Molecular Mechanisms of Protein Sorting by the Type II Secretion System

霍乱是一种急性腹泻疾病，仍然是人类健康的全球负担。主要负责的关键因素 这种毁灭性的疾病是霍乱毒素，一种由霍乱弧菌产生和分泌的AB5毒素。它的 细胞外分泌依赖于II型分泌系统(T2SS)，该系统也负责 蛋白水解酶、脂肪酶、核酸酶和几丁质酶的外膜易位。许多革兰氏阴性菌都有共性 病原体，它独特地将这些因子从周质间隔室输送到细胞外 它们完全折叠的构象中的环境。尽管对T2SS的结构知识有了显著的增长 以及T2S底物被识别和识别的机制 外膜转位的分类仍有待确定。缺乏有意义的顺序和结构 这些蛋白质之间的相似性使识别共同的分泌信号变得复杂。其他令人困惑的问题 这些因素包括T2SS支持可溶性蛋白质的胞外运输和 脂蛋白和T2S底物在最终目的地上存在差异。一些T2S衬底 例如霍乱毒素一旦通过外膜被释放到细胞外空间； 然而，其他细菌仍然是表面相关的，或者可能在细胞外之后重新附着到细菌细胞表面。 放手。类胰蛋白酶VesB是主要保留在细胞上的T2S底物的一个例子 浮出水面。VesB属于一类独特的胞外酶，它的C末端延伸由以下组成 两个突出的甘氨酸和一个疏水螺旋，后面是带正电荷的残基(GlyGly-CTERM 域)。一种新发现的膜内菱形蛋白水解酶家族成员--菱形酶 在VesB通过细胞膜的运输过程中关闭GlyGly-CTERM结构域，并在翻译后 修饰后的VesB定位于细胞表面。 本提案中描述的实验旨在测试假设T2S系统，在 与菱形糖苷酶协同作用，介导含GlyGly-CTERM的成熟和表面定位 蛋白质。具体地说，这项提议将确定用来生产的蛋白质的表面锚定机制 GlyGly-CTERM延伸，破译GlyGly-CTERM蛋白是如何被 T2SS，并评价T2SS/菱形糖苷酶系统用于GlyGly-CTERM标记的表面定位 异源蛋白质。这些发现将有助于理解菱形糖苷酶的功能和特异性。 以及更广泛的医学上相关的菱形蛋白水解酶，并可能找到操纵 预防性使用的T2 S/菱形核糖核酸酶系统。

Cholera, an acute diarrheal disease, remains a global burden to human health. The key factor chiefly responsible for this devastating disease is cholera toxin, an AB5 toxin that is produced and secreted by Vibrio cholerae. Its extracellular secretion is dependent on the type II secretion system (T2SS), which is also responsible for the outer membrane translocation of proteases, lipases, nucleases and chitinases. Common to many Gram-negative pathogens, it uniquely transports these factors from the periplasmic compartment to the extracellular environment in their fully folded conformations. Despite a dramatic increase in structural knowledge of the T2SS and its individual components in recent years, the mechanism by which T2S substrates are recognized and sorted for outer membrane translocation remains to be determined. A lack of significant sequence and structural similarity between these proteins complicates the identification of a common secretion signal. Other confounding factors include the findings that the T2SS supports the extracellular transport of both soluble proteins and lipoproteins and that there are differences in the final destination among T2S substrates. Some T2S substrates such as cholera toxin are released to the extracellular space once transported through the outer membrane; however, others remain surface associated or may reattach to the bacterial cell surface following extracellular release. The trypsin-like protease VesB is an example of a T2S substrate that is primarily retained on the cell surface. VesB belongs to a unique class of extracellular enzymes that have a C-terminal extension consisting of two prominent glycines and a hydrophobic helix followed by positively charged residues (GlyGly-CTERM domain). Rhombosortase, a newly discovered member of the intramembrane rhomboid protease family, cleaves off the GlyGly-CTERM domain during transit of VesB through the cell envelope, and the posttranslationally modified VesB is localized to the cell surface. The experiments described in this proposal are designed to test the hypothesis that the T2S system, in collaboration with rhombosortase, mediates the maturation and surface localization of GlyGly-CTERM containing proteins. Specifically, this proposal will determine the mechanism of surface anchoring of proteins produced with GlyGly-CTERM extensions, decipher how GlyGly-CTERM proteins are differentially recognized and secreted by the T2SS, and assess the T2SS/rhombosortase system for surface localization of GlyGly-CTERM-tagged heterologous proteins. The findings will facilitate understanding of the function and specificity of rhombosortase as well as the broader class of medically relevant rhomboid proteases and may identify ways to manipulate the T2S/rhombosortase system for preventative use.