Translocation of DNA across the Agrobacterium Envelope

描述(由申请人提供)：大分子在细胞之间的转移是生物医学感兴趣的一个主要领域。在细菌中，大分子的细胞间转移通常由被称为IV型分泌系统的表面细胞器来协调。许多细菌物种使用IV型系统通过一种称为接合的过程在物种、属甚至王国边界内或跨物种、属甚至王国边界输送DNA底物。结合DNA转移有助于基因组在进化时间内的可塑性，以及抗生素耐药性基因和其他毒力特征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运送到真核靶细胞。根癌农杆菌的VIRB/VirD4系统是详细研究IV型机械功能的重要模型。该系统由VirB1-VirB11和VirD4亚基组装而成，通过一种依赖于细胞间直接接触的机制将DNA和蛋白质转移到系统发育不同的细菌和真核细胞中。本实验室的总体工作目标是用完整的分子术语描述VIRB/VirD4机器生物发生途径、底物识别和跨细胞膜转移的基础以及VIRB/VirD4机器-靶细胞接触的性质。最近的研究发现了VirB10的三个新特征，使我们的注意力集中在这个通道亚基上：i)它横跨整个革兰氏阴性细胞膜，这是所有已描述的细菌蛋白中的一个新特性；ii)它在外膜形成一个β-螺旋孔，仅有第二个所描述的-螺旋外膜孔蛋白；以及iii)它经历了底物跨外膜转运所需的结构转变。我们假设VirB10在整个细胞被膜上形成了一个结构支架，用于组装两个结构，即VIRB/VirD4易位通道和细胞外T菌毛，后者对于建立靶细胞接触是重要的。我们建议：(A)定义与底物转移和菌毛生物发生有关的β-螺旋外膜孔的结构、组装动力学和功能，(B)通过含有VirB10的机器组件的生化和结构特征确定VirB10如何对通道功能起作用，以及(C)阐明位于内膜的VirB10跨膜区和VirB4 ATPase如何对菌毛生物发生起作用。对IV型分泌物的研究是必要的，至少有两个原因。首先，这些机器在大多数(如果不是全部)原核细胞中广泛使用，但它们的作用机制仍然知之甚少。其次，IV型分泌物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佳靶点。

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.