Cinnamic aldehydes affect hydrolytic enzyme secretion and morphogenesis in oral Candida isolates

Microbial Pathogenesis 52 (2012) 251e258

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Cinnamic aldehydes affect hydrolytic enzyme secretion and morphogenesis in oral Candida isolates Sheikh Shreaz a, Rimple Bhatia a, Neelofar Khan a, Indresh Kumar Maurya c, Sheikh Imran Ahmad b, Sumathi Muralidhar d, Nikhat Manzoor a, Luqman A. Khan a, * a

Department of Biosciences, Jamia Millia Islamia, New Delhi 110025, India Department of Applied Sciences & Humanities, Jamia Millia Islamia, New Delhi 110025, India School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India d Regional Sexually Transmitted Disease Centre, Safdarjung Hospital, New Delhi 110029, India b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 August 2011 Received in revised form 21 November 2011 Accepted 30 November 2011 Available online 20 December 2011

Effect of cinnamaldehyde (CD), 4-hydroxy-3-methoxy cinnamaldehyde (HMCD) and 3,5-dimethoxy-4hydroxy cinnamaldehyde (HDMCD) on growth and virulence factors of standard (Candida albicans 90028) and 26 oral isolates of C. albicans has been investigated. Growth was significantly inhibited by all three compounds in both solid and liquid medium, no systematic difference was observed between various isolates. MIC90 ranged from 125 to 450 mg/ml for CD, 100e250 mg/ml for HMCD and 62.5e125 mg/ml for HDMCD. All oral isolates were found to be proteinase and phospholipase secretors, both proteinase and phospholipase secretion was significantly inhibited by all the three tested molecules. No systematic difference in secretion or its inhibition was observed between standard and oral isolates as also between various isolates. Average drop in proteinase and phospholipase secretion caused by ½ MIC of CD was 33% and 28%, HMCD; 46% and 44%, HDMCD; 59% and 54%. The standard strain and all the 26 oral isolates displayed morphogenesis under triggering experimental conditions; no difference was seen between standard and various isolates. In the absence of test compounds hyphae development at 300 min was 83% for standard strain whereas average hyphae development for oral isolates was 85%. Average hyphal transition was suppressed by all tested compounds. At ½ MIC concentration at 300 min average hyphal transition of standard and oral isolates was CD; 49% and 57%, HMCD; 45% and 38%, HDMCD; 5% and 5%. Average haemolytic activity of the three tested compounds varied from 10 to 15% at their highest MIC compared to 20% shown by fluconazole at typical MIC of 30 mg/ml. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Cinnamaldehyde Proteinase Phospholipase Yeastehyphal transition Candida albicans

1. Introduction Candida species are the most common opportunistic fungal pathogens in humans, with Candida albicans being the most prevalent pathogen in mucosal and systemic fungal infections [1]. Oropharyngeal candidiasis is predominantly caused by C. albicans [2e4]. Current line of antifungals: polyenes and azoles have serious host toxicity and organisms are fast developing resistance towards them [5]. Several virulence factors contribute to the pathogenicity of Candida spp., including the production of hydrolytic enzymes: proteinases and phospholipases [6,7]. Phospholipases attack phospholipids of cell membranes and facilitates invasion of yeast. It has been demonstrated that C. albicans strains with increased

* Corresponding author. Tel.: þ91 11 2698 1717x3410; fax: þ91 11 2698 0229. E-mail address: [email protected] (L.A. Khan). 0882-4010/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2011.11.005

phospholipase activity are associated with higher virulence in murine models [8]. Secretory aspartyl proteinases (Sap) constitute a family of enzymes that are able to degrade several physiologically important substrates such as albumin, immunoglobulin and skin proteins [9]. Many investigators have suggested that the virulence of C. albicans could be attributed, atleast in part, to the filamentous form following a yeastehyphal transition and found a higher frequency of hyphae phenotype of C. albicans in fungal infection sites [10]. Hyphal form is involved in the early stages of invasion, penetrating into the tissues [11], adhering to epithelia [12], and avoiding of phagocytosis [13]. Given the increased resistance of pathogenic microorganisms to currently used antibiotics and chemotherapeutics, there is a need for alternative prevention and treatment products for oral infections. Natural products derived from plants are considered as a significant source of biologically active compounds. Cinnamaldehyde occurs naturally in the bark of Cinnamon trees and other species of genus Cinnamomum like camphor and cassia.

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Cinnamaldehyde is effective in inhibiting growth of bacteria, yeast and filamentous moulds and is tentatively thought to act by inhibiting ATPases [14], cell wall biosynthesis [15], and by changing membrane structure and integrity [16]. Functioning of PM-ATPase and membrane integrity is intimately associated with hydrolytic enzyme secretion and morphogenesis [17]. Cinnamaldehyde have been only curiously explored with respect to growth and no reports are available on its effect on pathogenecity of C. albicans [16,18]. Our previous study has shown that cinnamaldehyde possess a good in vitro antifungal activity against standard C. albicans and C. tropicalis strains [19]. In the present study, we have explored the effect of cinnamaldehyde (CD) and its two derivatives: 4-hydroxy-3-methoxy cinnamaldehyde (HMCD) and 3, 5-dimethoxy-4-hydroxy cinnamaldehyde (HDMCD) (Fig. 1 A, B, C) on standard strain C. albicans ATCC 90028 and 26 oral isolates with respect to growth, hydrolytic enzyme secretion and morphogenesis. Results obtained indicate that C.D and its derivatives significantly decrease hydrolytic enzyme secretion and yeast to hyphal transition in all isolates. 2. Results 2.1. Minimal inhibitory concentration Table 1 summarizes the in vitro susceptibilities of standard C. albicans ATCC 90028 and twenty six C. albicans oral isolates. The data are reported as MICs required to inhibit 90% growth as compared to control (absence of any test compound) for each isolate. The test compounds were found to be active against all Candida isolates. The MIC90 of CD against twenty six isolates of C. albicans ranged 125e450 mg/ml, HMCD ranged 100e250 mg/ml, and that of HDMCD ranged 62.5e125 mg/ml. 2.2. Spot assay Drug susceptibility testing by spot assay revealed that all the 26 oral isolates are highly sensitive towards CD, HMCD and HDMCD. Fig. 2 gives spot assay results of standard Candida strain and three randomly chosen oral isolates. Significant and pronounced effect is observed for other twenty one tested isolates (data not shown). HDMCD was most effective in growth inhibition followed by HMCD and CD. These results correlate very well with MIC90 determined in liquid medium. 2.3. Proteinase secretion When grown on plates for 2 days with bovine serum albumin, analysis of proteinase secreted by different C. albicans oral isolates in presence and absence of sub-MIC concentrations of CD, HMCD and HDMCD and respective mean PZs were determined. Fig. 3 gives typical photographs when five different C. albicans isolates were treated with sublethal concentration (½ MIC and ¼ MIC) of CD, HMCD and HDMCD. In all the tested isolates of C. albicans, compounds lead to increase in PZ in a concentration-dependent manner. No systematic difference was seen between various

CHO

CHO HO

A

CHO

MeO HO

OMe

OMe

B

C

Fig. 1. Chemical structures of (A) CD; (B) HMCD; (C) HDMCD.

isolates. Fig. 4 gives bar diagrams of proteinase secretion results of randomly selected ten of twenty six C. albicans isolates following exposure to two sub-inhibitory concentrations of the test compounds. The average control PZ was 0.35 cm. The PZ increased to 0.44, 0.51 and 0.68 cm, respectively, at ¼ MIC of CD, HMCD and HDMCD representing a drop of 21%, 31% and 49% in proteinase secretion. The respective drop in proteinase secretion at ½ MIC values of CD, HMCD and HDMCD was 33%, 46% and 59%, respectively. 2.4. Phospholipase secretion Fig. 5 gives typical photographs obtained when three different C. albicans isolates were tested for phospholipase activity in the presence and absence of sub-inhibitory concentrations of CD, HMCD and HDMCD. The opaque zones produced on plates containing 10% egg yolk enrichment were clearly seen. Fig. 6 summarizes the results of phospholipase secretion of randomly selected ten of twenty six isolates following exposure to two sub-inhibitory concentrations of CD, HMCD and HDMCD. The average control PZ value was 0.41 cm. Exposure to ¼ MIC of three compounds lead to significant increase in PZ value. The PZ increased to 0.47, 0.56 and 0.70 cm, representing a drop of 13%, 28% and 41% in phospholipase secretion. Respective figures at the ½ MIC values were 28%, 44% and 54%. HDMCD was found to be most effective in suppressing phospholipase secretion in all the tested yeast isolates. 2.5. Yeast to hyphal transition Fig. 7A and B shows a typical, randomly chosen field at zero time and 300 min following incubation of C. albicans STD No 05 in Lee’s media at 37  C without the addition of any test compound.

Table 1 Minimum inhibitory concentration (MIC90) of CD, HMCD and HDMCD against oral C. albicans isolates. Oral isolates

STD Noa

C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.

05 27 14 24 25 26 15 22 11 18 21 9 6 17 12 16 28 4 7 10 13 19 20 29 50 1

albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans

MIC90 (mg/ml) CD

HMCD

HDMCD

200 250 250 250 400 400 250 250 125 400 450 250 400 450 450 400 400 450 225 250 250 400 400 250 250 400

100 125 200 200 225 250 200 200 100 250 225 200 200 125 200 250 250 250 200 125 200 200 225 125 125 250

62.5 100 112.5 100 100 125 100 100 62.5 125 100 125 100 100 112.5 62.5 125 125 100 112.5 100 125 100 112.5 112.5 125

MIC90 (mg/ml) of CD, HMCD and HDMCD against standard strain C. albicans 90028 was 200,150 and 100 respectively. a Sexually Transmitted Disease.

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Fig. 2. Spot assay profiles of C. albicans cells in presence of different concentrations of CD, HMCD and HDMCD.

Fig. 7CeE shows randomly chosen field for hyphal development at 300 min following addition of ¼ MIC concentrations of CD, HMCD and HDMCD in culture medium of C. albicans STD No 05. Fig. 7 FeH gives typical photographs obtained for hyphal development at 300 min following addition of ½ MIC concentrations of the three

compounds in culture medium of tested C. albicans cells, respectively. Table 2 shows average percent transition from yeast to hyphae of five randomly selected isolates following exposure to two sub-inhibitory concentrations of CD, HMCD and HDMCD. Table 2

Fig. 3. Proteinase production by standard and oral isolates of C. albicans in absence (control) and presence of sub-MIC concentrations of CD, HMCD and HDMCD.

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Fig. 4. Average proteinase production (diameter of colony/Total diameter) by ten oral isolates of C. albicans when grown in ¼ and ½ MIC of CD, HMCD and HDMCD. PZ for standard C. albicans 90028 was control: 0.43, CD (¼ and ½ MIC); 0.46 and 0.53, HMCD (¼ and ½ MIC); 0.50 and 0.63, HDMCD (¼ and ½ MIC); 0.70 and 0.87 respectively.

Fig. 5. Phospholipase production by standard and oral isolates of C. albicans in absence (control) and presence of sub-MIC concentrations of CD, HMCD and HDMCD.

Fig. 6. Average phospholipase production (diameter of colony/Total diameter) by ten oral isolates of C. albicans when grown in ¼ and ½ MIC of CD, HMCD and HDMCD. PZ for standard C. albicans 90028 was control: 0.52, CD (¼ and ½ MIC); 0.60 and 0.69, HMCD (¼ and ½ MIC); 0.69 and 0.81, HDMCD (¼ and ½ MIC); 0.81 and 0.90 respectively.

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Fig. 7. Yeast phenotype observed in Lee’s media at 37  C of a representative strain STD No 05. Hyphae development in absence of test compounds (control): 0 min (A), 300 min (B). Hyphae development when grown with ¼ MIC of CD (C) ¼ MIC of HMCD (D) and ¼ MIC of HDMCD (E) at 300 min. Hyphae development when grown with ½ MIC of CD (F) ½ MIC of HMCD (G) and ½ MIC of HDMCD (H) at 300 min.

Table 2 Yeast to hyphal transition in randomly chosen five of twenty six oral isolates of C. albicans when treated with two sub-inhibitory concentrations of CD, HMCD and HDMCD.

Control CD

¼ MIC ½ MIC

HMCD

¼ MIC ½ MIC

HDMCD

¼ MIC ½ MIC

Ia Iaa Ia Iaa Ia Iaa Ia Iaa Ia Iaa Ia Iaa Ia Iaa

0 min

60 min

120 min

180 min

240 min

300 min

e

6.4 2.6  0.6 e e e e e e e e e e e e

7.0 22  0.7 5 11.6  0.6 e e 4.0 8.0  0.5 e e e e e e

7.6 44.98  0.8 5.3 50.7  0.6 3.7 32.2  1.4 4.4 35.8  0.86 3.2 24.8  1.1 2.5 5.2  0.5 e e

8.2 75.24  2.0 5.6 56.4  1.1 4.1 43.0  0.7 4.9 41.9  0.8 3.7 37.46  1.24 2.95 16.8  0.6 1.4 2.6  0.9

10 85.2  2.8 6.0 71.1  3.3 4.5 57.8  0.6 5.1 42.8  0.8 4 38.0  0.9 3.2 20.0  0.0 1.9 4.7  0.4

e e e e e e e e e e e e

(e): represents the absence of cells. Ia: represents average length (mm) of randomly selected C. albicans isolates on the scale of 10. Iaa: represents average % of cells showing hyphal growth of randomly selected C. albicans isolates. For standard strain C. albicans 90028 hyphal length and %age of population showing hyphal were control; 10 and 38%, CD (¼ MIC); 5.7 and 67%, CD (½ MIC); 3.2 and 49%, HMCD (¼ MIC); 4.3 and 52%, HMCD (½ MIC); 3.0 and 45%, HDMCD (¼ MIC); 2.5 and 30%, HDMCD (½ MIC); 2.3 and 5.1% respectively.

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also gives average hyphal length on relative basis where length of hyphae at 300 min in control experiment is taken as 10 units. All the three compounds inhibited the transition of tested Candida cells from yeast to hyphae. In control cells hyphae was observed at 60 min while in ¼ MIC treated CD, HMCD and HDMCD cells, time delays upto 120 min, 120 min and 180 min respectively were observed. In case of ½ MIC treated cells, time delay observed was upto 180 min, 180 min and 240 min. Hyphae percentage as well as length of hyphae goes on increasing in all the strains till it reaches to maximum at 300 min at which control cells shows 85% transition, respective figure for cells treated with ¼ MIC and ½ MIC of CD, HMCD and HDMCD was 71%, 42%, 20% and 57%, 38%, 4.7%, respectively. Hyphae continued to grow in length till 300 min beyond which no change was observed in control experiments. Hyphal length was profoundly affected by all three tested compounds. Compared to 10 units in control cells, maximum hyphal length in the presence of ¼ MIC values of CD, HMCD and HDMCD at 300 min was only about 6.0, 5.1 and 3.2. Respective figure in case of ½ MIC, single decimal hyphal length was 4.5, 4.0 and 1.9, respectively. 2.6. Hemolysis Toxicity of the tested compounds was checked on fresh human red blood cells. Table 3 gives hemolysis caused by tested compounds and fluconazole at their highest MIC90 value. These values are 10.10%, 13.10% and 14.50% for CD, HMCD and HDMCD, respectively. Fluconazole at it highest MIC 30 mg ml1 caused 20.0% hemolysis. 3. Discussion Cinnamaldehyde, benzaldehyde and hydroxy and methoxy derivatives of benzaldehydes are employed as preservatives and are reported to have strong antifungal activity [14,20,21]. This is significant finding as cinnamaldehyde is a normal component of food and has a high human consumption in India. As Candida is normal resident of oral cavity and genitourinary tract, it receives direct exposure of cinnamaldehyde. Our findings suggest options for expanding the utility of plant derived principles as antifungal agents against oral infections. In this study, we have explored the effect of cinnamaldehyde and its hydroxy and methoxy derivatives on oral clinical isolates of Candida. Cinnamaldehyde is tentatively thought to act by inhibiting ATPase and changing membrane structure and integrity. PM-ATPase and membrane integrity is linked to hydrolytic enzyme secretion and morphogenesis in Candida. Cinnamaldehyde and its derivatives are found to be effective against all tested Candida oral isolates. Range of MIC90 decreased with increase in methoxy groups: 125e450 mg/ml for parent cinnamaldehyde, 100e250 mg/ml for 4-hydroxy-3-methoxy cinnamaldehyde, and 62.5e125 mg/ml for 3,5-dimethoxy-4hydroxy cinnamaldehyde. Table 3 Hemolysis caused by different agents at their highest (MIC90) value: Hemolysis was determined by an absorbance reading at 450 nm and compared to hemolysis achieved with 1% Triton X-100 (reference for 100% hemolysis). Data is mean of three experiments. Test compounds

Concentration

% Hemolysis

Cinnamaldehyde 4-hydroxy-3-methoxy cinnamaldehyde 3, 5-dimethoxy-4-hydroxy cinnamaldehyde Fluconazole

450 mgml1 250 mgml1

10 13

125 mgml1

14

30 mgml1

20

C. albicans is capable of producing proteinase [22] and phospholipases [8] that cause tissue damage and allow invasion of yeast. Cinnamaldehyde and its derivatives have been shown to have strong inhibitory effect on PM-ATPase of C. albicans [19,23]. An ATPase-dependent efflux mechanism has been suggested in the transport of Sap into membrane bound vesicles [24], and upregulation of such a transport mechanism is argued to explain enhancement of Sap activity. Significant decrease in proteinase and phospholipase secretion by CD and its derivatives can be attributed to ATPase-dependent efflux mechanisms [24]. Yeast to hyphal transition is intimately associated with Candidal pathogenicity. Cinnamaldehyde is found to be significantly suppressing transition even at ¼ MIC. HDMCD, the most active derivative of CD almost completely inhibited transition at ½ MIC concentrations. Dimorphism in yeast is also associated with functioning of ATPase [25]. Hyphal length is another pathogenitic attribute in Candida which facilitates invasion, HDMCD at concentration that of ¼ MIC reduces hyphal length by about 70%. Our results are also supported by Xie et al. [16] who showed that 0.05 mg cinnamaldehyde/ml could inhibit the germination of spores in Aspergillus flavus and Taguchi et al. [18] who suggested that cinnamaldehyde in the cassia preparation was the principal component responsible for the inhibitory activity of Candida mycelial growth. To conclude we show in this study that cinnamaldehyde, 4-hydroxy-3-methoxy cinnamaldehyde and 3, 5-dimethoxy-4hydroxy cinnamaldehyde are active against all oral Candida isolates. These essential oil components are found to be substantially inhibit key pathogenicity factors: Hydrolytic enzyme secretion and morphogenesis even at sub-MIC concentrations. These results taken together with their limited toxicity make them eligible for further development as antifungals.

4. Materials and methods 4.1. Strains and media All oral Candida isolates were obtained from Regional STD (Sexually Transmitted Disease) Centre, Safdarjung Hospital New Delhi, India. Samples were harvested in Sabouraud glucose agar (SGA) with 1% chloramphenicol (Hi Media, India); identification of C. albicans species was carried out through germinative tube development test, sugar assimilation and fermentation, and growth within a chromogenic medium (Hi Media, India). For experimental purposes all of the strains were grown on yeast extract, peptone and dextrose (YPD) medium containing 2% (w/v) glucose, 2% peptone, and 1% yeast extract (Hi Media, India). YPD agar plates containing 2.5% agar (Hi Media) in addition were used to maintain the culture. Cinnamaldehyde (CD), 4-hydroxy-3-methoxy cinnamaldehyde (HMCD) and 3, 5-dimethoxy-4-hydroxy cinnamaldehyde (HDMCD) were purchased from Aldrich (USA), whereas all inorganic chemicals were of analytical grade and were procured from E. Merck (India). The stock solutions of the test compounds were prepared in 1% dimethyl sulphoxide (DMSO), which had no effect on fungal growth.

4.2. Assessment of the MIC90 For microtitre assays, the minimum inhibitory concentration (MIC), defined as the lowest concentration (highest dilution) of CD, HMCD and HDMCD that causes 90% decrease in absorbance (MIC90) compared with that of control, was as determined by the National Committee for Clinical Laboratory Standards (NCCLS), document M27-A2, 2002 [26].

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4.3. Spot assay The yeast cells were grown overnight on yeast nitrogen base (YNB) medium containing 2% glucose and the respective auxotrophic supplements. The cells were then suspended in normal saline to an OD600 of 0.1 (A600). Five microliters of five fold serial dilutions of each yeast culture was spotted onto YNB plates in the absence (control) and in presence of different concentrations of CD, HMCD and HDMCD respectively. Growth differences were recorded following incubation of the plates for 48 h at 30  C as reported previously [27].

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at pH 6.5 to induce hyphae formation. To see the effect of sun-inhibitory concentrations of CD, HMCD and HDMCD on transition from yeast to hyphae, their desired concentrations were added to Lee’s medium. External pH of the medium was checked using Consort C832 Multi-parameter analyzer and adjusted every half hour to the original setting. Cell divergence was observed microscopically using Motic AE31 (Germany) by taking aliquots at different time intervals. At least 60 cells were counted on each coverslip, and all experiment was performed in triplicate on atleast three separate occasions. Percentage hyphae were obtained by ratio of the number of hyphae with total number of cells as reported earlier [30].

4.4. Proteinase assay 4.7. Hemolytic activity C. albicans strains were transferred to flasks containing 5 ml YPD, alone and along with different sub-inhibitory concentrations of CD, HMCD and HDMCD and incubated at 37  C for 18 h. Following incubation, 1.5 ml of the yeast culture were transferred to an eppendrof tube and centrifuged at 3000 rpm, for 5 min. The pellets obtained were washed twice by resuspension in saline and centrifuged under the same conditions to remove the residual culture medium. After standardizing the suspensions (at index of 5 on the MacFarland scale), volumes of 1 ml were plated, at equidistant points, on proteinase agar-BSA fraction V, 2 g; yeast nitrogen base w/o amino acids; ammonium sulfate, 1.45 g; glucose, 20 g; distilled water added to 1000 ml respectively. The plates containing C. albicans cells treated with different concentrations of test compounds for the detection of proteinase were incubated at 37  C for 2 days [28]. The effect on proteinase synthesis was determined by the formation of the transparent halo around the yeast colonies and enzyme activity was measured by dividing the diameter of the colony by the diameter of the colony plus zone of clearance as done earlier [29].

The hemolytic activities of the test compounds were determined on human red blood cells as described earlier [32]. Human erythrocytes from healthy individuals were collected in tubes containing EDTA as anti-coagulant. The erythrocytes were harvested by centrifugation for 10 m at 2000 rpm at 20  C, and washed three times in PBS. To the pellet, PBS was added to yield a 10% (v/v) erythrocytes/PBS suspension. The 10% suspension was then diluted 1:10 in PBS. From each suspension, 100 ml was added in triplicate to 100 ml of a different dilution series of test compounds (or fluconazole) in the same buffer in eppendorf tubes. Total hemolysis was achieved with 1% Triton X-100. The tubes were incubated for 1 h at 37  C and then centrifuged for 10 m at 2000 rpm at 20  C. From the supernatant fluid, 150 ml was transferred to a flat-bottomed microtiter plate (BIO-RAD, iMark, US), and the absorbance was measured spectrophotometrically at 450 nm. The hemolysis percentage was calculated by following equation: % hemolysis ¼ [(A450 of test compound treated sample  A450 of buffer treated sample)/(A450 of 1% Triton X  A450 of buffer treated sample)]  100.

4.5. Phospholipase assay 5. Statistical analysis Phospholipase activity assay was performed using method described by [28] with minor modification. C. albicans strains grown alone and along with different sub-inhibitory concentrations of CD, HMCD and HDMCD were centrifuged at 3000 rpm for 5 min. The pellets obtained were washed twice by resuspension in saline and centrifuged under the same conditions to remove the residual culture medium. After standardizing the suspensions (at index of 5 on the MacFarland scale), volumes of 1 ml were plated, at equidistant points, on phospholipase agar media-peptone, 10 g; glucose, 30 g; NaCl, 57.3 g; CaCl2 0.55 g; distilled water added to 1000 ml and 10% egg yolk enrichment 100 ml (HiMedia). The plates were incubated at 37  C for 2e4 days. The presence of phospholipase was determined by an opaque zone around the yeast colonies and enzyme activity (Pz) was measured by dividing the diameter of the colony by the diameter of the colony plus precipitation zone as reported earlier [30]. 4.6. Yeast to hyphal transition To initiate the growth for experimental purposes, C. albicans cells from agar slants were grown in amino acid rich Lee’s simplified medium in plastic Erlenmeyer flask and grown at 25  C upto the late log phase of growth (5  108 cells/ml). For Zinc depletion [31], cells were inoculated into fresh flask and grown upto stationary phase at 25  C and maintained for 48 h resulting in a synchronous population at a final density of 2.5  108 cells/ml. Stationary phase G1 singlets (1.5  109 cells), maintained as described above, were transferred to fresh 300 ml nutrient Lee’s media in a 500 ml Erlenmeyer flask. Cells were transferred to 37  C

All the experiments were performed in triplicate and the results were determined as Mean  Standard Deviation. Acknowledgements Sheikh Shreaz greatly acknowledges the financial support by ICMR (India) grant 45/93/09-Pha/BMS, (Senior Research Fellowship). This work was also supported by UGC grant No. 33-223/2007 to Dr. LAK. The authors are also thankful to Regional STD (Sexually Transmitted Disease) Centre, Safdarjung Hospital New Delhi, India, for providing the oral C. albicans isolates used in this study. We thank Shageer Ahmad (Lab. Assistant) from Department of Biosciences, Jamia Millia Islamia, New Delhi, India for providing valuable technical assistance. References [1] Pfaller MA, Diekema DJ, Jones RN, Messer SA, Hollis RJ. Trends in antifungal susceptibility of Candida spp. isolated from pediatric and adult patients with bloodstream infections: Antimicrobial Surveillance Program, 1997 to 2000. J Clin Microbiol 2002;40:852e6. [2] Calderone RA, Fonzi WA. Virulence factors of Candida albicans. Trends Microbiol 2001;9:327e50. [3] Webb BC, Thomas CJ, Willcox MD, Harty DW, Knox KW. Candida-associated denture stomatitis. Aetiology and management: a review. Part 2. Oral diseases caused by candida species. Aust Dent J 1998;43:160e6. [4] Banerjee U. Progress in diagonosis of opportunistic infection in HIV/AIDS. Indian J Med Res 2005;121:395e406. [5] Sanglard D, Ischer F, Parkinson T, Falconer D, Bille J. Candida albicans mutations in the ergosterol biosynthetic pathway and resistance to several antifungal agents. Antimicrob Agents Chemother 2003;47:2404e12.

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