土地类芽胞杆菌(Paenibacillus terrae)NK3-4 EsxA结构与系统发育分析

前期研究在植物根际促生菌土地类芽胞杆菌(Paenibacillus terrae )NK3-4中发现一个EsxA编码基因,为明确该基因编码的蛋白的性质、结构及系统发生关系,对该基因进行了生物信息学分析。分析表明,该EsxA含有91个氨基酸,分子质量10 276.53 Da,理论pI 5.29,分子式为C₄₄₅H₇₁₁N₁₂₅O₁₄₆S₄,弱酸性,亲水,具有WEG保守基序,属于WXG超级家族成员;建模预测表明,自然状态下EsxA 形成不对称的同源二聚体,其中每个亚基都由一个β折叠连接两个α螺旋组成,两个α螺旋反向平行排列;二聚体中两个亚基的肽链呈反向排列,所有N末端和C末端均暴露在外,形成棒状表面形态,其中一个亚基的N端的不规则卷曲形成与棒状二聚体垂直的短柱形凸起;系统发育分析显示,EsxA在种内及种间进化关系虽是不保守的,但类芽胞杆菌源的EsxA与病原细菌的EsxA同源性较低,暗示类芽胞杆菌源EsxA可能与病原微生物的EsxA具有截然不同的功能。结果为包括NK3-4菌株在内的类芽胞杆菌属EsxA外源分泌表达条件,活性保持及功能的深入研究提供了理论依据。     

牛乳源金黄色葡萄球菌EsxA蛋白的表达及免疫原性分析

为分析牛乳源金黄色葡萄球菌(Staphylococcus aureus)EsxA蛋白的免疫原性,构建EsxA-p ET-28a重组表达质粒,重组质粒经诱导表达后进行SDS-PAGE和Western blotting鉴定。用纯化后重组EsxA蛋白免疫小鼠,用间接ELISA检测免疫小鼠血清中的IgG、IgG1和IgG2a水平;免疫小鼠经S.aureus菌株攻击后,检测小鼠肝、脾、肾组织荷菌数和免疫保护率,观察S.aureus菌株攻击后小鼠肝、脾、肾病理组织学变化。结果表明,成功诱导表达了EsxA重组蛋白,该重组蛋白免疫小鼠后血清抗体效价可达1∶900,与对照相比,重组蛋白免疫后可减少小鼠肝、脾、肾组织的荷菌数,减轻这些脏器的病理损伤,对免疫小鼠保护率达75%。上述结果表明,该重组Esx A蛋白具有良好的免疫原性。     

Characterization of Mycobacterium tuberculosis EsxA Membrane Insertion: ROLES OF N- AND C-TERMINAL FLEXIBLE ARMS AND CENTRAL HELIX-TURN-HELIX MOTIF*

EsxA (ESAT-6), an important virulence factor of Mycobacterium tuberculosis, plays an essential role in phagosome rupture and bacterial cytosolic translocation within host macrophages. Our previous study showed that EsxA exhibits a unique membrane-interacting activity that is not found in its ortholog from nonpathogenic Mycobacterium smegmatis. However, the molecular mechanism of EsxA membrane insertion remains unknown. In this study, we generated truncated EsxA proteins with deletions of the N- and/or C-terminal flexible arm. Using a fluorescence-based liposome leakage assay, we found that both the N- and C-terminal arms were required for membrane disruption. Moreover, we found that, upon acidification, EsxA converted into a more organized structure with increased α-helical content, which was evidenced by CD analysis and intrinsic tryptophan fluorescence. Finally, using an environmentally sensitive fluorescent dye, we obtained direct evidence that the central helix-turn-helix motif of EsxA inserted into the membranes and formed a membrane-spanning pore. A model of EsxA membrane insertion is proposed and discussed.