Genetic requirements for lipoteichoic acid synthesis in Staphylococcus aureus

描述（由申请人提供）：拟议的研究旨在进一步了解脂磷壁酸（LTA）合成的遗传水平，LTA是一种在许多革兰氏阳性细菌（包括人类病原体金黄色葡萄球菌）细胞壁包膜内发现的重要壁聚合物。LTA的功能是清除细胞壁相关酶正常功能所需的Mg+2离子，以及调节细胞生长和分隔所需的自溶素。最近，LTA被认为是革兰氏阳性细菌病原体的免疫刺激分子。尽管LTA的生化结构的详细知识，只有少数基因产物已被证明是LTA合成所需的，这不能完全解释聚甘油磷酸聚合物的合成。革兰氏阳性细菌中没有描述完全消除LTA生物合成的突变，并且推测LTA是细胞壁的必需组分。提出的实验旨在识别和表征额外的S。金黄色葡萄球菌LTA生物合成所需的基因。我们发现了两个S。金黄色葡萄球菌突变体与转座子插入以前未知的基因，产生LTA的改变结构的判断，通过蛋白质印迹分析。本文描述了使用已建立的生物化学测定来表征这些突变体的实验设计。此外，我们还构建了一个表达S.金黄色葡萄球菌基因。大肠杆菌，并筛选质粒克隆，其赋予异源宿主产生聚甘油磷酸聚合物的能力。LTA由许多革兰氏阳性病原体产生，包括A组和B组链球菌、粪肠球菌和B组。炭疽病通过研究这种保守的、可能是必需的表面分子在S.金黄色葡萄球菌，我们将进一步了解细菌包膜的一个重要元素的合成。 总之，细菌金黄色葡萄球菌引起广泛的人类疾病。细菌表面含有许多重要的分子，使细菌能够粘附和侵入宿主细胞并引起疾病。这项研究的目的是了解一种特定的表面分子是如何产生的，目的是抑制其合成，防止定植和疾病。

DESCRIPTION (provided by applicant): The proposed research aims to further the understanding on a genetic level of the synthesis of lipoteichoic acid (LTA), an important wall polymer found within the cell wall envelope of many Gram-positive bacteria, including the human pathogen Staphylococcus aureus. Functions of LTA are scavenging of Mg+2 ions required for the proper function of cell wall-associated enzymes and regulation of autolysins required for cell growth and septation. More recently, LTA has been recognized as immunostimulatory molecule of Gram-positive bacterial pathogens. Despite a detailed knowledge of the biochemical structure of LTA, only a few gene products have been shown to be required for LTA synthesis, which together cannot account fully for the synthesis of the polyglycerolphosphate polymer. No mutations that completely abrogate LTA biosynthesis have been described for Gram-positive bacteria and it has been speculated that LTA is an essential component of the cell wall. The experiments proposed aim to identify and characterize additional S. aureus genes required for LTA biosynthesis. We have identified two S. aureus mutants with transposon insertions in previously uncharacterized genes that produced LTA of altered structure as judged by western-blot analysis. An experimental design is described herein to characterize these mutants using established biochemical assays. In addition, we propose to construct a plasmid library for expression of S. aureus genes in E. coli and to screen for plasmid clones, which confer to the heterologous host the ability to produce polyglycerolphosphate polymers. LTA is produced by many Gram-positive pathogens, including Group A and B streptococci, Enterococcus faecalis, and B. anthracis. By studying the biosynthesis pathway of this conserved, possibly essential surface molecule in S. aureus, we will further our understanding of the synthesis of an important element of the bacterial envelope. In summary, the bacterium Staphylococcus aureus causes a wide range of human diseases. The bacterial surface harbors many important molecules that allow the bacterium to adhere and invade host cells and cause disease. The goal of this research is to understanding how a particular surface molecule is produced with the goal to inhibit its synthesis, prevent colonization and disease.