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胆酸（LTA）合成的遗传水平，脂磷胆酸是一种重要的壁聚合物，存在于许多革兰氏阳性细菌的细胞壁包膜中，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功能所需的Mg+2离子和调节细胞生长和分裂所需的自溶素。最近，LTA被认为是革兰氏阳性细菌病原体的免疫刺激分子。尽管对LTA的生化结构有详细的了解，但只有少数基因产物被证明是LTA合成所必需的，这些基因产物不能完全解释聚甘油磷酸聚合物的合成。在革兰氏阳性菌中没有描述完全取消LTA生物合成的突变，并且推测LTA是细胞壁的重要组成部分。该实验旨在鉴定和表征LTA生物合成所需的其他金黄色葡萄球菌基因。我们已经鉴定出两种金黄色葡萄球菌突变体，在以前未表征的基因中插入转座子，通过western-blot分析判断，这些突变体产生了结构改变的LTA。本文描述了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，我们建议构建一个质粒文库，用于在大肠杆菌中表达金黄色葡萄球菌基因，并筛选质粒克隆，使异源宿主能够产生聚甘油磷酸聚合物。LTA由许多革兰氏阳性病原体产生，包括A族和B族链球菌、粪肠球菌和炭疽杆菌。通过研究这种保守的、可能是金黄色葡萄球菌必需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重要元素的合成。

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.