The surface-exposed carboxyl region of Mycoplasma pneumoniae elongation factor Tu interacts with fibronectin
The surface-exposed carboxyl region of Mycoplasma pneumoniae elongation factor Tu interacts with fibronectin
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DOI：
10.1128/iai.00173-08
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发表时间：
2008-07-01
影响因子：
3.1
通讯作者：
Baseman, Joel B.
中科院分区：
文献类型：
--
作者：
Balasubramanian, Sowmya;Kannan, T. R.;Baseman, Joel B.
肺炎支原体是人类呼吸道的一种细菌病原体，可引起广泛的气道疾病以及肺外症状。它具有独特的、分化的末端结构，称为附着细胞器，介导对宿主呼吸上皮的粘附。以前，我们报道了表面相关的M。肺炎支原体延伸因子To（EF-Tu，也称为MPN 665）作为纤连蛋白（Fn）结合蛋白，促进支原体和细胞外基质之间的相互作用。在本研究中，我们确定了M.肺炎杆菌EF-Tu转化为Fn主要由EF-Tu羧基区介导。跨越羧基末端的179个氨基酸的区域（命名为EC;氨基酸192至394）以剂量依赖性方式结合Fn。对羧基构建体（ED 3和ED 4）及其缺失截短体（ED 3.1、ED 3.2和ED 4.1）的进一步分析显示，羧基区域具有两个不同的位点，具有不同的Fn结合效率。使用针对重组ED 3和ED 4提出的抗体的免疫金电子显微镜证明了EF-Tu羧基区域的表面可及性。使用完整的放射性标记的支原体和纯化的重组ED 3和ED 4蛋白的竞争性结合试验，沿着抗体阻断试验，加强了Fn结合中表面暴露的EF-Tu羧基区域的作用。支原体膜和Triton X-114分配的支原体组分的碱和高盐处理证实了支原体膜内EF-Tu的稳定缔合。这些观察结果突出了独特的，多方面的，和不可预测的作用，经典定义的细胞质蛋白EF-Tu相对于细胞功能，区室化，和地形。
Mycoplasma pneumoniae is a bacterial pathogen of the human respiratory tract that causes a wide range of airway diseases as well as extrapulmonary symptoms. it possesses a distinct, differentiated terminal structure, termed the attachment organelle, that mediates adherence to the host respiratory epithelium. Previously, we reported that surface-associated M. pneumoniae elongation factor To (EF-Tu, also called MPN665) serves as a fibronectin (Fn)-binding protein, facilitating interactions between mycoplasmas and extracellular matrix. In the present study, we determined that binding of M. pneumoniae EF-Tu to Fn is primarily mediated by the EF-Tu carboxyl region. A 179-amino-acid region spanning the carboxyl terminus (designated EC; amino acids 192 to 394) binds Fn in a dose-dependent manner. Further analysis of carboxyl constructs (ED3 and ED4) and their deletion truncations (ED3.1, ED3.2, and ED4.1) revealed that the carboxyl region possessed two distinct sites with different Fn-binding efficiencies. Immunogold electron microscopy using antibodies raised against recombinant ED3 and ED4 demonstrated the surface accessibility of the EF-Tu carboxyl region. Competitive binding assays using intact radiolabeled mycoplasmas and purified recombinant ED3 and ED4 proteins, along with antibody blocking assays, reinforced the role of the surface-exposed EF-Tu carboxyl region in Fn binding. Alkali and high-salt treatment of mycoplasma membranes and Triton X-114-partitioned mycoplasma fractions confirmed the stable association of EF-Tu within the mycoplasma membrane. These observations highlight the unique, multifaceted, and unpredictable role of the classically defined cytoplasmic protein EF-Tu relative to cellular function, compartmentalization, and topography.