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首页> 外文期刊>Infection and immunity >Mutagenesis and Functional Analysis of the Bacterial Arginine Glycosyltransferase Effector NleB1 from Enteropathogenic Escherichia coli
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Mutagenesis and Functional Analysis of the Bacterial Arginine Glycosyltransferase Effector NleB1 from Enteropathogenic Escherichia coli

机译：肠致病性大肠杆菌细菌精氨酸糖基转移酶效应子NleB1的诱变和功能分析

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Enteropathogenic Escherichia coli (EPEC) interferes with host cell signaling by injecting virulence effector proteins into enterocytes via a type III secretion system (T3SS). NleB1 is a novel T3SS glycosyltransferase effector from EPEC that transfers a single N-acetylglucosamine (GlcNAc) moiety in an N-glycosidic linkage to Arg117 of the Fas-associated death domain protein (FADD). GlcNAcylation of FADD prevents the assembly of the canonical death-inducing signaling complex and inhibits Fas ligand (FasL)-induced cell death. Apart from the DXD catalytic motif of NleB1, little is known about other functional sites in the enzyme. In the present study, members of a library of 22 random transposon-based, in-frame, linker insertion mutants of NleB1 were tested for their ability to block caspase-8 activation in response to FasL during EPEC infection. Immunoblot analysis of caspase-8 cleavage showed that 17 mutant derivatives of NleB1, including the catalytic DXD mutant, did not inhibit caspase-8 activation. Regions of interest around the insertion sites with multiple or single amino acid substitutions were examined further. Coimmunoprecipitation studies of 34 site-directed mutants showed that the NleB1 derivatives with the E253A, Y219A, and PILN(63–66)AAAA (in which the PILN motif from residues 63 to 66 was changed to AAAA) mutations bound to but did not GlcNAcylate FADD. A further mutant derivative, the PDG(236–238)AAA mutant, did not bind to or GlcNAcylate FADD. Infection of mice with the EPEC-like mouse pathogen Citrobacter rodentium expressing NleBE253A and NleBY219A showed that these strains were attenuated, indicating the importance of residues E253 and Y219 in NleB1 virulence in vivo. In summary, we identified new amino acid residues critical for NleB1 activity and confirmed that these are required for the virulence function of NleB1.
机译：肠致病性大肠杆菌（EPEC）通过通过III型分泌系统（T3SS）将毒力效应蛋白注入肠细胞来干扰宿主细胞信号传导。 NleB1是EPEC的新型T3SS糖基转移酶效应子，可将 N -糖苷键中的单个 N -乙酰葡糖胺（GlcNAc）部分转移至Arg 117 Fas相关死亡域蛋白（FADD）的表达。 FADD的GlcNAcylation可阻止规范性死亡诱导信号复合物的组装并抑制Fas配体（FasL）诱导的细胞死亡。除了NleB1的DXD催化基序外，对该酶的其他功能位点知之甚少。在本研究中，测试了22个基于随机转座子，符合读框的NleB1接头插入突变体的文库成员在EPEC感染过程中阻断caspase-8激活以响应FasL的能力。 caspase-8裂解的免疫印迹分析表明，NleB1的17个突变体衍生物（包括催化DXD突变体）不抑制caspase-8活化。进一步检查了具有多个或单个氨基酸取代的插入位点周围的目标区域。对34个定点突变体的免疫共沉淀研究表明，NleB1衍生物具有E253A，Y219A和PILN （63-66）AAAA （其中从残基63到66的PILN基序已更改为AAAA）突变与GlcNAcylate FADD结合但不结合。另一个突变体衍生物，PDG （236–238）AAA 突变体，不与GlcNAcylate FADD结合或结合。表达NleB E253A 和NleB Y219A 的EPEC样小鼠病原体柠檬酸杆菌对小鼠的感染表明这些菌株已减毒，表明E253和Y219残基对NleB1毒力的重要性体内。总之，我们确定了对NleB1活性至关重要的新氨基酸残基，并确认了这些对于NleB1的毒力功能是必需的。

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