Neddylation Alleviates Methicillin-resistant Staphylococcus Aureus Infection By Inducing Macrophage ROS Production

Objective:Methicillin-resistant Staphylococcus aureus(MRSA)is one of the most important pathogens in hospital infection,which is a serious threat to human health.The binding process of the ubiquitin-like protein NEDD8(Neural precursor cell-expressed developmentally downregulated 8)with the substrate protein is known as Neddylation.As a new type of protein post-translational modification,Neddylation participates in many physiological and pathological processes including cell proliferation,differentiation and apoptosis,signal transduction,inflammation.This study aims to explore the role and mechanism of Neddylation in MRSA infection.Methods:8-week-old female C57BL/6 mice were injected with MRSA via tail vein to establish the MRSA bloodstream infection model.The levels of Neddylation in various organs were detected by Real-time Quantitative polymerase chain reaction(qPCR)and western blot(WB).Next,we injected MRSA blood infection mice with clodronate liposome,Neddylation inhibitor(MLN4924)or Nrf2 inhibitor(ML385),the expression of IL-1β,IL-6 and TNF-α in serum was detected by ELISA,the bacterial load was counted after homogenization of liver,spleen,lung and kidney tissues,the ratio and production of reactive oxygen species(ROS)of macrophages in mice organs were examined by flow cytometry(FCM),the inflammatory infiltration and injury of liver,lung and kidney tissues were observed by hematoxylin-eosin staining,the survival time of mice was recorded to detect the effect of Neddylation on MRSA infection.Peritoneal macrophages were extracted in vitro and co-cultured with MRSA,treatment with Neddylation inhibitor(MLN4924,DI591),Neddylation E1 siRNA(UBA3 siRNA),Neddylation E2,E3 overexpression plasmids(UBE2M and DCN1),deneddylation enzyme siRNA(CSN5 siRNA),proteasome inhibitor(MG132),ROS inhibitor(NAC),Cullin3 and Nrf2 siRNA.Then,protein levels of NEDD8-Cullin3,Cullin3 and Nrf2 in macrophages were examined by WB,mRNA levels of Nrf2 downstream factors(Sodl,Cat,Gst)were detected by qPCR,the phagocytosis and killing ability of MRS A by macrophages were analyzed by FCM,immunofluorescence and bacterial plate count,apoptosis rate and the production of ROS of macrophages were tested by FCM.The above is for exploring whether the effect of neddylation on ROS production of macrophages is related to nedd8-cullin3-nrf2-ros axis.Results:We found that mouse MRS A bloodstream infection increases the level of NEDD8-Cullins in liver,spleen,lung and kidney tissues.In MRSA bloodstream infection model,MLN4924 inhibits the production of ROS in macrophages,increases the bacterial load of lung,liver,kidney and spleen,aggravates the liver and kidney injury,and increases the mouse mortality.Nrf2 inhibitor-ML385 improve the increase of organs bacterial burden and decrease of survival time of mice caused by MLN4924.In vitro,MRSA stimulation also increased the level of NEDD8-Cullins in mouse peritoneal macrophages.MLN4924 did not change the apoptosis rate and phagocytic ability of macrophages to MRSA,but inhibited their bactericidal ability by reducing the production of ROS.Blockade of Neddylation,either pharmacologically(MLN4924,DI591)or through the use of UBA3 siRNA,inhibited Cullin3 Neddylation and promoted Nrf2 accumulation,thus reducing ROS induction and bacterial killing ability.Overexpression of UBE2M and DCN1,or interference with CSN5,increasing the protein level of NEDD8-Cullin3,promote the degradation of Nrf2 and the production of ROS,and enhancing the bactericidal ability of macrophages.Conclusions:In summary,our findings suggest that activation of Neddylation in mouse peritoneal macrophages plays a critical protective role against MRSA infection by increasing ROS production,partially by signalling through the NEDD8-Cullin3Nrf2-ROS axis.Furthermore,our results may provide a new non-antibiotic treatment strategy for MRSA infection through targeting of Neddylation.