Iron Ions Regulated Gram-negative Bacteria OmpW To Stand Up Host’s Oxidative Stress Action

Gram-negative bacterium is a major pathogenic bacterium causing infectiousdiseases. Few of outer membrane proteins of gram-negative bacterium is a kind of virulencefactors and has the function that resists to innate immunity of host. These proteins play a vitalrole in virulence of bacterium and infection of host.Innate immunity depends on Fenton reaction mediated by ferrous iron and serves as a majorway to resist to pathopoiesia bacterium and infection. Low-level free radicals generated byorganism’s Fenton reaction form oxidative stress（OS） surroundings to kill and clear invadedpathogenic bacterium. Because of this action, organism is protected effectively. But it isdangerous that there are overdose of ferrous irons resulting from iron metabolic disorderbecause they involve in promoting to elevate bacterial virulence, accelerate growth and producelots of free radicals. OS is developed by lots of active group, if we can not clear these freeradicals due to physiology or pathology disorder. When this happen, we would encounterdiseases resulting from damaging normal cells and dysbolism. We would even lose our lifesometimes. Immunogenic OmpW belonging to a member of gram-negative bacterium outermembrane protein family acts as one of virulence factors and also has close correlations withbacterial infection. In view of iron ions and OS having effects on immunization and bacterialinfections, it is significance for us to investigate the relationship between infection andimmunization and reveal the pathogenesis of infection diseases by the respect of bacterialresisting to host immunization.In my experiment, I explored the effectiveness which iron ions have effects on outermembrane protein W（OmpW） from gram-negative bacterium in order to deeply comprehendthe interaction between bacteria and host’ OS. This work will offer an rationale for pathogenicmechanism research about gram-negative bacterium further and have guiding effects oncontrolling and curing munity diseases in theory and practice. First, the recombinant expressionvector pLLP-OmpA-ompW was constructed. The recombinant vector was then transferred intoE. coli K12to obtain the strain with up-expression of OmpW location in outer membrane（calledhigh-expression bacterium）. In order to verify the highly expressed OmpW whether located correctly, polyclonal antibody of OmpW was prepared by immunization of BAL B/c mice withOmpW protein extracted from membrane protein which were highly expressed in the bacterialcytoplasm by recombinant expression vector pET-28a-ompW. Second, wild-type bacterium（K12）, ompW-deletion mutant bacterium, and high-expression bacterium were exposed in OSunder deficient iron ions, then I quested to the survival ratio differences aiming to mirrorbacterial antioxidation variance. Meanwhile, this bacteriums differences were analysised byferrous iron uptake within the same period of time. The last, Western blot revealed the impactsiron ions and OS having on OmpW from wild-type bacterium.The following are results. When the three kinds of bacteriums were cultivated under nativesurroundings and were dealed with H₂O₂, high-expression bacterium had the strongest toleranceto OS, then the wild-type bacterium and ompW-deletion mutant bacterium had the leasttolerance. Further, total tendence remained the same under deficient iron ions but had littlelower than the native surroundings. The ability of wild-type bacterium reduced under deficientiron ions. Ferrous iron uptake reseach suggested that high-expression bacterium uptaked moreferrous iron and however, ompW-deletion mutant bacterium took on the reverse result. Iron ionscould regulated the level of OmpW in the following way. When wild-type bacterium wascultivated under deficient iron ions, expression of OmpW were not detected by Western blot.While overdose of iron ions up-regulated OmpW. So we had no difficulties to find thatbacterium up-regulated OmpW by uptaking iron ions to resist host’ killing action owing to OSand escaped from host immunization, then aquired survival.It can be seen that gram-negative bacterium OmpW regulated by iron ions could conduce tostand up OS, escape killing from host immunization and strengthen its existence ability andchances of infection. My research revealed a sort of new pathogenic mechanism resulted fromdisease caused by Gram-negative germ. This discover suplied a rationale to antiseptics andvaccinal research and development, thereby, had fundamental practical importance.