Molecular Mechanisms Of Innate Immune Evasion By A Novel Anti-inflammatory Effector TcpS Of Salmonella Enteritidis

Toll-like receptors(TLRs)are one kind of type I transmembrane pattern recognition receptors(PRRs),which as immune sentry molecules can recognize different pathogen-associated molecular patterns(PAMPs)and activate nuclear factor ?B(NF-?B),thereby activating innate immune responses,inducing the production of inflammatory cytokines and type I interferons,and playing a key role in resisting and removing pathogenic microorganisms.TLRs activation induces MyD88(Myeloid differentiation factor 88)-dependent NF-?B activation,and promotes the production of pro-inflammatory cytokines and chemokines.In MyD88-independent signaling,TLRs can directly recruit TRIF(TIR-domain-containing adapter-inducing interferon-?)which interacts with TRAF6 to activate NF-?B,and also interacts with TBK1 to activate IRF3(Interferon regulatory factor 3).TLR and downstream adaptors contain an evolutionarily conserved cytoplasmic region,the TIR(Toll/IL-1 receptor)domain,which can form homodimers or heterodimers,and is particularly important for TLR signaling.Abnormal or excessive activation of the TLR signaling can cause inflammation.Many infectious diseases,autoimmune diseases and malignancies can cause excessive inflammation.Therefore,drugs that target innate immune responses and signal transmission have potential medical prospects.Salmonella is an important foodborne zoonotic pathogen that can cause local infection and even severe systemic diseases in humans and animals,such as gastroenteritis,diarrhea and typhoid.Salmonella can secrete a variety of virulence effectors through the Type ?secretion system(T3SS)to promote its colonization.Salmonella pathogenicity island(SPI)-encoded effectors directly translocate into the host cells through T3SS,and regulate the biochemical process.SPI-1 and SPI-2 are particularly important during Salmonella infection.These effectors have multiple roles,including participation in rearrangement of host cytoskeleton,recruitment of immune cells,cell metabolism,humoral secretion,and host inflammatory responses.Salmonella has evolved a variety of strategies to escape or manipulate the host immune defenses to promote its survival.In this study,the TIR domain-containing gene tcpS of Salmonella Enteritidis was identified,and its structural characteristics,expression and secretion,and infection dynamics were explored.The effect of TcpS on the virulence of Salmonella Enteritidis was studied in vitro and in vivo.The molecular mechanism that the anti-inflammatory effector TcpS interferes with the TLR signaling to evade the host innate immunity was revealed.The anti-inflammation and anti-infection efficacy of the functional peptides of the TcpS TIR domain was identified.This study reveals a new mechanism by which Salmonella Enteritidis evades the host innate immunity,and provides new ideas for the design of novel small-molecule therapeutic drugs that inhibit inflammation.1.Bioinformatics analysis of Salmonella Enteritidis TcpS and its expression and secretion characteristicsBioinformatics analysis identified the presence of tcpS(TIR-and Coiled-coil-domain containing Protein of Salmonella)in Salmonella Enteritidis C50041.TcpS is located in the 5' asn tRNA island of ROD21(Region of difference 21),which contains phage integrase and conjugal transfer protein nearby.The PCR method was used to identify the distribution of tcpS gene in different Salmonella serotypes and other bacteria.The results showed that tcpS was only found in Salmonella Enteritidis,S.Pullorum/Gallinarum and S.Dublin.The two genes of Salmonella lygD and flhB were introduced and a tcpS-based multiplex PCR detection method was established to quickly and efficiently identify the three specific Salmonella serovars.TcpS contains 293 amino acids,and the C-terminal TIR domain is 137 amino acids The amino acid similarity of the TIR domain between TcpS and TLR4/MyD88 is about 40%.The TIR domain has three conserved motifs:Box 1,Box 2 and Box 3,which are particularly important for TLR signaling.Comparing the three boxes with various TLRs and adaptors,the sequence of TcpS Box 1 is highly similar.Bioinformatics analysis shows the structure of TIR-TcpS has high similarity and spatial overlap with that of other proteinsTcpS was expressed using a prokaryotic expression system and TcpS polyclonal antibody was prepared.The expression of TcpS was detected after culturing C50041 alone or co-cultured with RAW264.7 cells.The results showed that TcpS could be detected in the bacterial precipitate,indicating that Salmonella could express TcpS protein,and tcpS is not a putative gene.After co-culture with RAW264.7 cells,TcpS could be detected in the culture supernatant,indicating that co-culture can promote the secretion of TcpS.C50041?SPI-1 and ?SPI-2 mutants were constructed,and the expression and secretion of TcpS protein were identified by Transwell system.The results showed that TcpS could be detected in the upper compartment of the C50041 infection group,but DnaK could not be detected in the supernatant of the lower compartment,suggesting that TcpS was secreted by bacteria.After the deletion of SPI-1,the secretion level of TcpS was significantly reduced,while the absence of SPI-2 did not affect its secretion,indicating that TcpS can be secreted through T3SS1.C50041 ?tcpS,C50041 ?tcpS+pCX340 and C50041 ?tcpS+pCX340-TcpS were constructed.HeLa cells were infected with Salmonella expressing each fusion protein.The CCF2/TEM-1 reporter system was used to identify the secretion of Salmonella TcpS into the cells.The results showed that both TEM-1 and TcpS-TEM-1 fusion proteins were successfully expressed.Cells infected with Salmonella expressing TEM-1 showed green fluorescence,indicating that TEM-1 was not transported into the cells,while cells infected with Salmonella expressing TcpS-TEM-1 showed blue fluorescence,indicating TcpS-TEM-1 was successfully transported into the cells and the CCF2 substrate was decomposed.Mice were challenged intragastrically by Salmonella C50041,and livers were collected at different time points.The levels of Salmonella pro-inflammatory and anti-inflammatory effectors and mouse cytokines were detected by quantitative real-time PCR.The results showed that the expression of anti-inflammatory effectors(tcpS,avrA and sptP)was higher at 24 h-2 d after infection,and the pro-inflammatory effectors(sopB,sopE,srfA,and sopD)were highest at 24 h.The levels of cytokines(TNF-?.IL-1?,IL-6,IL-12p40,IFN-y.CXCL2,and CCL4)were higher at 12 h and 7 d.At the early stage of infection,Salmonella reduced the pro-inflammatory effectors and increased the anti-inflamatory effectors to inhibit the production of cytokines.Besides,the expression dynamics of TcpS is consistent with other anti-inflammatory effectors.2.The effect of Salmonella Enteritidis TcpS on bacterial virulenceThe Salmonella C50041 ?tcpS mutant and C50041 ?tcpS+pBR322-TcpS complementary strain were constructed.The growth rates of TcpS WT,mutant strain and complementary strain were relatively consistent.RAW264.7 cells were used as the infection model,and the bacterial invasion rate,proliferation rate and the expression of inflammatory cytokines were detected.The results showed that C50041 and TcpS complementary strains had higher invasion and proliferation rates than the ?tcpS mutant,while the inflammatory cytokines IL-6 and IL-1? were significantly lower.It shows TcpS can inhibit the production of cytokines and promote the bacterial colonization.The over-expression of TcpS in the complementary strain has a more obvious inhibitory effect on the TLR signaling,so it has lower levels of inflammatory cytokines and higher invasion and proliferation rates.BALB/c mice were infected with Salmonella C50041,?tcpS and ?tcpS+pTcpS,and the livers and spleens were collected 4 days after challenge.The levels of inflammatory cytokines,bacterial load and histopathological changes were detected.The results showed that the WT and the TcpS complementary strain had significantly higher bacteria load in tissues than the ?tcpS mutant group,indicating that TcpS can promote bacterial survival in vivo.Compared with the ?tcpS mutant,the WT and the TcpS complementary strain inhibited the production of CXCL15,TNF-?,IL-6,and IL-1?,indicating that TcpS inhibited the production of inflammatory cytokines in vitro and in vivo,and promote bacterial proliferation.Histopathological analysis showed that TcpS can inhibit the inflammatory responses at the early stage of infection.The inflammatory infiltration of livers and spleens in mice infected with AtcpS mutant was significantly higher than that in WT and TcpS complementary strain groups.While at the late stage of infection,the liver and spleen of?tcpS-infected mice recovered to normal.These results indicate that Salmonella effector TcpS can inhibit the inflammatory responses at the early stage of infection and evade the host innate immunity to promote the bacterial survival.Mice sera were collected 6 days after infection.The sera of Salmonella WT and TcpS complementary strain-infected mice were dark red,while the sera of ?tcpS-infected mice appeared normal light yellow.The hemoglobin in the sera was detected,and Salmonella WT and the TcpS complementary strain could cause hemocytolysis and the release of hemoglobin at the late stage of infection,and severe pathological damages were observed in the livers and spleens.The levels of inflammatory cytokines in the sera at the late stage of infection were detected by ELISA,and the inflammatory cytokines IL-12p40,TNF-?.IL-1?and IL-6 were significantly higher in the sera of mice infected with WT and the complementary strains.It showed that TcpS evaded the innate immunity and promotes bacterial survival at the early stage of infection,leading to inflammatory storms and histopathological damages in the late stage of infection.In the late stage of Salmonella challenge,the hairs of mice from the WT and TcpS complementary groups were messy and dull,with reduced appetite and no vigor.The survival curve was used to evaluate the effect of TcpS on bacterial virulence during the evasion of Salmonella from the innate immunity.The results showed that the mortality of mice infected with ?tcpS mutant was significantly reduced.The LD50 challenge experiment was performed in a mouse model.The LD50 results showed that the LD50 of the Salmonella WT and the TcpS complementary strains were 1.7×105 CFU and 4.5×105 CFU,respectively,while the LD50 of the ?tcpS mutant was 5.4×106 CFU,indicating that Salmonella virulence attenuated after TcpS is deleted,and tcpS is a new virulence effector that evades the host immune system.3.The molecular mechanisms of innate immune subvertion by Salmonella Enteritidis TcpSUsing HEK293T and RAW264.7 as cell models,the NF-?B luciferase reporter system was used to detect the inhibitory effects of TcpS on the TLR signaling.The results showed that TcpS can inhibit TLR2,TLR4,TLR5,TLR7 and TLR9-mediated NF-?B activation in a dose-dependent manner.In addition,TcpS can strongly inhibit TLR3-mediated activation of IFN-? and NF-?B,while TLR3 signaling is MyD88-independent,indicating that TcpS can inhibit both MyD88-and TRIF-mediated TLR activation.Overexpression of MyD88 and TRIF can significantly activate the NF-?B and IRF pathways,while TcpS can effectively inhibit MyD88 and TRIF-mediated TLR signaling.MyD88 and TRIF-specific siRNA experiments further demonstrated that the inhibitory effect of TcpS is mediated by MyD88 and TRIF.In order to prove that MyD88 is necessary for TcpS to exert its inhibitory function,C57BL/6J WT and MyD88-/-BMMs cells were infected with Salmonella C50041 WT,?tcpS mutant and TcpS complementary strain,respectively.The levels of inflammatory cytokines and bacterial invasion and proliferation rates were determined.The results showed that in WT BMMs cells,TcpS can promote bacterial invasion and proliferation,and inhibit the expression of IL-1?.In MyD88-/-BMMs cells,TcpS could not inhibit the inflammatory responses or promote bacterial proliferation,suggesting that MyD88 plays an important role in the anti-inflammatory effects of TcpS.The co-immunoprecipitation assay was used to identify the interaction between TcpS and MyD88.The results showed there was a direct interaction between TcpS and MyD88,indicating TcpS could directly interfere with the innate immunity through interaction with MyD88.Eukaryotic expression plasmid of TcpS was transfected into HeLa cells,and the localization of p65 subunit was observed by laser confocal microscopy after LPS stimulation.The results showed that TcpS can significantly inhibit the translation of p65 in a dose-dependent manner.TcpS/TLR4/MD2 plasmids were transfected into HEK293T cells,and the levels of inflammatory cytokines were detected after LPS stimulation.The results showed that TcpS can significantly inhibit LPS-induced IL-1?,IL-8,IL-17A and TNF-?expression.In addition,in HeLa cells,TcpS can inhibit the expression of the inflammatory cytokine IL-1? induced by LPS,indicating that TcpS can interfere with the TLR-NF-?B signaling and thereby block the innate immune system.In order to identify the key amino acids that play anti-inflammatory effects in the TcpS TIR domain,amino acids that may be sensitive to point mutations were selected and a series of key amino acid mutants were constructed.After transfection of TIR-TcpS and each mutant,the NF-?B activation and IL-8 secretion were detected.The results showed that E180A weakened its inhibitory effect,while Y191A and I284A completely abolished its inhibitory effect.Particularly,the inhibitory effect of TIR-TcpS on TLR signaling was significantly weaker than that of full-length TcpS,indicating that the N-terminus of TcpS plays an important role in its anti-inflammatory effect.Bioinformatics analysis shows that TcpS contains a Coiled-coil(CC)domain at the N-terminus in addition to the TIR domain at the C-terminus.pCMV-HA-TcpS and pCMV-HA-?CCTcpS were transfected into HEK293T cells,respectively,and the effect of CC domain on TcpS function was detected.Full-length TcpS can significantly inhibit the NF-?B activation and the IL-8 secretion,while the anti-inflammatory effect of ?CCTcpS was significantly reduced,indicating that the CC domain plays an important role for TcpS in its highly effective inhibition of TLR signaling.HEK293T cells were transfected with pMyc-TcpS and pFlag-TcpS/pFlag-?CCTcpS,respectively.Co-immunoprecipitation assays were performed to determine whether TcpS would form homodimers.The results showed that Myc-TcpS interacted with Flag-TcpS,and the interaction was achieved through the CC domain.The TcpS Coiled-coil domain is a supercoil formed by entanglement of two amphoteric alpha helices,which can enhance the binding ability of TcpS to TLRs or other TIR-containing adaptors.It is shown that TcpS can form homodimers through its N-terminal Coiled-coil domain,which can effectively block the TLR signaling pathway and thereby inhibit the inflammatory responses.4.Anti-inflammation and anti-infection effects of functional peptides of TcpS TIR domainThe TIR domain of TcpS was analyzed by bioinformatics,and TIR-TcpS was divided into six peptide regions.The inhibitory effects of each peptide on the TLR4 signaling were determined in mouse primary peritoneal macrophages.All peptides,especially SR2,SR3,SR4,SR5 and SR6 could significantly inhibit the production of IL-1? and TNF-?,which were mediated by MyD88.Peptides SR3 and SR5 moderately inhibited the production of IFN-?,while SR2,SR5 and SR6 can inhibit the production of RANTES.Different peptides have different degrees of inhibitory effects on the activation of p-ERK induced by LPS.Among them,the inhibition of SR3,SR5 and SR6 is particularly obvious,and SR6 even eliminates its activation.These peptides may be potential functional TIR-TIR binding sites in the TcpS TIR domain.The position of each peptide in the TcpS TIR structure was predicted using SWISS-MODEL.These peptides are located on the surface of the TIR structure and surround the TIR spherical structure,which also indicates that these sites can interact more directly with other molecules.The functional TIR-TcpS peptides SR4,SR5 and SR6 were injected into mice intraperitoneally,and then challenged with sub-lethal dose of LPS.The secretion levels of inflammatory cytokines in the sera were detected to determine the immune protection effect of TcpS peptide.It is noteworthy that after LPS challenge,the sera of mice in the PBS group showed a dark red color,while the sera of mice in the LPS non-challenge group showed normal light yellow.The three peptides SR4,SR5 and SR6 can alleviate the color deterioration,especially the SR4 treatment can prevent the change of sera color.In addition,LPS challenge can significantly increase the hemoglobin in the sera,while peptide treatment can significantly reduce the hemoglobin release.All three peptides can effectively inhibit the production of inflammatory cytokines IL-6,IL-12p40 and TNF-? in mouse sera,which were even similar to the normal levels.These results indicate that SR4,SR5,and SR6 peptides have potent anti-inflammatory effects and protective effects against LPS challenge.To detect the effect of peptides on cell viability,different TIR-TcpS peptides were incubated with mouse peritoneal macrophages,and cell viability was evaluated by MTT experiments.The results showed that after SR1,SR3 and SR4 peptide treatment,the cell viability was still greater than 90%,which was even comparable to that of the medium treatment group.Therefore,anti-inflammatory peptide SR4 can block TLR-mediated inflammatory responses in vivo and in vitro,and has less damage to cell viability.The protective effect of SR4 peptide against influenza virus was tested in a mouse model.The results showed that after H1N1 influenza virus infection,the body weight of mice in the PBS and control peptide groups dropped dramatically(27%and 29%),while the body weight of mice in the SR4-treated group decreased slightly(5.5%),indicating that the SR4 peptide can regulate the immune system in the pathological state of the body,thereby resisting influenza virus infection.TIR-TcpS peptide can inhibit LPS-mediated excessive inflammatory responses and has antiviral efficacy,indicating that it could be used as a potential therapeutic drug for excessive inflammation and autoimmune diseases.