Regulation Of Host Gene IFI204 And Virulence Factor OatA In The Extracellular Traps Induced By Staphylococcus Aureus

Staphylococcus aureus infection is one of the important problems affecting the development of aquaculture industry and human health.The infected final outcome mainly depends on the interaction between pathogen and host,and it is difficult to effectively prevent and control it only from the perspective of pathogen.In natural immune recognition,IFI204 is an intracellular DNA receptor that can bind to single-stranded or double-stranded DNA.It is a DNA sensor in pattern recognition receptors(PRRs).In the early stage,We found that IFI204 significantly inhibited the lethal infection of Staphylococcus aureus and restricted the tissue colonization of Staphylococcus aureus,indicating that IFI204 has an important role in host defense against Staphylococcus aureus.Next,We found that IFI204 did not affect the phagocytosis and intracellular clearance of Staphylococcus aureus by macrophages,but inhibited the extracellular proliferation of Staphylococcus aureus,and initially revealed that this may depend on Extracellular Traps(ETs).In this paper,IFI204 has conducted a more in-depth analysis on the role of IFI204 in the formation of ETs in Staphylococcus aureus infection.In order to explore the relationship between IFI204 and ETs,Wild-type(WT)and IFI204 gene-deletion(IFI204 KO)mice were constructed a Staphylococcus aureus infection pneumonia model.The results that the lung tissue and alveolar lavage fluid(BALF)of IFI204 KO mice bacteria loads significantly increased at 24 h,which was consistent with the previous results.By separating the cells in BALF and staining with SYTOX Green,we found that the formation of ETs in IFI204 KO mice was significantly reduced.At the same time,citrullinated histone H3(Cit H3)expression and the myeloperoxidase(MPO)level were also significantly reduced,indicating that IFI204 promoted ETs to form.ETs as a double-edged sword,while resisting pathogens,it will possible damage to the body,Therefore,For explore the role of ETs in Staphylococcus aureus pneumonia,We designed the DNase I treatment experiments,and the cell free DNA content in BALF was reduced significantly.The lung tissues and BALF bacterial load increased significantly,and the albumin content in BALF increased.It shows that ETs have a protective effect on the host in the respiratory tract infection of Staphylococcus aureus.In order to further explore the mechanism of IFI204 in the formation of ETs,the following in vitro experiments were performed.Isolation of WT and IFI204 KO mice bone marrow-derived macrophage(BMDM)and neutrophil(BMDN),Construct a model of METs and NETs induced by Staphylococcus aureus in vitro,The BMDM and BMDN cell free DNA was reduced significantly in IFI204 KO mice,which was consistent with the results in vivo.and the expression level of Cit H3 was significantly lower than WT.The co-localization of MPO,NE and DNA was observed by immunofluorescence,IFI204 KO was less than WT.Simultaneously infected with extracellular bacteria was higher significantly than WT,and there was no difference in intracellular bacteria.There was no difference in the total ROS level,After treatment with DPI,a NADPH-derived ROS inhibitor,the amount of extracellular DNA released decreased,but the difference did not disappear,proving that IFI204 promoted of ETs formation independ on ROS.And found that IFI204 promotes the formation of ETs induced by Staphylococcus aureus independent of the MAPK signaling pathway.The expression of Cit H3 requires the participation of protein arginine deiminase 4(PAD4).Western blotting showed that the expression level of IFI204 KO-BMDM PAD4 was significantly lower than that of WT.After treatment with PAD4 inhibitor GSK484,the amount of DNA released decreased and the difference disappeared.Therefore,IFI204 promotes the expression of PAD4 to promote the formation of ETs.The study also found that heat-inactivated Staphylococcus aureus could not induce BMDM to release METs,suggesting that the induction of ETs by Staphylococcus aureus requires the participation of virulence factors.Using Staphylococcus aureus acetyltransferase(OatA)gene deletion strain ?OatA-USA300 and Staphylococcus aureus wild strain USA300 respectively to infect WT-BMDM,We found that the Staphylococcus aureus virulence factor OatA inhibits the formation of ETs.To further explore whether the virulence factor OatA inhibits the formation of ETs by inhibiting IFI204,?OatA-USA300 and USA300 were used to infect WT and IFI204 KO-BMDM,respectively.The DNA release was quantitatively detected,and no difference.Therefore,OatA was inhibition of ETs formation is not mediated by IFI204.To further clarify the mechanism by which the virulence factor OatA inhibits the formation of ETs,important proteins related to ETs were detected by Western blotting.We found that ?OatA-USA300 can significantly activate GSDMD,and the GSDMD gene deletion BMDM has no difference in the amount of extracellular DNA released after ?OatA-USA300 and USA300 infection,and the Cit H3 expression level of BMDM after ?OatA-USA300 infection of GSDMD gene deletion is significantly lower than WT.Therefore,we infer that OatA inhibits the formation of ETs by inhibiting the activation of GSDMD.In summary,the research shows that IFI204 promotes the expression of PAD4 to promote the formation of ETs,thus limits the proliferation of Staphylococcus aureus.IFI204 does not mediate the Staphylococcus aureus virulence factor OatA inhibits the formation of ETs and virulence factor OatA inhibits the formation of ETs by inhibiting the activation of GSDMD.