Abstract: The influence of cultural conditions that affect GPX production in Candida albicans grown in Lee's medium was investigated. Optimum temperature and pH for GPX activity were 25°C and 7.2, respectively. Substrate specificity for C. albicans. Glutathion peroxidase was in the order of cumene hydroperoxide>t-butyl hydroperoxide> hydrogen peroxide>benzoyl peroxide. Aeration as well as large head space volume enhanced the growth of C. albicans and GPX production. Arabinose and ammonium sulphate significantly increased the GPX synthesis. Among nitrogen sources, polypeptone enhanced both the growth and GPX synthesis. Various cellular activities are regulated by the level of GSH. Therefore, the level of GPX might be used as one of the criteria in developing new drugs against Candida albicans.
INTRODUCTION
Glutathione peroxidase (GPX; EC 1.11.1.9) is one of the important antioxidant enzymes that protects the cell against oxidative damage. Glutathione peroxidase converts the reduced glutathione (GSH) to its oxidized form (GSSG) in the following reaction:
In animals, this enzyme was studied extensively and found in two forms namely selenium (Se) dependent GPX and selenium independent GPX (Wendel, 1980; Arthur, 2000; Li et al., 2004). The Se-dependent enzyme contains selenocysteine at the active site and catalyzes the reduction of peroxides as well as organic hydroperoxides with glutathione as its hydrogen donor (Brown et al., 2007; Fu et al., 2007). The Se-independent GPX is active with organic hydroperoxides only and is identical with some GSH transferase (Prohaska, 1980). Missall et al. (2005) reported two glutathione peroxidases in the fungal pathogen Cryptococcus neoformans are expressed in the presence of specific substrates. In mammalian studies selenium is known to protect the cells against the toxic effects of heavy metals (Nordberg et al., 1978). The activity of GPX in general, is known to be influenced by age, species and several environmental and nutritional factors (Manavathu et al., 1996).
Candida albicanis an opportunistic yeast belongs to the class fungi Imperfecti and is present as a part of normal flora. This opportunistic yeast is capable of invading and colonizing the body tissues when the host immunity is impaired (Prasad, 1991). This organism releases different metabolites into the bloodstream, causing varying symptoms such as lethargy, chronic diarrhea, yeast vaginitis, bladder infections, muscle and systemic candidiasis (Prasad, 1991).
Candida albicans possesses two distinct morphological forms as yeast and mycelium and this phase transition plays a major role in its pathogenicity (Prasad, 1991). These morphologic changes are due to various environmental and nutritional conditions such as pH and temperature, carbon and nitrogen sources (Gunasekaran et al., 1995). This study reports on the influence of cultural conditions on growth and GPX production in C. albicans cells grown in a synthetic medium amended with various testing compounds (such as nutrients, inducers and inhibitors) under different environmental (temperature and oxygen stress) conditions.
MATERIALS AND METHODS
Organism and Culture Medium
The strain of Candida albicans (3153A) was maintained in Sabouraud
dextrose agar (Difco) at 37°C and utilized in the present investigation
(Gunasekaran et al., 1995). Each 250 mL Erlenmeyer
flask containing Lee synthetic medium (Lee et al.,
1975) was seeded with 1.0 mL of yeast suspension and incubated in a rotary
shaker at 100 rpm and 250C for 72 h or the time specified in each
set of experiments.
Preparation of Cell Free Extract
Yeast cells grown in Sabouraud dextrose broth were harvested at 72 h of
growth by centrifugation at 7,000x g washed with 0.5 M of potassium phosphate
buffer, pH 7.5 containing phenylmethylsulfonyl fluoride (1 mM), EDTA (4 mM)
and 2-mercaptoethanol (5 mM) and resuspended in the same buffer. Cells were
homogenized in the same buffer and the homogenate was centrifuged at 20,000x
g for 20 min at 4°C. The supernatant was used as the cell free extract for
enzyme assay.
Enzyme Assay
Glutathione Peroxidase Activity
Glutathione-peroxidase activity was measured in a coupled enzyme method
by measuring the decrease of NADPH at 340 nm (Aisaka et
al., 1983). The reaction mixture, which initially contained 570 μL
of 0.1 M potassium phosphate buffer with 1 mM of EDTA (pH 7.0), 100 μL
of crude enzyme, one hundred microlitters of 10 mM GSH and 30 μL of glutathione
reductase from Baker's yeast (50 units of lots [129H7465 and 78H7430]) was incubated
at 37°C for 10 min. One hundred microliters of pre-warmed 12 mM t-butyl
hydroperoxide was incubated accordingly following incubation. One hundred microliter
of 1.5 mM NADPH were added to the pre-warmed assay mixture and the peroxide
independent consumption of NADPH was monitored every 30 sec for 3 min with a
Hitachi U-3000 spectrophotometer. The reaction was initiated by adding pre-warmed
12 mM t-butyl hydroperoxide to the assay mixture. The decrease in absorption
was monitored every 30 sec for 5 min. The GPX activity in the enzyme sample
was determined using the appropriate extinction coefficient and the liner slope
of the absorbance values obtained from the t-butyl hydroperoxide consumption
of NADPH (Flohe and Gunzler, 1984). Protein was measured
by the method of Bradford (1976). All the experiments
were repeated 3 times and the values represent the mean of triplicates and these
values are given in the Fig. 1-6.
Effect of Carbon and Nitrogen Sources on GPX Production
The organism was grown in Lee medium for 72 h at 25°C to study the influence
of four types of carbon sources arabinose, sucrose, glucose and gluconic acid
at two concentrations (1.25 and 10%, 86 and 690 mM, respectively) on GPX production
was tested. To determine the effect of different nitrogen source, C.
albicans was grown at 25°C for 72 h in Lee medium substituted for
ammonium sulphate, with potassium nitrate, urea, glutamic acid and the media
were prepared as previously described, with the addition of the respective nitrogen
at 38 mM concentration.
Effect of Temperature on GPX Production
To determine the optimum temperature for GPX production, the organism was
grown at four different temperatures (15, 25, 37 and 45°C) for 72 h in Lee
medium, harvested and the crude enzyme extracts were assayed as described earlie.
Effect of Oxygen Tension on GPX Production
The effect of oxygen tension on GPX production, four sets of 250 mL flasks
containing 15, 25, 50 and 100 mL of Lee medium were prepared and inoculated
with yeast cells and grown as a shake culture in yeast form at 25°C for
72 h.
Effect of Peroxides on GPX Production
To determine the substrate specificity of GPX, five types of peroxides (cumene
peroxide, t-butyl peroxide, hydrogen peroxide, potassium peroxide and benzoyl
peroxide) at 12 mM concentration were measured.
Kinetics of Growth on GPX Activity
For determination of the changes in GPX activity at different growth phases,
flasks with Lee medium were inoculated with C. albicans and incubated
at 25°C for 120 h. At 24 h interval cell growth was measured.
Effect of pH on GPX Production
To determine the influence of pH on GPX production, the yeast was grown
in Lee media with pH values ranging from 3.0 to 8.5 at 25°C for 72 h. At
the end of growth period, cells were harvested and the dry weight was determined.
Effect of Media
To study the influence of media on the growth and GPX activity in C.
albicans , the organism was grown in three types of media, Lee synthetic
medium, Sabouraud Dextrose Broth (SDB) and Tryptic Soy Broth (TSB) were prepared
as per manufacturer's instructions (Difco) and grew the organism at 25°C
for 72 h. The growth and GPX activity were measured.
RESULTS AND DISCUSSION
Among the four tested carbon source, glucose supported the maximum growth followed by sucrose. However, maximum GPX production was observed from the cells grown in arabinose followed by gluconic acid (Fig. 1). At higher concentration GPX production was reduced regardless of the type of carbon source.
The influence of various nitrogen sources on GPX production revealed that polypeptone supported the maximum growth and enzyme production (Fig. 2). Among the other nitrogen sources, the least amount of growth and enzyme production was found in cells grown in potassium nitrate.
| Fig. 1: | Effect of carbon source on the GPX synthesis in C. albicans |
| Fig. 2: | Effect of nitrogen source on the GPX synthesis in C. albicans |
| Fig. 3: | Effect of temperature on the GPX synthesis in C. albicans |
Under assay conditions, optimum temperature for growth and GPX production was found to be 25°C (Fig. 3). Very little difference was observed in enzyme activity between the cells grown at other temperatures.
The influence of growth of C. albicans on GPX activity revealed that the enzyme activity reached its maximum at 48 h followed by a steady decrease in later growth periods. The highest observed GPX activity during the early part of C. albicans growth could be due to its active metabolism.
The study on substrate specificity of Candida GPX, on different hydroperoxides showed that cumene peroxide was the preferred substrate for the enzyme as compare to other substrates tested (Fig. 4). The least effective substrate for GPX was found benzoyl peroxide.
| Fig. 4: | Substrate specificity of GPX in C. albicans |
| Fig. 5: | Effect of oxygen tension on the GPX synthesis in C. albicans |
The results on the influence of oxygen tension on GPX are shown in Fig. 5. Maximum GPX activity was observed from cells grown in flasks containing 25 mL of medium in either shaking or stationary conditions. However, more enzyme activity was observed from the shake culture. The enzyme production was reduced as the volume of medium increased regardless of the type of culture. Similar findings were observed with GPX from Mucor hiemalis (Aisaka et al., 1983).
In order to determine the inductive effect of the substrates of GPX, cumene peroxide, t-butyl hydroperoxide and hydrogen peroxide were added to the warm medium under sterile conditions. For comparison, cells were grown in Lee medium without any of the tested with compounds. Maximum induction was observed by cumene peroxide as compared to other hydroperoxides (Fig. 6). Although, an increase in enzyme activity was found at higher concentration (1 mM), except in the case of cumene peroxide, there is no correlation between the concentration and the activity.
Maximum growth occurred between pH 6.0-6.5 and the GPX activity was found to be maximum at pH 6.0. Among the three tested media, maximum growth found maximum in SDB followed by Lee medium. On the other hand GPX activity was maximum in Lee medium followed Sabouraud dextrose broth and GPX activity were maximum in Lee medium followed by TSB. The factors that influence the synthesis and the properties of C. albicans GPX are very similar to other organisms reported earlier (Sundquist and Fahley, 1988; Stohs, 1990).
| Fig. 6: | Effect of peroxide on the GPX synthesis in C. albicans |
CONCLUSION
In the present study, we found that GPX production was influenced by various cultural and nutritional factors. Future experiments are planned to study on the mechanism(s) of antioxidants on GPX as well as the purification and characterization of GPX from Candida albicans.
ACKNOWLEDGMENTS
The authors wish to acknowledge support from the US Department of Education (P120A060075), National Institute of Health (K01 GM080578) National Science Foundation (Grant HRD 9253037), (Grant HRD 0927876) NASA (Grant NAG 2-6015), Howard Hughes Medical Institute (Grant 71194-527-802) and UNCF.