Protein Export and Surface Anchoring in Gram+ Bacteria

描述（由申请方提供）：在低G+C革兰氏+细菌中，有许多重要的人类病原体，包括一些被归类为潜在生物恐怖主义制剂的病原体（即炭疽芽孢杆菌，A类和单核细胞增生李斯特菌，B类）。这些生物体中的许多在其表面上具有缺乏典型的Sec依赖性N-末端分泌信号的蛋白质，因此使用未知的机制通过细胞膜（cm）输出。这些蛋白质中的一些显然参与发病机制，因为它们对小鼠的被动免疫是有效的。这项工作的目的1和2是针对在这些生物体中的新的Sec-独立的蛋白质分泌系统的鉴定和表征。一旦在革兰氏阳性生物体中通过cm分泌，蛋白质被释放到细胞外环境中或锚定到细胞表面。在革兰氏阳性细菌中将蛋白锚定到细胞壁（cw）的一般机制需要称为分选酶的转肽酶，其识别并切割C-末端疏水区和带电尾部之前的短氨基酸基序。我们最近的特点是两个分选酶在A组链球菌（GAS），锚不同的蛋白质与LPXTG基序的子集。在其他GAS菌株基因组中的类似位置，我们已经鉴定了编码与分选酶具有同源性的其他蛋白质的基因。在目标3中，我们将描述这些提出的新分选酶在GAS中的功能，并测试它们锚它们预测的底物蛋白的能力，我们发现它们在附近编码。我们还将表征与信号肽酶I（在相同区域编码）同源的预测蛋白，我们提出这些蛋白的分泌将需要该蛋白。从这项工作中产生的革兰氏阳性细菌的分泌和细胞壁锚定的更好的理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.