Study On Antioxidant Enzymatic Activities Of T.asahii

BackgroundFollowed with aerobic respiratory metabolism and energy generation systems, ROS take shape in the cells. ROS mainly include superoxide anion(O2?ˉ),hydrogen peroxide(H2O2),hydroxyl radical(?OH)),and so on[1]. ROS have highly mass destruction which not only affect many cellular functions by damaging nucleic acids, oxidizing proteins and causing lipid peroxidation, but also are coming the important factors of apoptosis,mutagenesis, tumorigenesis and ageing[1]. Many studies(including bacterium and fungus) have demonstrated that other sources of ROS include microsomal metabolism and the respiratory burst produced by phagocytes as part of the process of killing bacteria and fungus during their infection[1]. Some research about antifungals drugs also proved that the azole, polyene and echinocandin could induce the information of intracellular ROS[2].In order to servive from the deleterious ROS, organisms have evolved a variety of ways to either escape or repair these oxidant damage which contribute to the process of infection and pathopoiesis. Previous studies have shown that SOD and CAT were the most important antioxidant enzymes which protect fungus from the oxidant damage of ROS.SOD converts O2?ˉinto less damaging H2O2 and O2;CAT converts H2O2 into H2 O and O2,further decrease the production of ?OH,which reacts indiscriminately with most cellular constituents[3].These antioxidant enzymes have been proved extensively in the studies of common fungus including Candida albicans(C. albicans),Aspergillus fumigatus(A. fumigatus),Cryptococcus neoformans(C. neoformans) and so on[4-9]. Some research show that the two antioxidant enzymes may have a potential role as virulence factors[4,5].In recent years, the incidence of disseminated trichosporonosis caused by T.asahii trends to rise obviously, accounting for 5%~10% of deep infection of fungus in patients[10]. Also, T.asahii is resistant to most of antifungal drugs. Once T.asahii caused disseminated or systemic infection in the body, its mortality could be more than 80% [11], leading to the therapy and administration of T.asahii infection become more and more troublesome. Therefore, it will be of great significance to explore the infection mechanism and pathogenic mechanism of T.asahii from the perspective of antioxidant defense. In our previous studies, Zong lina, et al [12] found that three different oxidants(diamide, H2O2 and menadione) induced different levels of oxidative damage to T.asahii.Therefore, we hypothesize that: like other fungal pathogens [4-9], T.asahii also have relative antioxidant defense system; since T.asahii could invade the host and cause serious infection, we felt that there may be a connection between its pathogenicity and their capacity of resistance or evade the attack of host immune system(especially the antioxidant defense capacity).To test this hypothesis, we collected our laboratory existing 44 strains that from different sources,and investigated the activity of SOD and CAT, then compared and analyzed the results. Yet, this study also explored antioxidant defense mechanism preliminarily and lay foundation for further study of the fungus infection mechanism. Objective1. To observe and compare the two antioxidant enzymatic activities of T.asahii from source of environment and clinic.2.To observe the influence of internal passage in mice on the two antioxidant enzymatic activities of T.asahii.3.To observe the influence of fluconazole on the two antioxidant enzymatic activities of T.asahii. Methods1.There were 44 strains of T.asahii collected and they were divided into four groups according to their sources: Environmental group, Clinical group, Internal passage group and Fluconazole-resistant group. The strains were cultured on PDA for 48 h at 35℃.The cell suspensions were prepared that contained about 1.5 x 108 cfu/ml T. asahii and 0.9% sterile saline. For each strain, 1ml volume of cell suspension was transferred to 50 ml Sabouraud glucose broth(SDB) containing 4% glucose and 1% yeast peptone. These cultures were incubated at 37℃ with shaking(150 r/min) for 48 h. After centrifugation, the cell mats were collected for enzyme analysis.2. Total superoxide dismutase activity assay kit and catalase activity assay kit were employed to determine the SOD activity and CAT activity according to the protocol of the manufacturer. Total protein quantitative assay kit was used by BCA method, which were compared with standard serum protein. Repeated three times.3. SPSS 13.0 statistical software analysis was employed in this study.Results1. Environmental group:the SOD activity of strains CBS8904、CBS7137、CBS8520 were 3.828、3.46、4.75U/mgprot, the mean value was 4.01±0.66U/mgprot.The CAT activity of strains CBS8904、CBS7137、CBS8520 were 45.833、42.61、57.082U/mgprot, the mean value was 48.51±7.60U/mgprot.2. Clinical group: the clinical isolated strains of different sources had different antioxidant enzymatic activity. The SOD activity of Clinical group ranged from 6.221U/mgprot to 16.826U/mgprot, the mean value was 10.45±3.87 U/mgprot,the CAT activity ranged from 61.956U/mgprot to 164.552U/mgprot, the average was 110.56±35.77U/mgprot.3. Internal passage group: the two antioxidant enzymatic activities of T.asahii strains CBS2479、CBS8904、CBS7137 and CBS8520 were gradually promoted after passage in mice. The SOD activities of strain CBS2479 before passage and after the 5th passage were 6.221 U/mgprot and14.610 U/mgprot(P<0.05);its CAT activities before passage and after the 5th passage were 61.956 U/mgprot and 89.045 U/mgprot( P <0.05),respectively.The SOD activities of strain CBS8904 before passage and after the 5th passage were 3.828U/mgprot and17.854 U/mgprot(P<0.05);its CAT activities before passage and after the 5th passage were 45.833 U/mgprot and 61.418U/mgprot(P<0.05),respectively.The SOD activities of strain CBS7137 before passage and after the 5th passage were 3.460U/mgprot and15.016 U/mgprot(P<0.05);its CAT activities before passage and after the 5th passage were 42.610U/mgprot and 66.025U/mgprot(P<0.05),respectively.The SOD activities of strain CBS8520 before passage and after the 5th passage were 4.750U/mgprot and 7.115 U/mgprot(P<0.05);its CAT activities before passage and after the 5th passage were 57.082U/mgprot and 68.964 U/mgprot(P<0.05),respectively.4. Fluconazole-resistant group: The SOD activities of strain CBS2479 before drug treatment and after the10 th generation of Fluconazole-resistant induction were 6.221 U/mgprot and 9.126 U/mgprot(P<0.05);its CAT activities before drug treatment and after the10 th generation of Fluconazole-resistant induction were 61.956 U/mgprot and 86.738 U/mgprot U/mgprot(P<0.05),respectively.The SOD activities of strain CBS8904 before drug treatment and after the10 th generation of Fluconazole-resistant induction were3.838 U/mgprot and 6.921 U/mgprot(P<0.05);its CAT activities before drug treatment and after the10 th generation of Fluconazole-resistant induction were 45.833 U/mgprot and 74.128 U/mgprot U/mgprot U/mgprot(P<0.05),respectively. For reply induction strains, their antioxidant enzymatic activity gradually returned to normal levels.Conclusion1. This study shows that the SOD and CAT activities of Clinical group were significantly higher than those of Environment group. The two antioxidant enzymatic activities of T. asahii in Internal passage group went up gradually after passage in mice;the SOD and CAT activities of the 5th generation of Internal passage group were significantly higher than before passage;and its CAT activity in 5th generation strains was significantly higher than Environment group. The two antioxidant enzymatic activities of T. asahii in Fluconazole resistant group also went up gradually after the drug treatment; the research proves the SOD and CAT activities were significantly higher in the 10 th generation of Fluconazole-resistant group than untreatment; both SOD and CAT activities of strains in Fluconazole-resistant group were higher than those of Environmental group.2. The strains of T.asahii from different sources have different antioxidant abilities,which mainly manifest in the difference of antioxidant enzymatic activities.Clinical group strains has the strongest antioxidant capacity;Internal passage group strains and Fluconazole resistant group strains better;Environmental group strains the lowest.