Protein Export and Surface Anchoring in Gram+ Bacteria

描述(由申请人提供)：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因子的细菌(即炭疽芽孢杆菌，A类和单核细胞增生性李斯特菌，B类)。其中许多生物表面的蛋白质缺乏典型的SEC依赖的N末端分泌信号，因此使用未知的机制通过细胞膜(Cm)输出。其中一些蛋白质显然与发病有关，因为它们对小鼠的被动免疫是有效的。这项工作的目标1和2旨在鉴定和表征这些生物体中新的SEC非依赖性蛋白质分泌系统。在革兰氏阳性生物中，一旦通过cm分泌，蛋白质要么释放到细胞外环境中，要么固定在细胞表面。在革兰氏阳性细菌中，蛋白质锚定到细胞壁(CW)的一般机制需要一种名为Sortase的转肽酶，它识别并切割C末端疏水区和带电尾巴之前的一个短氨基酸基序。我们最近鉴定了A组链球菌(GAS)中的两种分类酶，它们锚定了具有LPXTG基序的不同蛋白质亚集。在其他GAS菌株基因组中的类似位置，我们已经确定了编码与索酸酶同源的额外蛋白质的基因。在目标3中，我们将表征这些建议的新分类酶在气体中的功能，并测试它们锚定预测底物蛋白的能力，我们在附近发现了编码的底物蛋白。我们还将鉴定一个预测的具有同源性的蛋白质，以信号肽酶I(编码在同一区域)，我们认为这些蛋白质的分泌将需要它。这项工作对革兰氏阳性细菌的分泌物和细胞壁锚定的更多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.