Selenium-mediated Protection And SIPI-8294-induced Damage In Oxidative Stress Response Of Bacteria

?The oxidative stress response and antibiotics tolerance of se-enrich bacteriaDeeper understanding of antibiotic-induced physiological responses is critical to developing new“weapons”to kill bacteria.Recently,the damaging reactive oxygen species?ROS?formation induced by bactericidal antibiotics has been proved to involve in numerous metabolic processes in bacteria,including oxidative stress response,extensive oxidative damage to biological macromolecules,cell death and so on.Therefore,how to effectively prevent or reduce the damage of ROS in cells is not just an important challenge,but as immediate areas of research focus.Instead,seleno-enzymes is generally recognized to be a powerful antioxidant in maintaining the redox balance and defending the oxidative stress,so that protecting the cells from the harmful effects of ROS.Therefore,we took selenium-rich E.coli as research objects to systemly study the selenium metabolism in bacteria,the effect of the high selenium content on growth of bacteria,the alteration of Trolox-Equivalent Antioxidant Capacity?TEAC?and ROS in cell,and evaluate the relationship between TEAC and the tolerance of bacterium to bactericidal antibiotics.The research contents and results as follows:The cellular antioxidant activity of selenium-pretreated bacteria were analyzed,as well as intracellular ROS production and elimination when selenium-pretreated bacteria in the presence of various antibiotics.The results showed that selenium-rich E.coli could significantly improve the bactericidal antibiotic resistance and reduce their intracellular ROS levels.Compared to complete inhibition of the parental strain by bactericidal antibiotics,the bactericidal antibiotics at the same conditions only exhibited slight and reversible inhibition of selenium-pretreated Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923,which indicated that intracellular seleno-enzymes provided substantial protection against antibiotics.ROS generation caused by bactericidal antibiotics was confirmed by fluorescence spectrophotometry using 2',7'-dichloro-uorescein diacetate?DCFH-DA?as substrate.The time course experiments of pretreatment with selenium showed significant decrease of ROS level at 2h.In summary,the present study provides experimental evidence supporting seleno-enzymes has good scavenging effect to ROS and can protect bacteria from oxidative stress injury induced by bactericidal antibiotics.This study found and proved that selenium-pretreated bacteria could scavenge the damaging reactive oxygen species?ROS?induced by bactericidal antibiotics so that protecting itself from the harmful effects of antibiotics.The research will be benefited to understand the stress response mechanisms against oxidative stress in selenium-enriched bacteria and enrich and improve the bacterium tolerance mechanism.Morphology and bacterial growth changes of E.coli in M9+2 mM H₂SeO₃-S conditions.From the selective medium of M9?Se-rich and S-free?,10 species well grown E.coli of hign-selenic tolerance type have been isolated.Especially,E.coli C5 could be continuously cultured in the high-selenium and trace-sulfur environments.We determined the content of selenium and sulfur in E.coli C5 which cultured in M9+2 mM H₂SeO₃-S media with ICP-MS methods,the results showed that the ratio of sulfur to selenium in E.coli C5?wet weight?was 0.059/1,while the ratio of sulfur to selenium reach up to 105/1 when E.coli C5 cultured in the high-sulfur and trace-selenium environments.Results revealed that through adaptive domesticated,there were still some bacterial cells gradually adapted to the high-selenium and trace-sulfur environment and sustained growth even in such harsh environments.Ultrastructure observation of selenium-rich bacteria found that selenium-rich bacteria preserved cell integrity.Although the cell wall was loosely,some obvious particles aggregated in cells,the E.coli C5 still had some proliferating ability in M9+2 mM H₂SeO₃-S condition.? Functional proteomic research of MRSA by the association of oxidative stress response when suffered a new combination drugsBacterial resistance is one of the most important culprits which threat the human health in 21 century.Traditional mechanisms of bacterial resistance could not explain why the resistant bacteria spread so quick all over the world.Recently,the proteome research of bacterial resistance opens a window for us to be able to insight the changes of proteins network when the bacteria exposure to sub-lethal concentration of antibiotics.Afterwards,the more important step is to find out which protein dominates the resistance among the network except those classical resistant proteins clarified before and verify it with the aid of biological methods.This study found and proved that novel synthesized erythromycin derivatives were screened for synergy with oxacillin and other?-lactam antibiotics against Methicillin-resistant Staphylococcus aureus?MRSA?.MRSA ATCC43300 and some clinical isolated MRSA were used.Several erythromycin derivatives were found to possess high synergism with oxacillin against MRSA.The newly synthesized erythromycin derivatives were aslo tested for their inhibitory effects against MRSA,either separately or in combination with oxacillin,using serial broth dilution,disc diffusion,Etest strips,growth curves and time-kill curves.A representative derivative,SIPI-8294,could potentiate almost allb-lactam antibiotics tested against the model strain MRSA ATCC43300 from 4 to 128 times and had synergism with oxacillin against 12 of 16 clinical isolates of MRSA under one-fourth of the minimum inhibitory concentration?MIC?of the compounds.This is the first report on the synergistic activity of these new erythromycin derivatives.These findings provide a new choice for treatment of infection caused by MRSA and lead us to further study the synergistic mechanism.Next,to identify cellular functions significantly perturbed by the combination drugs,functional annotations were statistically analyzed for the proteins that showed significant expression level changes.Further,the expression levels of the proteins that underwent the functional annotation analysis were compared with those suffered SIPI-8294 alone or oxacillin alone.These functional annotations originate from KEGG pathways,gene ontology?GO?terms and clusters of orthologous groups of proteins?COG?that describe cellular components,molecular functions and biological processes.The results showed statistically significant GO terms?p<0.05,with multiple testing corrections?and 7 most statistically significant KEGG pathways?p<0.05?.The functional annotation analysis suggested that the cellular responses perturbed by the combination drugs were represented by the following annotations:“Alanine,aspartate and glutamate metabolism”,“Pyrimidine metabolism”,“Purine metabolism”,“Citrate cycle,TCA cycle”,“Two-component system”,“Pyruvate metabolism”,and“Amino sugar and nucleotide sugar metabolism”.The expression level changes of the above proteins were in agree with genes reportedly involved in oxidative stress response under aerobic conditions.Especially,most of the genes involved in TCA cycle were up-regulated by the combination drugs.The essentiality of fumarase C?fum C;malate to fumarate?was further supported by prior studies reporting that fum C is involved in regulation of various cellular functions such as oxidative stresses,tricarboxylic acid?TCA?cycle and iron homeostasis.