The Molecular Mechanism Of Toll/IL-1Receptor-like Domain Containing Protein Of Brucella(TcpB)’s Effection On TLR

Brucellosis is a worldwide spread zoonotic disease and caused by Brucella.Brucella is an intracellular parasite, the main features of the virulence of Brucella ishost’s chronic infection which caused by brucella’s survival and replication in host’scells. Brucella type IV secretion system (T4SS) is a multi-protein complex andcapable of secreting bacterial virulence factors out of bacteria. It could transferBrucella proteins (also known as effectors) into the host cell, these effectors make iteasy for brucella to survive and reproduct in host cells. Brucella TIR-containingdomain protein (TcpB) is one of T4SS’s substrates, it can destroy the natural immuneresponse of the host in many ways, an important one of which is throughTLRs’competitive inhibitor：the TIR domain. It can bind with the TLRs downstreamadaptors competitively which also contain the TIR domain. While how exactly TcpBaffect host innate immune response by TIR domain is still unclear.In this study, we designed and synthesized13TcpB TIR-derived decoy peptides,and had a research on theimpact of these13peptides on TLR4signal whichinducedby lipopolysaccharide (LPS). Each peptiderepresents a non-fragmented patch of TcpBTIR surface, andthe entire set encompasses the TIR surface domains of differentsurface area location. In order to find these peptides which have a significantinhibition on TLR4-mediated cytokine, we tested the cytokines on three differentlevels after cells were incubated with peptides. In the next we explored whether thesepeptides affect the activity of TLR4-mediated NF-κB signaling pathway, and whetherthey could cause cytotoxicity on the cell level. Our research would provide basis for abetter understanding of the mechanism of TcpB.1Impact on cytokine expression and secretionTo evaluate the effect of TcpB TIR-derived peptides on cytokines’ expression andsecretion. we first incubated cells or intraperitoneal injected with peptide molecular, and then detected the cytokine mRNA and protein expression inlipopolysaccharide(LPS) induced inflammation models. In this study we choseRAW264.7cell and C57BL/6J mice as modelsThe results showed that after80min of the stimulation of LPS, the mRNAexpression level of IFN-γ, IL-6, TNF-α were significantly higher than the blankcontrol group. Compared with the negative control, the cytokine mRNA expressionof cells incubated with TF-8,9was inhibited apparently， no matter on theMyD88-dependent cytokine or not.Experiments on the protein level in vitro show that,after the stimulation of LPS, cytokine secretion is significantly increased comparedwith the blank control, while the secretion was significantly inhibited if they wereincubated with TF8initially, the secretion of IL-1and IFN-γ also got controlled withthe incubation of TF9, while the power was much weaker than TF8. Experiments onthe protein level in vitro show that,, after the stimulation of LPS, cytokine in miceserum is significantly increased compared with the blank control. The TNF-α andIFN-γ protein level in TF8and TF9experimental group decreased significantlycompared with the negative group, in addition the inhibition of TF8was strongerthan TF9.2The impact on the NF-κB signaling pathway activityIn this study, to evaluate the effect of TcpB peptides on NF-κB pathway activity，we incubated cells with peptides,and then the I-κB level change was determined bywesternblot in LPS-induces inflammation models. The results showed that due to theactivation of NF-κB signaling pathway the I-κB was greatly reduced in the negativecontrol group, while cells incubated with TcpB TIR-derived peptides TF2,3,4,5,8,9,10,11,12,13, this reduction got reversed in some degree.3CytotoxicityIn this study, we used the lactate dehydrogenase (LDH) release assay to detect thecytotoxicity of TcpB TIR-derived peptides. The results showed that, TF1, TF5, TF7and TF8have a significant cytotoxicity compared with the negative control group,however, the control peptide also appeared cytotoxicity, and there’s no statisticaldifferences with TF1, TF5, TF7and TF8. Hypothesize that the cytotoxicityof peptide molecules due to peptides as a small molecule proteins that may be interfere withspecific metabolism in cell, the cytotoxicity that TcpB protein exhibited may beassociated with the fold, modification or other process after TcpB was translated,oneseparate domain of TcpB is not able to exhibie cytoxicity.In summary, TcpB peptides TF8and TF9could significantly decrease theTLR4-mediated cytokines secretion through MyD88-dependent andMyD88-independent ways; Peptides TF2,3,4,5,8,9,10,11,12,13showed asignificant inhibitory effect on NF-κB signaling pathway activity; Peptide TF0,1,5,7，8could obviously reduce the cell survival rate of RAW264.7. Through this study,we found DD loop、αD、DE、βE plays an important role in TcpB’s inhibition on TLR4pathway in MyD88-dependent and MyD88-independentways,which provides a basis for further research on the mechanism of TcpB.