Antioxidant Activities Of Ferritin And MsrA Protein From Ureaplasma Urealyticum

Background and objects:Ureaplasma urealyticum is one of the smallest self-propagating prokaryotes that can cultured in vitro. U. urealyticum, a common pathogen of human urinary tract, is associated with many diseases including urethritis, prostatitis, rheumatoid arthritis, and pelvic inflammation. And several abnormal sperm characteristics such as tail defects, decreased motility and elevated levels of reactive oxygen species(ROS) have been reported in connection with U. urealyticum infections, which can lead to infertility for males. ROS are a inevitable by-product during the normal aerobic metabolism for host. Human macrophages were able to use ROS to kill invading pathogens. In order to survive, bacteria have developed several mechanisms to combat the stress associated with ROS. U. urealyticum confronts oxidative stress during colonization, thus the ability of U. urealyticum to protect itself from oxidative stress is likely to be important for its pathogenesis and survival. However, the antioxidant mechanisms of U. urealyticum are still unclear. This study aim to provide experimental basis for exploring the antioxidant mechanisms of U. urealyticum by studying the antioxidant activity and mechanisms of Ferritin and Msr A protein of U. urealyticum.Methods:1. U. urealyticum was co-cultured with THP-1 cells according to multiplicity of infection by the ratio of 1 to 200(bacteria ratio cell)which were collected at 0, 3, 6, 12 or 24 h respectively. The ROS in THP-1 cells were detected by using fluorescent probe DCFH-DA.2. The Ferritin and Msr A gene in U. urealyticum were analyzed by bioinformatics.3. U. urealyticum was treated by H2O2, CHP or t-BHP for 20, 40, or 60 min. The relative m RNA expression levels of Ferritin and Msr A gene were measured by q-PCR.4. Ferritin and Msr A genes were amplified by PCR and then were inserted into p ET28 a to construct p ET28a-Ferrtin and p ET28a-Msr A recombinant plasmids. The recombinant plasmids were transformed into the E. coli BL21 and the recombinant proteins were induced by IPTG and then purified by Ni2+-affinity chromatography.5. The interaction between Ferritin and Fe2+ was assessed via immobilized metal ion affinity chromatography(IMAC) and intrinsic fluorescence assay. Kinetics of Fe2+ oxidation which catalyzed by Ferritin protein was determined by spectrophotometric analysis. The antioxidant activity of Ferritin was examined through in vitro experiment. The DNA binding and protection ability of Ferritin were analyzed by electrophoretic mobility shift assays.6. The Ferritin gene was ligated into p Trc99 a to construct the p Trc99a-Ferritin recombinant plasmid which was transformed into oxidation-sensitive mutant E. coli(Δdps JW0797). The survival ratios of the recombinant bacteria and control(with empty p Trc99a) under oxidative conditions were detected.7. The function of Msr A protein reduce Met-O into Met was determined.8. The Msr A gene was ligated into ptrc99 a to obtain p Trc99a-Msr A recombinant vector, and the recombinant vector was then transformed into E. coli mutant(ΔMsr A, JW4178-3). The survival rates of the recombinant strain and the control strain under different oxidative stress were determined.Results:1. U. urealyticum infection can induce THP-1 cells to produce ROS.2. Ferritin and Msr A genes were found in the genome of U. urealyticum by bioinformatics software analysis.3. Ferritin transcription was significantly increased at 20, 40, and 60 min when treated with H2O2(3.5-, 4.4-, and 5.1-fold, respectively), CHP(4.4-, 4.5-, and 4.7-fold, respectively), or t-BHP(2.4-, 3.2-, and 3.1-fold, respectively). Msr A transcription was significantly increased at 20, 40, and 60 min when treated with H2O2(4-, 6-, and 6-fold, respectively), CHP(11fold, respectively), or t-BHP(3-, 3.5-, and 4-fold, respectively).4. The recombinant expression vector p ET28a-Ferritin and p ET28a-Msr A were successfully constructed. The recombinant Ferritin protein with molecular weight of about 22 k Da and Msr A protein with molecular weight of about 21 k Da were successfully induced to express by IPTG and purified by Ni2+ affinity chromatography.5. Ferritin has Fe2+ binding ability and addition of Fe2+ dramatically quenched the intrinsic fluorescence of Ferritin. The Ferritin has ferroxidase activity which converts Fe2+ into Fe3+. Ferritin protein has the antioxidant function and its IC50 value was lower than that of BSA protein(P < 0.05). DNA interact with Ferritin to form a Ferritin-DNA complex under Fe2+ or H2O2 conditions which could protect DNA from oxidative damage.6. In comparison with the control group(transformed p Trc99 a null vector), p Trc99a-Ferritin transformed Δdps E.coli showed high tolerance to oxidative stress(P < 0.05).7. Msr A protein has the ability to reduce Met O into Met.8. The survival ratios of p Trc99a-Msr A transformed ΔMsr A E.coli which were much higher than the control(with empty p Trc99a) Conclusions:1. The expression of Ferritin in U. urealyticum is elevated in response to oxidative stress. Ferritin protein has Fe2+ binding property and ferrous oxidation activity. H2O2 and Fe2+ can induce Ferritin to combine with DNA and protect the DNA from oxidative damage. And Ferritin protein of U. urealyticum has antioxidant ability both in vitro and in vivo and plays an important role in the anti-oxidation stress.2. Oxidative stress can increase the expression of Uu Msr A gene of U. urealyticum; Oxidized met(L-Met O) can be reduced into Met by Msr A protein; The expression of Msr A protein can improve the antioxidant ability of oxidation-sensitive E. coli(ΔMsr A); When U. urealyticum is in the oxidative stress, Msr A protein can repair the oxidative damage of proteins, and thus play a role in the fight against oxidative stress.