more adherent and invasive. In addition, mannoproteins acting as principle adhesion proteins are associated with the cell wall [22] and hemolytic factors produced by C. albicans are also believed to be mannoproteins. [30] Therefore, interference in the production of these proteins upon damage of cell walls in fungal cells treated with oils may result in reduced cell adherence and low hemolytic activity. Furthermore, essential oils being hydrophobic in nature will aggregate more hydrophobic cells and severe action of oils might be achieved onto cell walls and membranes of Candida cells. This could result in the damage of hydrophobic interaction bonds, and therefore, lowering of cell wall associated CSH. However, the exact enzymatic or genetic interferences that lead to inhibition of tested virulence factors by essential oils are unknown and need to be explored. Moreover, inhibition of virulence in Candida sp. by oils at lower concentrations would result in no survival pressure of killing in cells, therefore minimizing the chances of developing resistance. This approach will be especially effective in immunosuppressed patients, where drug resistant strains lead to relapses of infection due to their virulence potential and result in high mortality rate accounting for nosocomial fangemia. [31],[32]
Our in vitro study has highlighted the broad spectrum antivirulence potential of test essential oils especially S. aromaticum, C. verum, eugenol, and cinnamaldehyde against the strains of C. albicans. The antivirulence property of these oils forms a new basis of validation for their traditional uses against fungal infections. However, further in vivo studies are needed to uncover their antipathogenic efficacy against the strains of C. albicans.
[Figure 1]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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