Translocation of DNA across the Agrobacterium Envelope

描述（由申请人提供）：大分子在细胞间的易位是生物医学关注的主要领域。在细菌中，大分子的细胞间转移通常由称为IV型分泌系统的表面细胞器协调。许多细菌物种使用IV型系统通过称为接合的过程在种、属或甚至界的边界内或跨种、属或甚至界的边界递送DNA底物。接合DNA转移有助于基因组在进化过程中的可塑性，以及抗生素抗性基因和其他毒力性状在更直接的时间尺度上的传播。许多医学上重要的细菌病原体也使用IV型系统在感染过程中将效应蛋白递送至真核靶细胞。根癌农杆菌VirB/VirD 4系统是深入研究IV型机器功能的重要模型。该系统由亚基VirB 1-VirB 11和VirD 4组装而成，通过依赖于细胞与细胞直接接触的机制将DNA和蛋白质易位到遗传多样性不同的细菌和真核靶细胞。本实验室工作的总体目标是以完整的分子术语描述VirB/VirD 4机器生物发生途径、底物识别和跨细胞包膜转移的基础以及VirB/VirD 4机器-靶细胞接触的性质。最近的研究确定了VirB 10的三个新特征，将我们的注意力集中在这个通道亚基上：i）它跨越整个革兰氏阴性细胞包膜，这是所有描述的细菌蛋白质中的一个新特性，ii）它形成一个？螺旋孔在外膜，只有第二个描述？螺旋外膜孔蛋白，和iii）其经历底物跨外膜易位所需的ATP介导的结构转变。我们假设，VirB 10形成一个结构支架在整个细胞包膜组装两个结构，VirB/VirD 4易位通道和细胞外T菌毛，后者是重要的建立靶细胞接触。我们建议：（A）定义的架构，装配动力学，和功能的？螺旋外膜孔与底物转移和菌毛生物发生的关系，（B）通过含VirB 10的机器翻译的生物化学和结构表征定义VirB 10如何有助于通道功能，和（C）阐明位于内膜的VirB 10跨膜结构域和VirB 4 ATP酶如何有助于菌毛生物发生。对IV型分泌物的研究至少有两个原因。首先，这些机器在大多数（如果不是所有）原核细胞中广泛使用，但它们的作用机制仍然知之甚少。第二，IV型分泌是抗生素耐药性扩散以及许多细菌病原体成功感染的主要贡献者;因此，这些系统是旨在抑制疾病进展的治疗的极好靶点。

DESCRIPTION (provided by applicant): Translocation of macromolecules between cells is a major area of biomedical interest. In bacteria, the intercellular transfer of macromolecules is commonly orchestrated by surface organelles termed type IV secretion systems. Many bacterial species use type IV systems to deliver DNA substrates within or across species, genus, or even kingdom boundaries through a process termed conjugation. Conjugative DNA transfer contributes to genome plasticity over evolutionary time, and the spread of antibiotic resistance genes and other virulence traits on a more immediate time scale. Many medically-important bacterial pathogens also use type IV systems to deliver effector proteins to eukaryotic target cells during infection processes. The VirB/VirD4 system of Agrobacterium tumefaciens serves as important model for detailed mechanistic studies of type IV machine function. This system, assembled from subunits VirB1 - VirB11 and VirD4, translocates DNA and proteins to phylogenetically-diverse bacterial and eukaryotic target cells by a mechanism dependent on direct cell-to-cell contact. The overall goal of work in this laboratory is to describe in complete molecular terms the VirB/VirD4 machine biogenesis pathway, the basis for substrate recognition and transfer across the cell envelope, and the nature of the VirB/VirD4 machine - target cell contact. Recent studies identified three novel features of VirB10 that focus our attention on this channel subunit: i) it spans the entire Gram-negative cell envelope, a novel property among all described bacterial proteins, ii) it forms an ?-helical pore at the outer membrane, only the second described ?-helical outer membrane pore protein, and iii) it undergoes an ATP-mediated structural transition required for substrate translocation across the outer membrane. We hypothesize that VirB10 forms a structural scaffold across the entire cell envelope for assembly of two structures, the VirB/VirD4 translocation channel and the extracellular T pilus, the latter being important for establishment of target cell contact. We propose to: (A) define the architecture, assembly dynamics, and function of the ?-helical outer membrane pore with respect to substrate transfer and pilus biogenesis, (B) define how VirB10 contributes to channel function through biochemical and structural characterization of VirB10-containing machine subassemblies, and (C) elucidate how the VirB10 transmembrane domain and the VirB4 ATPase located at the inner membrane contribute to pilus biogenesis. Studies of type IV secretion are essential for at least two reasons. First, these are widely used machines among most if not all prokaryotic cells and yet their mechanisms of action remain poorly understood. Second, type IV secretion is a major contributor to the proliferation of antibiotic resistance as well as successful infection by many bacterial pathogens; these systems are therefore excellent targets for therapies aimed at suppressing disease progression.