Objective To establish a high-throughput screening system to obtain key Staphylococcus aureus (S.aureus)secretory proteins which required for S.aureus survival in macrophages.Methods Based on our validated eukaryotic expression vector library of S.aureus secretory proteins,DNA transfection was used to obtain an RAW264.7 macrophage array expressing S.aureus secretory proteins.After the RAW264.7 cells were infected with S.aureus,the extracellular bacteria were removed to observe the intracellular surviving situation of S.aureus.Finally,the screening results were validated by the overexpression and knockout S.aureus of corresponding secretory proteins.Results The optimal transfection dose (1.0 μg/well)of plasmids for RAW264.7,multiplicity of infection (MOI,1 .0 ),and infection time (4 h after removing extracellular bacteria of S.aureus ) were established respectively.To validate the screening results,the corresponding overexpression and knockout strains were constructed.And hypothetical protein and Serine protease E were found to promote the survival of intracellular S.aureus.Conclusion We successfully construct a screening system for key secreted secretory proteins which required for S.aureus surviving in macrophages,which may advance the study of the intracellular surviving mechanism of S.aureus.

Objective To explore the mechanism which drives nimbolide(NIM)in treating acute pneumonia caused by Staphylococcus aureus(S.auteus).Methods A mouse model of acute pneumonia caused by S.auteus was constructed through endotracheal intubation.After NIM treatment,the survival rate was observed,the amount of bacteria in the lung was tested by plate culture,and the expression of inflammatory cytokines in the lung tissues was detected with ELISA.After primary cultured peritoneal macrophages(PM)were infected with S.auteus,the effect of NIM on the expression of inflammatory cytokines and activation of inflammatory pathway were studied with ELISA and Western blotting,respectively.The effect of RNF114 knockdown by lentiviral shRNA infection on inflammation responses in PM was explored with ELISA and Western blotting.Results Acute infection of S.auteus in the lung could cause acute death in the mice,while NIM treatment significantly improved the survival rate and down-regulated the levels of inflammatory cytokines in the lung.However,it had no effect on the lung colonization of S.auteus in the short term.The results of in vitro experiments indicated that NIM may regulate RNF114 function to down-regulate the phosphorylation level of ERK,inhibit the activation of MAPK pathway,and thus suppress the expression of inflammatory cytokines.Conclusion NIM may inhibit the activation of MAPK pathway by regulating the function of RNF114,and thus suppress the expression of inflammatory cytokines in the lung,and finally inhibit the death of mice with acute pulmonary hyperinflammation caused by S.auteus.

Objective To investigate the regulatory effect and mechanism of new skeleton small molecule compound ZHZ-33-1 on the inflammatory response induced by primary macrophages in mice infected by Staphylococcus aureus.Methods qRT-PCR was used to detect the mRNA expression levels of inflammatory factors Tnf,Illb,116,Il12p40 in macrophages cells.The protein levels of TNF-α,IL-1 β,IL-6 and IL-12 in cell supernatant were determined by ELISA.Western blot was used to analyze the phosphorylation of key signaling pathway proteins such as NF-κB and MAPK.CCK8 detection was used to evaluate the effect of ZHZ-33-1 on macrophages activity.The OD600nm method was used to investigate the effect of ZHZ-33-1 on the growth of Staphylococcus aureus.Results 80 pmol/L ZHZ-33-1 had no significant effect on bacterial proliferation(P＞0.05)and cytotoxicity of macrophages(P＞0.05).Compared with the DMSO group,ZHZ-33-1 significantly inhibited the transcription and expression levels of inflammatory cytokines(TNF-α,IL-iβ,IL-6,and IL-12)in primary macrophages infected by Staphylococcus aureus(USA300)(P＜0.05).Further studies showed that ZHZ-33-1 was able to inhibit the NF-κB and MAPK pathways of wild macrophages,but was not TLR2-/-macrophages.Conclusion Through inhibiting NF-κB and MAPK signaling pathways,ZHZ-33-1 may inhibit the macrophages inflammatory response caused by Staphylococcus aureus.Therefore,ZHZ-33-1 is expected to become a new drug candidate for the treatment of Staphylococcus aureus-induced hyperinflammatory response.

Objective To investigate the regulatory effect and mechanism of new skeleton small molecule compound ZHZ-33-1 on the inflammatory response induced by primary macrophages in mice infected by Staphylococcus aureus.Methods qRT-PCR was used to detect the mRNA expression levels of inflammatory factors Tnf,Illb,116,Il12p40 in macrophages cells.The protein levels of TNF-α,IL-1 β,IL-6 and IL-12 in cell supernatant were determined by ELISA.Western blot was used to analyze the phosphorylation of key signaling pathway proteins such as NF-κB and MAPK.CCK8 detection was used to evaluate the effect of ZHZ-33-1 on macrophages activity.The OD600nm method was used to investigate the effect of ZHZ-33-1 on the growth of Staphylococcus aureus.Results 80 pmol/L ZHZ-33-1 had no significant effect on bacterial proliferation(P＞0.05)and cytotoxicity of macrophages(P＞0.05).Compared with the DMSO group,ZHZ-33-1 significantly inhibited the transcription and expression levels of inflammatory cytokines(TNF-α,IL-iβ,IL-6,and IL-12)in primary macrophages infected by Staphylococcus aureus(USA300)(P＜0.05).Further studies showed that ZHZ-33-1 was able to inhibit the NF-κB and MAPK pathways of wild macrophages,but was not TLR2-/-macrophages.Conclusion Through inhibiting NF-κB and MAPK signaling pathways,ZHZ-33-1 may inhibit the macrophages inflammatory response caused by Staphylococcus aureus.Therefore,ZHZ-33-1 is expected to become a new drug candidate for the treatment of Staphylococcus aureus-induced hyperinflammatory response.