转燕麦噬酸菌蛋白激发子基因flagellin的链霉工程菌株构建及鉴定

利迪链霉菌A01可以产生大量的抗生素——纳他霉素,其通过结合病原真菌细胞膜上的甾醇分子而起到抑制病原菌生长的作用。燕麦噬酸菌的鞭毛蛋白组分FLAGELLIN可以诱导植物抗性,激发活性氧及水杨酸防御反应途径。本研究将燕麦噬酸菌蛋白激发子基因flagellin克隆,接合转移入利迪链霉菌A01中,使工程菌增加诱导植物抗性的功能。PCR验证表明:成功获得了含flagellin基因的阳性工程菌株,证明通过链霉工程菌构建可实现抗生与诱抗的协同作用,预计会提高防治植物病害的效果。

AEG-1肺归巢域与Flagellin融合基因的杆状病毒制备及鉴定

目的通过重叠PCR方法获得AEG-1C-Flic融合基因,采用Bac-To-Bac杆状病毒表达系统获得重组AEG-1C-Flic杆状病毒,为后续AEG-1C-Flic病毒样颗粒疫苗的制备奠定基础。方法从鼠伤寒沙门菌的基因组中PCR扩增细菌鞭毛蛋白Flagellin,采用重叠PCR的方法将AEG1肺归巢域段基因与Flagellin融合,并添加猴免疫缺陷病毒(simian immunodeficiency virus,SIV)包膜蛋白gp41信号肽与跨膜区,构建AEG-1C-Flic pFastBacTM重组表达载体,转座大肠杆菌E.coliDH10Bac感受态细胞后获得重组杆粒,重组杆粒转染昆虫细胞Tn5,获得AEG-1CFlic重组杆状病毒,并采用Western Blot方法对该病毒进行鉴定。结果构建的AEG-1C-Flic杆状病毒表达载体经双酶切及测序正确,通过转化E.coli DH10Bac感受态细胞获得重组Bacmid,经PCR鉴定大小正确,Western Blot结果显示Bacmid转染Tn5细胞后成功获得重组杆状病毒rBv AEG-1C-Flic。结论成功构建的AEG-1C-Flic杆状病毒可用于新型AEG1-病毒样颗粒疫苗的制备。

激动剂Flagellin对中性粒细胞凋亡和Bax蛋白表达的影响

目的:研究TLR激动剂flagellin影响中性粒细胞凋亡及凋亡调控蛋白Bax的表达。方法:分离培养正常人的外周血中性粒细胞,分为实验组(flagellin刺激组)、凋亡抑制组(LPS刺激组)和自然凋亡组。用流式细胞术检测不同组中性粒细胞的凋亡发生率,通过Western blotting技术检测各组中性粒细胞Bax蛋白的表达。结果:flagellin使中性粒细胞自发性凋亡的发生率增高,与凋亡抑制组和自然凋亡组比较差异有统计学意义(P<0.01);接受flagellin刺激的中性粒细胞表达Bax蛋白高于凋亡抑制组与自然凋亡组(P<0.01)。结论:在TLR激动剂诱发的中性粒细胞生存效应中,flagellin可能通过上调中性粒细胞胞内Bax蛋白的表达来缩短中性粒细胞的生命期限。

Molecular cloning and functional identification of an apple flagellin receptor MdFLS2 gene

The leucine-rich repeat receptor kinase flagellin-sensing 2 gene(MdFLS2; Gene ID: MDP0000254112) was cloned from Royal Gala apple(Malus×domestica Borkh.). This gene contained a complete open reading frame of 3 474 bp that encoded 1 158 amino acids. The phylogenetic tree indicated that Prunus persica FLS2 exhibited the highest sequence similarity to MdFLS2. The PlantCare database suggests that the promoter sequence of MdFLS2 contains several typical cis-acting elements, including ethylene-, gibberellin-, salicylic acid-, and drought-responsive elements. Quantitative real-time PCR analysis showed that MdFLS2 was widely expressed in the different tissues of the apple and most highly expressed in the leaves. Furthermore, MdFLS2 was significantly induced by the flagellin elicitor peptide flg22. Treatment of the apple seedling leaves with flg22 resulted in an increase in leaf callose levels with increased treatment duration. An increase in the production of Oalong with the expression of disease-related genes was also observed. An oxidative burst was detected in the treated seedlings, but not in the control seedlings, indicating that flg22 had stimulated the expression of the MdFLS2 gene and its downstream target genes. Furthermore, the ectopic expression of MdFLS2 complemented the function of the Arabidopsis fls2 mutant and conferred enhanced flg22 tolerance to the transgenic Arabidopsis, suggesting that MdFLS2 acts as a positive regulator in the response to pathogens in apple.

Polymorphism of flagellin A gene in Helicobacter pylori

AIM: To study the polymorphism of flagellin A genotype and its significance in Helicobacter pyiori ( H. Pylori).METHODS: As the template, genome DNA was purified from six clinical isolates of H. Pylori from outpatients, and the corresponding flagellin A fragments were amplified by polymerase chain reaction. All these products were sequenced. These sequences were compared with each other, and analyzed by software of FASTA program.RESULTS: Specific PCR products were amplified from all of these H. Pylori isolates and no length divergence was found among them. Compared with each other, the highest ungapped identity is 99. 10%, while the lowest is 94.65%.Using FASTA program, the alignments between query and library sequences derived from different H. Pylori strains were higher than 90%.CONCLUSION: The nucleotide sequence of flagellin A in H.pylori is highly conservative with incident divergence. This information may be useful for gene diagnosis and further study on flagellar antigen phenotype.

Relationship between genotype and phenotype of flagellin C in Salmonella

AIM: To discover the relationship between the genotype and antigen serotype of flagellin C among Salmonella strains.METHODS: Fragment of Salmonella flagellin C in plasmid pLS408 was cloned, sequenced and compared with the corresponding sequence in other strains. Salmonella strains including two typhi strains, one paratyphoid strain, one enteritidis and one typhimurium strain were isolated from outpatients. Genome DNA was purified respectively from these clinical isolstes, then the corresponding flagellin C fragment was amplified by polymerase chain reaction, and the amplification products were analyzed by agarose gel electrophoreeis.RESULTS: The cloned fragment includes 582 nucleotides encoding the variable region and partial conservative region of Salmonella flagellin C in plasmid pLS408. With comparison to the corresponding sequences reported previously, there is only a little difference from other strains with the same flagellar serotype in both nucleotide and amino acid level. Specific PCR products were amplified in Salmonella strains with flagellar eerotype H-1-d including S. Muenchen, typhi and typhimurium, but not in S.paratyphoid C or S. Enteritidis strains.CONCLUSION: In this experiment, the specificity of nucleotide sequence could be found in flagellin C central variable regions as it exists in flagellar serotypes in Salmonella. It may be helpful to developing a rapid,sensitive, accurate and PCR-based method to detect Salmonella strains with serotype H-1-d.

Effect of Acacetin on flagellin induced NLRC4 inflammasome activation in mouse bone marrow-derived macrophages

Objective:To investigate the effect of acacetin on flagellin induced NLRC4 inflammasome activation in mouse bone marrow-derived macrophages(BMDMs).Methods:Mouse BMDMs were divided into control group,LPS group,LPS+flagellin group and LPS+acacetin+flagellin group.All groups were added with complete medium,then primed with LPS(50 ng/mL)for 3 hrs except the control group,whereafter,LPS+flagellin group was treated with flagellin(10μmol/L)for 0.5 hr and LPS+acacetin+flagellin group was treated with acacetin(10μmol/L)for 0.5 hr following by flagellin(10μmol/L)for 0.5 hr.Pro-caspase-1,pro-IL-1βin cell lysate and caspase-1,IL-1βin supernatant were detected by Western blot(WB).IL-1β,IL-18 and TNF-αin supernatant were measured by enzyme-linked immunosorbent assay(ELISA).And the activity of LDH in supernatant was assessed by LDH test kit.Results:Compared with the control group,in LPS+flagellin group,the expression of caspase-1,IL-1βprotein in supernatant were significantly increased(all P-values<0.05),but the differences of the expression of pro-caspase-1 and pro-IL-1βprotein in cell lysate were not significant.Compared with LPS+flagellin group,in LPS+acacetin+flagellin group,the expression of caspase-1,IL-1βprotein in supernatant were significantly reduced(all P-values<0.05),while the differences of the expression of pro-caspase-1 and pro-IL-1βprotein in cell lysate were not significant.ELISA showed that compared with the control group,the levels of IL-1β,IL-18,and TNF-αand the activity of LDH in supernatant of LPS+flagellin group were significantly increased(all P-values<0.05).Compared with LPS+flagellin group,in LPS+Acacetin+flagellin group,the level of IL-1βin supernatant was significantly decreased(P<0.05),meanwhile,the decreases of the levels IL-18,TNF-αand the activity of LDH were not significant.Conclusions:We found that Acacetin can effectively inhibit flagellin induced NLRC4 inflammasome activation and reduce cell damage in mouse BMDMs.

Calcium phosphate nanoparticles show an effective activation of the innate immune response in vitro and in vivo after functionalization with flagellin

For subunit vaccines,adjuvants play a key role in shaping the magnitude,persistence and form of targeted antigen-specific immune response.Flagellin is a potent immune activator by bridging innate inflammatory responses and adaptive immunity and an adjuvant candidate for clinical application.Calcium phosphate nanoparticles are efficient carriers for different biomolecules like DNA,RNA,peptides and proteins.Flagellin-functionalized calcium phosphate nanoparticles were prepared and their immunostimuiatory effect on the innate immune system,i.e.the cytokine production,was studied.They induced the production of the proinflammatory cytokines IL-8(Caco-2 cells) and IL-1β(bone marrow-derived macrophages;BMDM) in vitro and IL-6 in vivo after intraperitoneal injection in mice.The immunostimulation was more pronounced than with free flagellin.

Gene cloning and prokaryotic expression of recombinant flagellin A from Vibrio parahaemolyticus

The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals.Bacteria flagellins play an important role during infection and induction of the host immune response.Thus,flagellin proteins are an ideal target for vaccines.We amplified the complete flagellin subunit gene(flaA) from V.parahaemolyticus ATCC 17802.We then cloned and expressed the gene into Escherichia coli BL21(DE3) cells.The gene coded for a protein that was 62.78 kDa.We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting,respectively.Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus.In addition,the purified FlaA protein can be used for further functional and structural studies.

Helicobacter pylori specific immune response induced by conservative flagellin linear B-cell epitope

AIM:To testify the immunogenicity of a conservative B-cell linear epitope of Helicobacter pylori ( H pylori) flagellin A. METHODS: Different programs were used to analyze the secondary structure, molecular hydropathy, and surface accessibility of Hpyloriflagellin A. Linear B-cell epitopes were estimated based on the structural and physiochemical information. Analysis of residue divergence was proposed to screen a conservative linear epitope. The 29-peptide (Pep29mer) synthesized by chemical method, including the predicted conservative B-cell epitope and a known K^2d compatible T-cell epitope, was used to immunize mice, and then H pylori-specific antibodies were detected by ELISA. RESULTS: Based on the analyses of divergent amino acid residues, structural and physiochemical characteristics, it was strongly suggested that the short fragment NDSDGR was the core of a conservative linear epitope in flagellin A. Animals immunized by Pep29mer acquired efficient immune response. In detail, serum Hpylori-specific IgA and IgGl increased significantly in immunized group, while IgG2a only had an insignificant change. Hpylori-specific IgA in gastrointestinal flushing fluid also increased significantly. CONCLUSION: The conservative short fragment NDSDGR is the core of a linear B-cell epitope of flagellin A.

Over-activation of TLR5 signaling by high-dose flagellin induces liver injury in mice

Flagellin is a potent activator of a broad range of cell types that are involved in innate and adaptive immunity. Therefore, it is a good adjuvant candidate for vaccines, and it might function as a biological protectant against both major acute radiation syndrome during cancer radiotherapy and a mitigator of radiation emergencies. However, accumulating evidence has implicated flagellin in the occurrence of some inflammatory diseases, such as acute lung inflammation, cardiovascular collapse and inflammatory bowel disease. The aim of this study was to elucidate whether only flagellin-TLR5 signaling activation plays a role in the pathophysiology of liver or whether some other flagellin activity also contributes to liver injury either via bacterial infections or during clinical applications. Recombinant flagellin proteins with or without TLR5-stimulating activity were used to evaluate the role of flagellin-TLR5 signaling in liver injury in wild-type and TLR5 KO mice. Gross lesions and large areas of hepatocellular necrosis were observed in liver tissue 12 h after the intraperitoneal administration of 100 or 200 pg flagellin （FliC） in a dose-and time-dependent manner in wild-type mice, but not in TLR5 KO mice. Deletion of the N-terminal or TLR5 binding domain of flagellin inhibited flagellin-induced inflammatory responses and the subsequent acute liver function abnormality and damage. These data confirmed that flagellin is an essential determinant of liver injury and demonstrated that the over-activation of TLR5 signaling by high-dose flagellin caused acute inflammatory responses, neutrophil accumulation and oxidative stress in the liver, which contributes to the progression and severity of flagellin-induced liver injury.

Rice OsFLS2-Mediated Perception of Bacterial Flagellins Is Evaded by Xanthomonas oryzae pvs. oryzae and oryzicola

Bacterial flagellins are often recognized by the receptor kinase FLAGELLIN SENSITIVE2 （FLS2） and activate MAMP-triggered immunity in dicotyledonous plants. However, the capacity of monocotyledonous rice to recognize flagellins of key rice pathogens and its biological relevance remain poorly understood. We demonstrate that ectopically expressed OsFLS2 in Arabidopsis senses the eliciting fig22 peptide and in vitro purified Acidovorax avenae （Aa） flagellin in an expression level-dependent manner, but does not recognize purified flagellins or derivative fig22x~ peptides ofXanthomonas oryzae pvs. oryzae （Xoo） and or- yzicola （Xoc）. Consistently, the fig22 peptide and purified Aa flageUin, but not Xoo/Xoc flagellins, induce various immune responses such as defense gene induction and MAPK activation in rice. Perception of flagellin by rice does induce strong resistance to Xoo infection, as shown after pre-treatment of rice leaves with Aa flagellin. OsFLS2 was found to differ from AtFLS2 in its perception specificities or sensitivities to different fig22 sequences. In addition, post-translational modification of Xoc flagellin was altered by dele- tion of glycosyltransferase-encoding rbfC, but this had little effect on Xoc motility and rpfC mutation did not detectably reduce Xoc virulence on rice. Deletion of flagellin-encoding fliC from Xoo/Xoc blocked swim- ming motility but also did not significantly alter Xoo/Xoc virulence. These results suggest that Xoo/Xoc carry flg22-region amino acid changes that allow motility while evading the ancient flagellin detection sys- tem in rice, which retains recognition capacity for other bacterial pathogens.

鞭毛素蛋白联合雷帕霉素对4T1乳腺癌细胞及荷瘤小鼠的抑制作用

目的 在乳腺癌4T1细胞构建的荷瘤小鼠模型中,探讨鞭毛素蛋白(flagellin,FliC)与雷帕霉素(rapamycin,RAPA)联用对肿瘤生长和转移的影响,并探讨其对部分免疫细胞的调节作用。方法 用小鼠乳腺癌4T1细胞建立Balb/c肿瘤模型,分别注射鞭毛素蛋白和(或)雷帕霉素,并定期记录肿瘤大小,计算肿瘤抑制率。用MTT法检测二者联用对乳腺癌4T1细胞的抑制作用。用流式细胞术检测荷瘤小鼠肿瘤组织中CD11b+Gr-1+骨髓来源的抑制性细胞(MDSCs)和CD11b+F4/80+肿瘤相关巨噬细胞(TAMs)的百分比。用HE染色检测肝、肺组织中转移情况。结果 体内实验中,RAPA和FliC对肿瘤生长均有抑制作用,但RAPA+FliC组抑制效果最好(F=5.593,P<0.05)。在体外实验中,给药组对4T1细胞增殖的抑制率均高于Control组,但RAPA+FliC组的抑制率最高。肿瘤组织中,与FliC组和Control组相比,RAPA+FliC组和RAPA组组织细胞中CD11b+Gr-1+MDSC和CD11b+F4/80+TAM百分比基本相同且均为最低(P<0.05)。与Control组相比,RAPA组和RAPA+FliC组肝、肺组织肿瘤转移情况较轻。结论 鞭毛素蛋白与雷帕霉素联合作用可抑制4T1细胞的增殖活性,并增强乳腺癌荷瘤小鼠抗肿瘤效应,抑制肿瘤生长和转移。其机制可能与肿瘤微环境中免疫细胞浸润有关,但具体相关性还有待进一步证实。

NLRC4 downregulates 的激活对 flagellin 的调停 TLR5 的抗体免疫者回答

Bacterial flagellin is a unique pathogen-associated molecular pattern （PAMP）, which can be recognized by surface localized Toll-like receptor 5 （TLR5） and the cytosolic NOD-like receptor （NLR） protein 4 （NLRC4） receptors. Activation of the TLR5 and/or NLRC4 signaling pathways by flagellin and the resulting immune responses play important roles in anti-bacterial immunity. However, it remains unclear how the dual activities of flagellin that activate the TLR5 and/or NLRC4 signaling pathways orchestrate the immune responses. In this study, we assessed the effects of flagellin and its mutants lacking the ability to activate TLR5 and NLRC4 alone or in combination on the adaptive immune responses against flagellin. Flagellin that was unable to activate NLRC4 induced a significantly higher antibody response than did wild-type flagellin. The increased antibody response could be eliminated when macrophages were depleted in vivo. The activation of NLRC4 by flagellin downregulated the flagellin-induced and TLR5-mediated immune responses against flagellin.

Salmonella flagella confer anti-tumor immunological effect via activating Flagellin/TLR5 signalling within tumor microenvironment

Salmonella:mediated cancer therapy has achieved remarkable anti-tumor effects in experimental animal models,but the detailed mechanism remains unsolved.In this report,the active involvement of the host immune response in this process was confirmed by comparing the tumor-suppressive effects of Salmonella in immunocompetent and immunodeficient mice bearing melanoma allografts.Since flagella are key inducers of the host immune response during bacterial infection,flagella were genetically disrupted to analyse their involvement in Salmonella-mediated cancer therapy.The results showed that flagellum-deficient strains failed to induce significant anti-tumor effects,even when more bacteria were administered to offset the difference in invasion efficiency.Flagella mainly activate immune cells via Flagellin/Toll-like receptor 5(TLR5)signalling pathway.Indeed,we showed that exogenous activation of TLR5 signalling by recombinant Flagellin and exogenous expression of TLR5 both enhanced the therapeutic efficacy of flagellum-deficient Salmonella against melanoma.Our study highlighted the therapeutic value of the interaction between Salmonella and the host immune response through Flagellin/TLR5 signalling pathway during Salmonella-mediated cancer therapy,thereby suggesting the potential application of TLR5 agonists in the cancer immune therapy.

Salmonella enterica Flagellin Is Recognized via FLS2 and Activates PAMP-Triggered Immunity in Arabidopsis thaliana

Infections with Salmonella enterica belong to the most prominent causes of food poisoning and infected fruits and vegetables represent important vectors for salmonellosis. Recent evidence indicates that plants recognize S. enterica and raise defense responses. Nonetheless, the molecular mechanisms controlling the interaction of S. enterica with plants are still largely unclear. Here, we show that flagellin from S. enterica represents a prominent pathogenassociated molecular pattern （PAMP） in Arabidopsis thaliana, which induces PAMP-triggered immunity （PTI） via the recognition of the fig22 domain by the receptor kinase FLS2. The Arabidopsis fls2 mutant shows reduced though not abolished PTI activation, indicating that plants rely also on recognition of other S. enterica PAMPs. Interestingly, the S. enterica type III secretion system （T3SS） mutant prgH- induced stronger defense gene expression than wild-type bacteria in Arabidopsis, suggesting that T3SS effectors are involved in defense suppression. Furthermore, we observe that S. enterica strains show variation in the fig22 epitope, which results in proteins with reduced PTI-inducing activity. Altogether, these results show that S. enterica activates PTI in Arabidopsis and suggest that, in order to accomplish plant colonization, S. enterica evolved strategies to avoid or suppress PTI.

Chimeric flagellin expressed by Salmonella typhimurium induces an ESAT-6-specific Th1-type immune response and CTL effects following intranasal immunization

The flagellin component FliC of Salmonella typhimurium is capable of activating the innate immune system via specific interactions with TLR5 and can also act as a carrier of foreign antigen to elicit antigen-specific immune responses.Thus,we constructed an attenuated Salmonella strain SL5928(fliC/esat)expressing chimeric flagellin that contained the ESAT-6 antigen coding sequence of Mycobacterium tuberculosis inserted into the highly variable region of the Salmonella flagellin coding gene fliCi.The chimeric flagellin functioned normally,as demonstrated using a flagella swarming assay and electron microscopy.To analyze the effects of chimeric flagellin,the cell-mediated immune response and cytotoxic T lymphocyte(CTL)effects specific for ESAT-6 antigen were tested after intranasal immunization of mice with flagellated Salmonella SL5928(fliC/esat).The results showed that SL5928(fliC/esat)intranasal immunization can strongly elicit an ESAT-6-specific T helper(Th)1-type immune response in mucosal lymphoid tissues,such as nasopharynx-associated lymph nodes,lung and Peyer’s patches,and a Th1/Th2 response was elicited in spleen and mesenteric lymph nodes.Furthermore,intranasal immunization of SL5928(fliC/esat)produced efficient CTL effects,as demonstrated using a 5-and 6-carboxyfluorescein diacetate succinimidyl ester(CFSE)assay.Thus,our study revealed that Salmonella flagellin acts as a carrier for foreign antigen and triggers strong Th1 and CTL responses during intranasal immunization.Chimeric flagellin is potentially an effective strategy for the development of novel vaccines against tuberculosis in humans and animals.

大肠杆菌Nissle 1917鞭毛蛋白FliC的结构特性及刺激Caco-2细胞作用的研究

试验旨在分析大肠杆菌Nissle 1917鞭毛蛋白FliC(FliC_(EcN))的结构特征及其对Caco-2细胞的刺激作用。试验通过大肠杆菌BL21(DE3)表达FliCEcN蛋白、经NTA柱纯化FliC_(EcN)蛋白并通过SDS-PAGE和Westernblot验证,利用SOPMA和Alphofold2预测FliC_(EcN)蛋白的结构模型、表面增强拉曼光谱和圆二色谱解析FliC_(EcN)蛋白的结构,使用FliC_(EcN)蛋白刺激Caco-2细胞后,检测免疫相关因子的分泌水平。结果显示,FliC_(EcN)蛋白的大小为64kDa,能够被抗His单克隆抗体特异识别;FliC_(EcN)蛋白的氨基和羧基末端主要由α-螺旋组成,中间结构域主要由β-折叠组成。FliC_(EcN)蛋白酰胺Ⅰ区最大吸收峰均位于1656 cm^(-1)处,主要二级结构为α-螺旋和β-折叠;FliC_(EcN)的α-螺旋占比44.4%,β-折叠占比23.4%,β-转角占比13.5%,无规则卷曲占比19.0%;FliC_(EcN)蛋白能够有效刺激Caco-2细胞分泌白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)和白细胞介素-10(IL-10);随着刺激时间延长,IL-6的分泌水平呈下降趋势,IL-10和TNF-α的分泌水平呈增加趋势。研究表明,α-螺旋和β-折叠是构成FliC_(EcN)的主要结构,可促进其构象的正确折叠和维持其三维结构稳定,FliC_(EcN)蛋白刺激Caco-2细胞分泌IL-6、IL-10、TNF-α的水平存在差异。

Flagellin of Pseudomonas aeruginosa induces transforming growth factor beta 1 expression in normal bronchial epithelial cells through mitogen activated protein kinase cascades

Background Acute lung infection due to Pseudomonas aeruginosa （P. Aeruginosa） is a serious problem, especially in patients with structural lung conditions or immune compromised hosts, leading to an overwhelming threat with a high risk of morbidity and mortality. As an outcome of infection, fibrosis can be linked with chronic lung diseases. But some fibrotic manifestations, such as an irreversible decrease of lung function and fibrous bands seen on chest imaging, have been found after an acute infection with P. Aeruginosa. Fibrogenesis/remodeling resulting from acute lung infection by P.aeruginosa is rarely reported. This study was designed to explore the relation between fibrogenesis/remodeling and acute infection by P. Aeruginosa in vitro. We used flagellin protein from P. Aeruginosa, a key initiator of acute P.aeruginosa lung infection, to elucidate mechanisms by which acute lung infection with P. Aeruginosa can cause fibrogenesis/remodeling.Methods We studied the effect of flagellin from P. Aeruginosa （flagellin for short） on the transforming growth factor beta 1 （TGF-β1） and interleukin-8 （IL-8） expression, and the possible involvement of the signaling pathway, tumor necrosis factor receptor-associated factor 6 （TRAF6）/mitogen activated protein kinase （MAPK） pathway. Flagellin was purified from the P. Aeruginosa standard strain, PAO1. Normal bronchial epithelial cells BEAS-2B were challenged with different concentrations of flagellin, and cell viability assessment was performed by cell counting kit-8. BEAS-2B cells were incubated with flagellin with the specific MAPK inhibitors or TRAF6 siRNA. Cell lysates and the cultured supernatant were collected. The level of TGF-β1 and IL-8 were detected by enzyme-linked immunosorbant assay （ELISA）. Western blotting was used to detect the protein levels of MAPK signal proteins p38, c-Jun NH2-terminal kinase （JNK） and extracellular regulated kinase （ERK）.Results Expression of TGF-β1 in BEAS-2B cells was elevated by flagellin vs. Control groups （（104.3±20.8） vs.（44.6±4.4） pg/ml （P 〈0.01）） and was ablated by either p38 or JNK inhibitors compared with flagellin treatment （（45.1±18.8）vs. （104.3±20.8） pg/ml and （48.1±20.8） vs. （104.3±20.8） pg/ml, respectively （P 〈0.05））. Flagellin also elevated the expression of IL-8 in BEAS-2B cells vs. The control groups （（554.9±57.7） vs. （51.4±2.2.9） pg/ml （P 〈0.01））, and p38 MAPK inhibitors weaken the expression by flagellin （（301.1 ±155.1） vs. （554.9±57.7） pg/ml （P 〈0.05））. Western blotting revealed that all three MAPK proteins, p38, JNK and ERK were activated by flagellin challenge in an early phase, respectively in 15 minutes （P 〈0.01）, 30 minutes （P 〈0.01） and 15 minutes （P 〈0.01）. TRAF6 siRNA which decreased expression of TRAF6, altered the activation of JNK, p38, and ERK following flagellin treatment, but its influence on the expression of TGF-β1 and IL-8 has no statistical significance.Conclusions Flagellin from P. Aeruginosa PAO1 induces TGF-β1 expression in normal bronchial epithelial cells,BEAS-2B, through the MAPK signal cascade in vitro. It suggests that the fibrogenesis/remodeling process may be initiated from an early stage of acute lung infection due to P. Aeruginosa.

鞭毛蛋白诱导表达差异基因的分析

试验旨在分析鞭毛蛋白刺激机体基因表达谱的特征,揭示鞭毛蛋白佐剂的作用机制。从GEO数据库筛选目标微阵列GSE46421和GSE72016及其差异表达基因(DEGs),经DAVID分析DEGs参与的GO功能注释和KEGG信号通路、String构建DEGs的蛋白质-蛋白质相互作用(PPI)网络、Cytoscape可视化PPI网络并筛选高连通度的PPI子模块和hub基因。结果显示,共筛选出182个DEGs,包含上调基因161个、下调基因121个。DEGs参与GO生物过程主要有信号转导、免疫应答、炎症反应、细胞凋亡等,参与分子功能包含激酶激活、受体结合、细胞因子激活、膜信号转导。DEGs参与KEGG通路涵盖细胞因子-细胞因子受体相互作用、TNF信号通路、NF-κB信号通路、Toll样受体信号通路、NOD样受体信号通路等免疫相关途径。分析DEGs的PPI网络发现,2个重要的PPI子模块和10个关键基因(IL-10、IL-6、CCL2、CXCL2、NFKBIA、MyD88等)。研究表明,鞭毛蛋白通过识别TLR5和NOD样受体,触发MyD88依赖性和MyD88非依赖性途径发出信号,激活转录因子NF-κB,这些信号通过级联反应激活机体免疫系统、诱导机体产生多种细胞因子和趋化因子,发挥佐剂效应。