Introduction
Sporothrix schenckii (S. schenckii), a dimorphic fungus, is distributed worldwide and known as the etiological agent of sporotrichosis. Various clinical manifestations of sporotrichosis are recognized viz. lymphocutaneous, localized cutaneous, disseminated, mucosal, skeletal and visceral. 1 Pathogenic potential of S. schenckii is not as much known on mammalian host due to scarcity of enzymatic information. Transaminases or aminotransferases being a part of protein is found in various fungi. Fungal aspartate aminotransferase is very active in free-living mycelium.2 Chalot et al. 3 explained that synthesis of amino acid precursors during TCA cycle operation is an essential step for aspartate and alanine synthesis through aminotransferase activities in ectomycorrhizal fungus Paxillus involutus. No study has been found that shows the activity of transaminases enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in S. schenckii, the causative agent of sporotrichosis. Potassium iodide (KI) has been used in the treatment of sporotrichosis, however, the exact mechanism of action remains unknown.4 Therefore, it was planned to undertake the present study to estimate the transaminases enzymes in the mycelial form of S. schenckii and to study the effect of KI with increasing concentration on the production of these enzymes in-vitro.
Material and Methods
This experimental study was conducted in the department of Microbiology in a tertiary care hospital. A standard strain of S. schenckii (ATCC 14284 / MTCC 1359) was procured from Institute of Microbial Technology, Chandigarh, India. A master culture was prepared by doing the subculture of S. schenckii from slope of Sabouraud’s dextrose agar (SDA) in 50 mL of YNB (Yeast nitrogen base, HiMedia, Mumbai) medium in a screw-capped bottle and was incubated at 25ºC. On the seventh day the suspension of YNB medium with S. schenckii was adjusted to 90% transmission at 540 nm on the photo-colorimeter. 4 Master culture thus prepared was used for subsequent analysis.
YNB medium was prepared and dispensed in 50 mL aliquots into 150 screw capped (160 mL capacity) bottles. Potassium iodide was added into the YNB medium in increasing concentrations in such a way so as to have final concentrations of 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and 12.8 gram percent of the medium. One bottle of YNB without KI was served as a control. Out of 150 bottles, 50 bottles each were used for three different days, i.e. 4th day (early-log period), 9th day (mid-log period), 14th day (peak of growth). Out of 50 bottles, 5 bottles were used as control (without KI) and rest were used for various concentration of KI. To reduce the error 5 bottles were used for each concentration of KI. One mL suspension of master culture was inoculated into each bottle and incubated at 25ºC for different time period respectively. The content of respective bottle was centrifuged at 6000 rpm for half an hour on the 4th, 9th and 14th day. The deposit thus obtained was suspended in 5.0 mL citrate buffer saline (0.15 mol/L Sodium chloride, 0.015 mol/L Sodium citrate, pH 7) and was centrifuged again at 6000 rpm for half an hour. The process was repeated twice to ensure proper washing. The deposit thus obtained was taken, dried in the folds of filter paper, weighed and was crushed finely in a tissue homogenizer. A 5% homogenate was prepared from each weighed tissue in ice-cold distilled water and was used for subsequent enzyme assay.1, 4 The transaminases enzymes, aspartate aminotransferase and alanine aminotransferase were determined by the method of Reitman and Frankel.5 The enzymes AST and ALT were estimated in per mg wt. of homogenate by following formula:
Statistical analysis
The statistical analysis was done using SPSS (Statistical Package for Social Sciences) Version 15.0 statistical Analysis Software. ANOVA (Analysis of Variance) test was used to compare the within group and between group variances amongst the study groups. Dunnett’s "t"-Test was used for comparing each experimental mean with the control mean.
Results
The enzymes aspartate aminotransferase and alanine aminotransferase were determined for three different days, 4th day (early-log period), 9th day (mid-log period) and 14th day (peak of growth) respectively.
(a) Aspartate aminotransferase (AST)
On day 4, an irregular yet generally regressive trend of mean aspartate aminotransferase values was observed with increasing concentrations. The trend was polynomial in nature with higher values at blank, KI 0.1 and 1.6 gram % and values were of middle order at KI 0.05, 0.2, 0.4 and 0.8 gram % concentrations. The values were of lower order at KI 3.2 and 6.4 gram % concentrations (Table 1). On day 9, a generalized declining trend of aspartate aminotransferase values was observed with increasing concentration. Mean values were 22.42 and 29.51 IU at blank and KI 0.05 gram % which declined to reach at 3.44 and 2.63 IU at KI 3.2 and 6.4 gram % concentrations respectively (Table 2). On day 14, a generalized declining trend of aspartate aminotransferase values was observed with increasing concentration. Mean values were 53.57 and 38.54 IU at blank and KI 0.05 which declined to reach at 5.41 and 2.55 IU at KI 3.2 and 6.4 gram % concentrations respectively (Table 3).
On day 4, mean aspartate aminotransferase level of control specimen was 12.4 ± 3.30 IU. The aspartate aminotransferase level of test specimens was ranged from 3.5 ± 0.80 (KI 6.4 gram %) to 12.4 ± 4.66 IU (KI 0.1 gram %) (Table 4). On day 9, mean aspartate aminotransferase level of control specimen was 22.4 ± 3.69 IU. Among different test concentrations mean aspartate aminotransferase levels were ranged from 2.6 ± 0.21 (KI 6.4 gram %) to 29.5 ± 7.31 IU (KI 0.05 gram %) (Table 4). On day 14, mean aspartate aminotransferase level of control specimen was 53.6 ± 8.46 IU. For test specimens, mean values were ranged from 2.5 ± 0.23 (KI 6.4 gram %) to 42.3 ±3.70 IU (KI 0.2 gram %) (Table 4). By comparing all the three days, in general there was an increase in the activity of enzyme with increase in duration of incubation 4th to 14th day (Table 5). The mean value was lower significantly for all the test concentrations as compared to control. No deposit obtained at the concentration KI 12.8 gram%.
Table 1
Expression of AST in S. schenckii (mould) on 4th day
Table 2
Expression of AST in S. schenckii (mould) on 9th day
Table 3
Expression of AST in S. schenckii (mould) on 14th day
Table 4
Expression of mean AST in S. schenckii (mould) at different time intervals (n=5 for each concentration)
Table 5
Comparison of change in mean AST levels in S. schenckii (mould) at different concentrations
(b) Alanine aminotransferase (ALT)
On day 4, an irregular but regressive trend of mean alanine aminotransferase levels with increasing concentration was observed with higher mean values at blank and KI 0.1 concentration and lower at KI 3.2 and 6.4 gram % concentrations (Table 6). On day 9, an irregular but regressive trend of alanine aminotransferase levels with increasing concentration was observed with higher mean values at KI 1.6 and 0.1 gram % and lower at KI 3.2 and 6.4 gram % concentrations (Table 7). On day 14, an irregular but declining trend of alanine aminotransferase levels was observed with increasing concentrations with peak values at blank and KI 0.2 gram % and minimum values at KI 3.2 and 6.4 gram % concentrations (Table 8).
On day 4, mean alanine aminotransferase level of control specimen was 17.5 ± 5.93 IU. The alanine aminotransferase levels of test specimen were ranged from 2.2 ± 0.00 (KI 6.4 gram %) to 15.4 ± 2.36 IU (KI 0.1 gram %) (Table 9). On day 9, mean alanine aminotransferase level of control specimen was 24.6 ± 3.59 IU. Among different test concentrations mean alanine aminotransferase levels were ranged from 2.7 ± 0.81 (KI 6.4 gram %) to 29.5 ± 2.75 IU (KI 1.6 gram %) (Table 9). On day 14, mean alanine aminotransferase level of control specimen was 32.6 ± 7.54 IU. For test specimens, mean values were ranged from 3.5 ± 1.37 (KI 6.4 gram %) to 29.6 ± 2.82 IU (KI 0.2 gram %) (Table 9). By comparing all the three days, an increase in mean alanine aminotransferase levels of control together with all test concentrations was observed (Table 10). The mean value of test specimens was lower significantly for all the test concentrations as compared to control. No deposit obtained at the concentration KI 12.8 gram%.
Table 6
Expression of ALT in S. schenckii (mould) on 4th day
Table 7
Expression of ALT in S. schenckii (mould) on 9th day
Table 8
Expression of ALT in S. schenckii (mould) on 14th day
Table 9
Expression of mean ALT in S. schenckii (mould) at different time intervals
Table 10
Comparison of change in mean ALT levels in S. schenckii (mould) at different concentrations
Discussion
Transamination is a process in which an amino group is transferred from an amino acid to an alpha-ketoacid. It is an important step in the metabolism of amino acids. Amino acids are synthesized in tissues by amination of ketoacids derived from carbohydrate, protein and fat metabolism. The enzymes responsible for transamination are called transaminases or aminotransferases. Botton and Dell6 had determined the glutamate dehydrogenase and aspartate aminotransferase enzyme activities in various fungal isolates (Laccaria laccata, Hebeloma westraliense, Scleroderma verrucosum and Pisolithus tinctorius) by electrophoresis when the fungal cultures were in the exponential growth phase, after 10-15 days incubation. In addition, two electrophoretic isozyme patterns, esterase and glutamate oxalate transaminase (GOT), were determined to measure variability among 10 isolates of Fusarium species. The activity of GOT was observed in the samples of Fusarium solani and Fusarium oxysporum and not detected in other isolates of Fusarium.7 They studied the activity of transaminases enzyme in several fungus. Neither they studied the activity of transaminases in S. schenckii nor studied the effect of KI with varied concentration on it.
In the present study, the activity of transaminases enzymes AST and ALT was estimated on 4th, 9th and 14th day of incubation for mycelial form respectively that included various phases of growth i.e. early log, mid log and exponential phase.
(a) Aspartate aminotransferase (AST)
In the mycelial phase, on day 4, mean aspartate aminotransferase level of control specimen was 12.4 ± 3.30 IU. At all the test concentrations mean value was lower than control. Statistically, significant difference in mean AST levels as compared to control was observed only at KI 3.2 and 6.4 gram % (p<0.05) (Table 4). On day 9, mean aspartate aminotransferase level of control specimen was 22.4 ± 3.69 IU. A continuous decline was observed at all the test concentrations and this decline was statistically insignificant (p > 0.05) for concentrations of KI 0.05, 0.1, 0.2, 0.4, and 0.8 gram %. A significant difference between test groups and control group was observed at concentrations of KI 1.6, 3.2 and 6.4 gram % respectively (Table 4). On day 14, mean aspartate aminotransferase level of control specimen was 53.6 ± 8.46 IU. A decline in mean AST for concentrations of KI 0.05 and 0.1 gram % followed by instant incline at KI 0.2 gram % and again followed by a continuous decrease in mean AST level was observed. Mean value of test specimens was significantly lower as compared to control for all the test concentrations (Table 4 & Figure 1).
Graph 1
Expression of mean aspartate aminotransferase (AST) in S. schenckii (mould) at different time intervals
By comparing all the three days, in general there was an increase in the activity of enzyme aspartate aminotransferase with increase in duration of incubation 4th to 14th day except concentration of KI 6.4 gram %. Between day 4 and day 9, an increase in mean aspartate aminotransferase levels of control as well as all test concentrations except KI 3.2 and 6.4 gram % was observed. This change was significant statistically too for all control as well as all the test concentrations except KI 1.6 and 6.4 gram % (Table 5). Between day 4 and 14, an increase in mean aspartate aminotransferase levels (p < 0.05) was observed for control as well as all the test concentrations except KI 6.4 gram %. The mean change was ranged from -0.96 ± 0.73 to (KI 6.4 gram %) to 41.20 ± 8.13 IU (control). The change was significant statistically too for all the concentrations except KI 3.2 gram %. Between day 9 and day 14 too, an increase in mean aspartate aminotransferase levels was observed for control and all the test groups except KI 6.4 gram %. At concentration of KI 6.4 gram %, a decline in mean AST levels was observed during the period. The change was also significant statistically for control, KI 0.2, 0.4 and 0.8 gram % (Table 5).
(b) Alanine aminotransferase (ALT)
In the mycelial phase, on day 4, mean alanine aminotransferase level of control specimen was 17.5 ± 5.93 IU. For all test concentrations mean value was lower as compared to control. Statistically, significant difference in mean alanine aminotransferase levels as compared to control was observed at concentration of KI 0.05, 0.4, 0.8, 3.2 and 6.4 gram % respectively (p<0.05) (Table 9). On day 9, mean alanine aminotransferase level of control specimen was 24.6 ± 3.59 IU. A continuous decrease in the mean levels was observed in test concentrations from KI 0.05 gram % to onward except KI 0.8 and 1.6 gram %. A significant difference between test groups and control group was observed at concentrations of KI 0.4, 3.2 and 6.4 gram % respectively. On day 14, mean alanine aminotransferase level of control specimen was 32.6 ± 7.54 IU. A continuous increase in the mean test level was observed for KI 0.05, 0.1 and 0.2 gram % followed by a decrease in the mean test values. Mean value of test specimens was lower as compared to control for all the test concentrations (Table 9 &Figure 2).
Graph 2
Expression of mean alanine aminotransferase (ALT) in S.schenckii (mould) at different time intervals
By comparing all the three days, in general there was an increase in the activity of enzyme alanine aminotransferase with increase in duration of incubation 4th to 14th day in control and test concentrations except KI 0.05, 0.1, 0.8 and 1.6 gram % (Table 10). Between day 4 and day 9, an increase in mean alanine aminotransferase levels of control as well as all test concentrations was observed. This change was significant statistically too for all the groups except control, KI 0.4, 3.2 and 6.4 gram %. Between day 4 and 14, an increase in mean alanine aminotransferase levels was observed for control as well as all the test concentrations. The change was significant statistically too for control, KI 0.05, 0.2 and 0.4 gram %. Between day 9 and day 14 too, an increase in mean alanine aminotransferase levels was observed. The change was also significant statistically for all the groups except KI 0.1 and 6.4 gram % (Table 10).
Conclusion
In this study, an increase in mean aspartate and alanine aminotransferase levels of control as well as all test concentrations was observed but at all these concentrations mean value of test concentrations was lower as compared to control. It indicates that KI has inhibitory effect on the growth of S. schenckii and this has led to decrease in activity of transaminases. This effect along with other defense mechanisms of the body may be the mode of action of KI in the treatment of sporotrichosis.
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