Mechanisms Of Infectious Oxidative Stress And Major ROS Of Leptospira Interrogans And ROS-induced Protein Denaturation/DNA Damage And Protection Of Antioxidases

Background:Stress is one of vital signs and oxidative stress is a main stress manner of organisms.Oxidative stress in cells can produce a great quantity of reactive oxygen species?ROS?that cause protein oxidized denaturation and oxidative damage of DNA and lipids.Infection is a process of interaction between microbial pathogens and hosts.Recent literatures reported that the host cells infected with bacteria present oxidative stress reaction to produce a great quantity of ROS.Our laboratory reported that several different host cells produced a great quantity of ROS during infection with pathogenic Leptospira interrogans to cause the damage of DNA and mitochondrial membrane and apoptosis of the cells.However,the ROS production by prokaryotic microbes such as L.interrogans during infection of cells,ROS-based oxidative stress,major types of ROS,ROS-induced protein oxidized denaturation and DNA oxidative damage and leptospiral death as well as protection of leptospiral catalase,glutathione?GSH?peroxidase andcytochrome C?CytC?peroxidase have been not reported yetMethods:Bioinformatic softwares were used to analyze the structure,location and functional domains in the products of the genes annotated as KatE catalase?No LA 1859?,BtuE GSH peroxidases?No.LA 1007 and LA 4299?and CytC peroxidases?No.LA₀666?LA₂974 and LA₃139?of pathogenic L.interrogans serogroup Icterohaemorrhagiae Serovar Lai,a predominant leptospiral serovar epidemic in China The prokaryotic expression systems of the leptospiral genes were generated using routine genetic engineering techniques and the expressed target recombinant proteins,rLepKatE,rLepBtuE1/2 and rLepMauG1/2/3 were extracted by Ni-NTA affinity chromatography.The enzymatic activities,Km and Kcat values of the target recombinant proteins catalyzing specific substrates were determined by spectrophotometry.The change of ROS levels in the leptospires during infection of human THP-1 monocytes and umbilical vein endothelial cell?HUVEC?was detected by laser confocal miacroscropy and the major types of ROS was determined by electron paramagnetic resonance?EPR?spectrum method.Laser confocal miacroscropy,spectrofluorometry and ELISA were applied to detect the roles of ROS in leptospiral viability and protein denaturation and DNA damage.The change of ROS levels of the leptospires during infection of cells were detected by laser confocal miacroscropy before and after inhibition of catalase,GSH and CytC peroxidases,the products of the leptospiral genes mentioned above.The changes of the expression of L.interrogans strain Lai antioxidase genes were measured by real-time fluorescent quantitative RT-PCR.Results:LA₁859 gene contains a heme-binding Clade-3 catalase domain while LA₁007 and LA₄299 genes contain GSH peroxidase domains.LA 0666 gene,but not LA₂974 and LA₃139 genes,contains a di-haem cytochrome C peroxidase domain.The generated prokaryotic expression systems could express the target recombinant proteins and each of the extracted target recombinant proteins showed a single band in gel after SDS-PAGE.rLepKatE,rLepBtuE1/2 and rLepMauGl displayed the enzymatic abilities to catalyze the substrates H2O2,t-Bu-OOH and cumene hydroperoxide with Km values of 21.137-92.190 ?mol·L-1 and Kcat values of 37.620-850.602 s-1.rLepMauG2/3 did not present any enzymatic activities.The ROS levels of the leptospires during infection of human THP-1 monocytes and HUVEC were significantly elevated?p<0.05?with a peak level at 8 h post-infection,in which ·OH was acted as the major type of ROS?p<0.05?.However,the leptospires during infection of cells did not produce nitric oxide?NO?.In the process of L.interrogans strain Lai infecting human THP-1 monocytes and HUVEC,27.6%and 7.5%of extracellular leptospires were dead,37.8%and 11.3%leptospiral protein were denatured and 18.61 ng/mL and 5.56 ng/mL DNA oxidative damage marker 8-OHdG were generated.After treatment of H2O2 at the dose corresponding to the highest level of ROS of the leptospires during infection of cells,the leptospiral growth and propagation were significantly decreased?p<0.05?.When the leptospiral catalase,GSH and CytC peroxidases were inhibited by inhibitors,the ROS levels in the leptospires were rapidly elevated?p<0.05?.The LepKatE-,rLepBtuEl/2-and LepMauGl-mRNA levels of L.interrogans strain Lai during infection of cells were significantly increased?p<0.05?.Conclusion:L.interrogans can generate an oxidative stress to produce a great quantity of ·OH-based ROS,but not produce NO.The high level of ROS causes leptospiral protein denaturation,DNA damage and death.The leptospiral catalase,GSH and CytC peroxidases can eliminate the infectious ROS to benefit the leptospiral survival,growth and propagation that indicates these antioxidases as virulence factors of of L.interrogans.Induction of L.interrogans-self infectious ROS and lethal oxidative damages may be a novel antibacterial mechanism of hosts.