| Brucellosis is a zoonotic disease caused by Brucella,which is widely prevalent in the world and seriously threatens public safety.Animal diseases can cause reproductive system diseases and cause major economic losses to animal husbandry.Human diseases can cause chronic infections and severe loss of labor ability.The infection cycle and pathogenicity of Brucella mainly depend on its intracellular survival mode.Brucella containing vacuoles(BCVs)are formed after Brucella enters cells.The secretion of Brucella type IV secretion system(T4SS)and its effector proteins help BCVs reach the endoplasmic reticulum to achieve intracellular replication of Brucella by regulating host immune response and affecting the transport of BCVs in cells.Therefore,T4 SS is the key for Brucella to survive in cells and inhibit the host ’s innate immune response.At present,16 T4 SS effectors and two hypothetical effectors have been identified.Among them,Bsp F plays an important role in the intracellular survival of Brucella,which specifically promotes Brucella replication in BCVs by interfering with vesicle transport between trans-Golgi network(TGN)and circulating endocytic compartments,but other functions of Bsp F need to be further studied.In this study,the effector protein Bsp F of T4 SS was analyzed by omics to explore its role in the host ’s innate immune response,and to reveal the mechanism of Brucella effector protein Bsp F affecting innate immune response and provide a theoretical basis for the treatment of diseases..It has been reported that Bsp F has the ability to inhibit the reverse transport of the trans-Golgi network,specifically promote the replication of Brucella in r BCV,and promote the growth of intracellular bacteria.In this study,Brucella T4 SS effector protein Bsp F has GCN5-Related N-acetyltransferases family domain,suggesting that it had crotonyltransferase activity.The Bsp F was expressed and purified by E.coli expression system,and an in vitro crotonyltransferase activity identification system was established to verify the enzyme activity of Bsp F.Results showed that Cro-Co A could enhance the level of histone crotonylation,while the addition of Bsp F weakened the level of histone crotonylation.Subsequently,the histones in the experimental group and the control group were analyzed by mass spectrometry.The results showed that the addition of Bsp F removed the crotonylation of H2AK37,H2BK117,H2BK21,H3K19,and H4K13.The above results confirmed that Bsp F had histone de-crotonyltransferase activity.However,it was found in the experimental results that Bsp F and its immune complexes in cells could enhance histone crotonylation.It was speculated that Bsp F could regulate the crotonylation of proteins in cells in other ways after entering cells.Therefore,Bsp F was overexpressed in HEK-293 T cells,and the whole protein in the cells was detected by pan-crotonylation antibody.It was found that Bsp F could cause changes in the overall crotonylation level in the cells.In order to further explore the specific effect of Bsp F on intracellular protein crotonylation,this study used label-free quantitative and crotonylation enrichment techniques and LC-MS/MS quantitative proteomics research strategies to quantitatively study the crotonylation of proteins in HEK-293 T cells caused by Bsp F,and the obtained protein data were subjected to bioinformatics analysis.The difference of more than 2times was considered to be significantly up-regulated,and less than 1 / 2 was considered to be significantly down-regulated.In this project,178 sites of 140 proteins were significantly up-regulated,and 153 sites of 125 proteins were significantly downregulated.The proteins increased by crotonylation are involved in amino acid biosynthesis,oocyte meiosis and Hippo signaling pathway.Proteins with reduced crotonylation are involved in biological processes such as antigen processing and presentation.In the crotonylation proteome data,it was found that Bsp F crotonylated TRIM38 on K142,and TRIM38 was found to be an important immunomodulator according to relevant reports.Therefore,it is speculated that Bsp F may be involved in the regulation of host innate immunity during Brucella infection.Subsequently,RAW264.7 cells were infected by 2308 WT and 2308Δbsp F,and Bsp F was overexpressed in He La cells to detect the effect of Bsp F on the secretion of pro-inflammatory factors IL-1β,IL-6,IL-8 and inflammatory pathways NF-κB and MAPK signaling pathways.The results showed that Bsp F inhibited the secretion of IL-1β,IL-6 and IL-8 by inhibiting the activation of NF-κB,p38 MAPK and JNK MAPK signaling pathways.TRIM38 is an E3 ubiquitin ligase of TRAF6,which mediates the ubiquitination of TRAF6 leading to protein degradation and plays its immunomodulatory function.Therefore,it is speculated that Bsp F has an effect on TRAF6 protein expression.Subsequently,to further explore the effect of Bsp F on TRAF6 protein,RAW264.7 cells were infected with 2308 WT and 2308Δbsp F,respectively,and Bsp F was overexpressed in He La cells.It was found that Bsp F could reduce the protein expression of TRAF6 in cells.After that,the mechanism of Bsp F affecting the expression of TRAF6 was explored.The effect of Bsp F on the expression of TRIM38 and the possibility of interaction between Bsp F and TRIM38 were excluded.Finally,it was determined that Bsp F enhanced TRIM38-mediated TRAF6 degradation by mediating TRIM38K142 Cr,affecting the amount of TRAF6 protein in cells,so as to achieve the purpose of regulating NF-κB,p38 MAPK and JNK MAPK signaling pathways in cells.In summary,Brucella T4 SS effector protein Bsp F has histone decrotonyltransferase activity in vitro and can regulate the level of crotonylation modification in cells.In the crotonylation modification omics data of HEK-293 T cells caused by Bsp F,we screened and verified RAB9 A,RAP1 and TRIM38 proteins.Bsp Fmediated TRIM38K142 Cr promotes the ubiquitination of TRAF6 by TRIM38,leading to the degradation of TRAF6,thereby inhibiting the transduction of NF-κB,p38 MAPK and JNK MAPK signaling pathways.This study provides a new theoretical basis for Brucella to achieve intracellular survival by regulating host innate immunity through T4 SS,and provides an important reference for the study of non-histone crotonylation modification. |
