Mode Of Action Of Cyadox On Escherichia Coli

Cyadox is a new member of QdNO drugs, attracting widespread attention because of its low toxicity and wide range of use. The antibacterial mechanism of cyadox has not been studied yet. To study the mode of action of cyadox can better explain the antibacterial mechanism of QdNO’s, speed up the development of QdNO’s, and guide more reasonable and safer use of cyadox.In this study, selected the most sensitive E.coli C84010for experiment, A large number of studies have shown that QdNO’s cause DNA damage and inhibit DNA synthesis. QdNO’s have significantly anaerobe-selective activity, and are reduced in vivo to yield free radicals. Now, it remains unclear which free radicals are produced and how they act in the mode of action of QdNO’s. Meanwhile, whether receptor(s) of QdNO’s exist in bacteria have not been studied so far.The free radicals which produced after the action of cyadox in bacteria will be detected in aerobic and anaerobic condition. And study the effect of all kinds of radicals in the antibacterial process of cyadox, the morphological changes and DNA damage of bacteria will also be studied. At last, we will find the mode action and target of cyadox.The binding of drug to receptors is one of the mode action of drugs, whether exist the receptor in E.coli has not been studied, we will use radioactive labeling method to study the receptor of cyadox.1. Study of free radicals in the antibacterial action of cyadoxThe experiment will study the antibacterial activity of cyadox and it’s metabolites under aerobic and anaerobic conditions, detect the free radicals which produced by cyadox, determine the role of free radicals in the anti-bacterials process.Then, the DNA damage and the relationship with free radicals will also be studied, what’s more, the membrane damage and morphological changes of bacteria will be detect.The MIC and MBC of cyadox against E. coli C84010was1and4μg/ml under anaerobic conditions respectively, while MIC and MBC was16and128μg/ml under aerobic conditions respectively, indicating that cyadox had anaerobe-selective activity like other QdNO’s. The MIC of cyadox metabolites (Cy10, Cy2, Cy1, Cyl7and CY6) were determined and only CY17had antibacterial activity (MIC=1μg/ml). The two cyadox-monoxides (CY10and CY2) and bisdesoxycyadox (CY1) had no antibacterial activity, indicating that two Nâ†'O was necessary for the activity of cyadox. The metabolites of cyadox were identified in the medium supernatant and bacterial cells under aerobic and anaerobic conditions. CY10and CY2were detected in the medium supernatant under aerobic conditions; CY10, CY2and CY6were found in the medium supernatant under anaerobic condition; CY19, CY2and CY6could be detected in bacterial cells under both aerobic and anaerobic conditions. This means cyadox can enter the bacterial cells and be metabolized to cyadox-monoxide and bisdesoxycyadox under aerobic and anaerobic conditions.Under anaerobic conditions,0.5MIC, MIC, MBC of cyadox caused a release of ROS in E. coli cell. The production of ROS increased with the increase of drug concentration, and the amount of ROS reached the highest level at1h after adding of cyadox. Under aerobic conditions,0.5MIC, MIC, MBC of cyadox caused high level of superoxide radicals in E. coli cells. The production of superoxide radicals increased with the increase of drug concentration and extension of time, and the level of superoxide radicals at4h was16times to that at Oh. Only the metabolites CY17produced the same amount of superoxide radicals as cyadox, other metabolites (CY10, CY2, CY1and CY6) had no superoxide radicals produced. Under anaerobic condition, hydroxyl radicals were not detected when the bacteria were treated by cyadox and it s metabolites at the concentration of0.5MIC, MIC and MBC.Under anaerobic condition, the free radical scavengers of mannitol, thiourea, DMSO, acetone, β-mercaptoethanol, ethanol, methanol, and sodium azide inhibited the antibacterial action of cyadox to a certain extent, indicating that free radicals may played an important role in the action of cyadox. Under aerobic condition, tiron as a superoxide radical scavenger inhibited the antibacterial activity of cyadox, and the effect of the inhibition was strengthened with the increase of concentration of tiron, showing that superoxide radicals played a major role in the action of cyadox under aerobic conditions. After E. coli containing plasmid pUC-19were treated by different concentration of cyadox for2h, the plasmid degradated significantly. The level of the degradation of plasmid increased with the increase of drug concentration and extension of incubation time. β-mercaptoethanol and sodium azide significantly inhibited the degradation of plasmid pUC-19, suggesting cyadox cause oxidative radical-mediated degradation of DNA strand by producing aromatic radicals acting at the site of C1of DNA.No obvious membrane damage of E. coli C84010treated by cyadox was found. The morphology of bacterial cells treated by cyadox under anaerobic condition was observed by scanning electron microscopy. After the cells were treated by0.5MIC, MIC and MBC of cyadox for0.5h, the change of bacterial shape was not remarkable, appearing normal shot rods. After the cells were treated by MIC of cyadox for4h, the bacterial became longer; aftrer24h, the bacterial showed long strip; after48h, the bacterial appearred a form of filament, some appearred fractured and cytoplasm leakage. All of these morphological changes may subject to serious damage of DNA.2. Investegation of binding sites of cyadox in E. coliCurrently, whether exist the receptor in E.coli has not been studied, we will use radioactive labeling method to study the receptor of cyadox. We studied from two aspects:first, the subcellular fragments of bacterial were separated and then the binding of subcellullar fractions with tritium labeled cyadox (H3-cyadox) was detected using radioactive dialysis; second, E. coli cells were treated with different concentration of Hj-cyadox and whether the saturation binding of various subcellular fractions existed were observed.Method for the separation of the cell wall, cell membrane and cytoplasm by ultracentrifugation were established, and the binding of subcellular fraction with the cyadox using radioactive dialysis were detected. When subcellular fraction of E. coli was dialyzed against0.01,0.1,1μg/ml of tritium labeled H3-cyadox for20h, the ratio of radioactivity of inside to outside of dialysis bag were1approximately, indicating that the drug was not specific bingding to various subcellular fractions. After E. coli was treate by H3-cyadox, the radioactivity of subcellular fractions was detected. The ratio of cyadox in cell wall, membrane and cytoplasm is5.89%,16.22%and77.8%, respectively, showing the drug distributed mainly in the cytoplasm. The binding sites of cyadox in bacterial were identified by radioactive filtration. Saturation binding of various sub-cellular fractions to the drug had not been found at the durg concentration of0.125-16μg/ml for0.5-2h, demonstrating that cyadox had no binding site in bacteria.In summary, cyadox has anaerobic selective activity as other QdNO’s do; under aerobic condition, superoxide radical plays an important role in the antibacterial action of cyadox; under anaerobic conditions, a release of intracellular ROS but no hydroxyl radicals were produced after the metabolization of cyadox; DNA damage caused by the free radicals produced by cyadox is one of most important mode of action of cyadox under anaerobic condition; E. coli cells treated by cyadox form filamentation but there is no obvious damage of membrane; there is no specific receptors of cyadox existing in the bacterial cells.