Molecular Mechanisms of Protein Sorting by the Type II Secretion System

霍乱是一种急性肠道疾病，仍然是人类健康的全球负担。主要负责的关键因素 霍乱毒素是霍乱弧菌产生和分泌的一种AB 5毒素。其 细胞外分泌依赖于II型分泌系统（T2 SS），其也负责细胞外分泌。 蛋白酶、脂肪酶、核酸酶和几丁质酶的外膜易位。常见于许多革兰氏阴性 在病原体中，它独特地将这些因子从周质区室转运到细胞外， 环境在其完全折叠的构象。尽管对T2 SS的结构知识有了显著的增加， 及其各个组成部分，T2 S底物被识别的机制， 外膜移位的分类仍有待确定。缺乏重要的顺序和结构 这些蛋白质之间的相似性使共同分泌信号的鉴定复杂化。其他混杂 这些因素包括发现T2 SS支持可溶性蛋白质的细胞外转运， T2 S底物之间的最终目的地存在差异。一些T2 S底物 如霍乱毒素一旦通过外膜转运就释放到细胞外空间; 然而，其他的则保持表面结合，或者在细胞外粘附后可以重新附着到细菌细胞表面。 release.胰蛋白酶样蛋白酶VesB是主要保留在细胞上的T2 S底物的实例 面VesB属于一类独特的胞外酶，其具有由以下组成的C-末端延伸： 两个突出的甘氨酸和一个疏水螺旋，随后是带正电荷的残基（GlyGly-CTERM 域）。菱形分选酶是膜内菱形蛋白酶家族的新成员， 在VesB通过细胞被膜的转运过程中， 修饰的VesB定位于细胞表面。 本提案中描述的实验旨在测试T2 S系统的假设， 与菱形分选酶合作，介导含有GlyGly-CTERM的成熟和表面定位 proteins.具体地说，这项建议将确定产生的蛋白质的表面锚定机制， GlyGly-CTERM延伸，破译如何GlyGly-CTERM蛋白质的差异识别和分泌的 的T2 SS，并评估T2 SS/菱蛋白分选酶系统的表面定位的甘氨酸-CTERM标记的 异源蛋白这一发现将有助于理解菱蛋白分选酶的功能和特异性 以及更广泛的医学相关的菱形蛋白酶，并可以确定操纵 T2 S/菱形分选酶系统用于预防性使用。

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.