Protein Export and Surface Anchoring in Gram+ Bacteria

描述（由申请人提供）：在低G+C革兰氏菌中有许多重要的人类病原体，包括一些被列为潜在生物恐怖制剂的细菌（如A类炭疽杆菌和B类单核增生李斯特菌）。许多这类生物的表面蛋白质缺乏典型的依赖于sec的n端分泌信号，因此使用未知的机制通过细胞膜（cm）输出。其中一些蛋白对小鼠的被动免疫有效，显然参与了发病机制。本工作的目的1和2是针对这些生物中新的不依赖于sec的蛋白质分泌系统的鉴定和表征。在革兰氏阳性生物体中，一旦通过cm分泌，蛋白质要么被释放到细胞外环境中，要么被固定在细胞表面。在革兰氏菌中，将蛋白质锚定到细胞壁（cw）的一般机制需要一种转肽酶，称为分选酶，它识别并切割c端疏水区和带电尾部之前的短氨基酸基序。我们最近在A组链球菌（GAS）中鉴定了两种排序酶，它们用LPXTG基序锚定不同的蛋白质亚群。在其他GAS菌株基因组的类似位置，我们已经确定了编码与分选酶同源的其他蛋白质的基因。在Aim 3中，我们将描述这些被提出的新分选酶在GAS中的功能，并测试它们锚定预测底物蛋白的能力，我们发现这些底物蛋白在附近编码。我们还将描述一个与信号肽酶I（编码在同一区域）同源的预测蛋白，我们认为这些蛋白的分泌将需要该蛋白。这项工作对革兰氏+细菌分泌和细胞壁锚定的更深入了解应该为广谱抗菌治疗和潜在的新疫苗载体提供新的靶点。此外，它应该为大规模生产蛋白质提供商业上有用的新方法。

DESCRIPTION (provided by applicant): Among the low G+C Gram+ bacteria are many important human pathogens, including some classified as potential bioterrorism agents (i.e. Bacillus anthracis, category A and Listeria monocytogenes, category B). Many of these organisms have proteins on their surface that lack typical Sec-dependent N-terminal secretion signals and thus use unknown mechanisms for export through the cell membrane (cm). Some of these proteins are apparently involved in pathogenesis since they are effective for passive immunization of mice. Aims 1 and 2 of this work are directed at identification and characterization of new Sec-independent protein secretion systems in these organisms. Once secreted through the cm in Gram+ organisms, proteins are either released into the extracellular milieu or anchored to the cell surface. The general mechanism for anchoring proteins to the cell wall (cw) in Gram+ bacteria requires a transpeptidase, called sortase, that recognizes and cleaves a short amino acid motif preceding a C-terminal hydrophobic region and charged tail. We recently characterized two sortases in the group A streptococcus (GAS) that anchor distinct subsets of proteins with the LPXTG motif. At a similar location in the genome of other GAS strains, we have identified genes encoding additional proteins with homology to sortase. In Aim 3 we will characterize the function in the GAS of these proposed new sortases and test their ability to anchor their predicted substrate proteins, which we find encoded nearby. We will also characterize a predicted protein with homology to signal peptidase I (encoded in the same region) that we propose will be needed for secretion of these proteins. The greater understanding of secretion and cell wall anchoring in Gram+ bacteria that results from this work should provide new targets for broad spectrum antibacterial therapy and potential new vaccine vectors. In addition, it should provide commercially useful new methods for large-scale production of proteins.