Genetic requirements for lipoteichoic acid synthesis in Staphylococcus aureus

描述(由申请人提供)：拟议的研究旨在加深对脂磷壁酸(LTA)合成的遗传水平的了解，LTA是许多革兰氏阳性细菌细胞壁包膜中发现的一种重要的壁聚合物，包括人类病原体金黄色葡萄球菌。LTA的功能是清除细胞壁相关酶正常运作所需的镁离子，以及调节细胞生长和分离所需的自溶素。最近，LTA被认为是革兰氏阳性细菌的免疫刺激分子。尽管对LTA的生化结构有详细的了解，但只有几个基因产物被证明是合成LTA所必需的，这些基因产物加在一起不能完全解释聚甘油磷酸聚合物的合成。目前还没有关于革兰氏阳性菌LTA生物合成完全丧失的突变的描述，推测LTA是细胞壁的重要组成部分。这些实验的目的是识别和鉴定LTA生物合成所需的其他金黄色葡萄球菌基因。我们已经鉴定了两个金黄色葡萄球菌突变体，它们在先前未描述的基因中插入了转座子，通过蛋白质印迹分析判断，它们产生了结构改变的LTA。本文描述了一种使用已建立的生化分析来表征这些突变体的实验设计。此外，我们还建议构建一个在大肠杆菌中表达金黄色葡萄球菌基因的质粒库，并筛选出能够使异源宿主产生聚甘油磷酸聚合物的质粒克隆。LTA由许多革兰氏阳性病原体产生，包括A组和B组链球菌、粪肠球菌和炭疽杆菌。通过研究金黄色葡萄球菌中这种保守的、可能是必不可少的表面分子的生物合成途径，我们将进一步了解细菌包膜的一个重要成分的合成。 综上所述，金黄色葡萄球菌可引起多种人类疾病。细菌表面含有许多重要的分子，这些分子使细菌能够黏附和入侵宿主细胞并导致疾病。这项研究的目的是了解特定的表面分子是如何产生的，目的是抑制其合成，防止定居和疾病。

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.