Translocation of DNA across the Agrobacterium Envelope

描述（由申请人提供）：细胞间大分子的易位是生物医学的一个主要领域。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。许多细菌使用IV型系统通过称为共轭的过程在种、属甚至界边界内或跨种递送DNA底物。结合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在感染过程中也使用IV型系统将效应蛋白传递给真核靶细胞。农杆菌的VirB/VirD4系统是IV型机器功能详细机理研究的重要模型。该系统由VirB1 - VirB11和VirD4亚基组成，通过依赖于细胞间直接接触的机制，将DNA和蛋白质易位到系统发育多样化的细菌和真核靶细胞。本实验室工作的总体目标是用完整的分子术语描述VirB/VirD4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/VirD4机器的性质-靶细胞接触。最近的研究发现了VirB10的三个新特征，使我们关注这个通道亚基：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的一个新特性；ii)它形成一个？-螺旋孔外膜，只有第二个描述？-螺旋外膜孔蛋白，iii)它经历atp介导的结构转变，这是底物跨外膜易位所必需的。我们假设VirB10在整个细胞包膜上形成一个结构支架，用于两种结构的组装，VirB/VirD4易位通道和细胞外T菌，后者对于建立靶细胞接触很重要。我们建议：(A)定义？的体系结构、装配动力学和功能。(B)通过对含有VirB10的机器组件的生化和结构表征，确定VirB10如何促进通道功能，(C)阐明VirB10跨膜结构域和位于内膜的VirB4 atp酶如何促进菌毛的生物发生。研究IV型分泌是必要的，至少有两个原因。首先，这些机器在大多数（如果不是全部的话）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型分泌物是抗生素耐药性扩散的主要因素，也是许多细菌病原体成功感染的主要因素；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.