Studies on Macrophage Resistance to Anthrax Lethal Toxin

描述(申请人提供)：致命毒素是炭疽芽孢杆菌的主要毒力因素。致死毒素由保护性抗原(PA)和致死因子(LF)两种蛋白质组成。巨噬细胞是抵御炭疽感染的第一道防线。小鼠品系和从这些品系衍生的原代巨噬细胞或巨噬细胞系对毒素介导的致死效应的敏感度存在显著差异。某些小鼠品系及其巨噬细胞对LT敏感，而其他小鼠品系及其巨噬细胞对LT高度耐药。耐药的潜在机制仍不清楚，由于这一信息对治疗学的发展具有潜在的用处，因此是炭疽领域的一个非常感兴趣的主题。本项目的主要目的是研究巨噬细胞对LT的耐药机制。针对这一目标，提出了三个具体目标：(1)使用免疫荧光/共聚焦显微镜来研究细胞转运和定位的变化，以检查LF在敏感和耐药巨噬细胞中的定位；(2)研究潜在的LF细胞内底物在耐药巨噬细胞中的作用；以及(3)差异芯片分析以评估LT敏感和耐药巨噬细胞中的基因表达，重点是评估细胞因子的表达，MAPK激酶效应分子的表达，以及最近被描述的与LT耐药相关的小鼠基因产物Kif1C的表达。这些研究的成功完成将对巨噬细胞抵抗LT的机制有一个基本的了解。

DESCRIPTION (provided by applicant): Lethal toxin is a major virulence factor of Bacillus anthracis. Lethal toxin (LT) is comprised of two proteins, protective antigen (PA) and lethal factor (LF). Macrophages serve as the first line of defense against anthrax infection. There is a striking difference in susceptibility of mouse strains and the primary macrophages or macrophage cell lines derived from those strains to lethal, toxin-mediated effects. Certain mouse strains and their macrophages are sensitive to LT whereas other mouse strains and their macrophages are highly resistant to LT. The underlying mechanism of resistance remains unknown and is a subject of great interest in the anthrax field due to the potential usefulness of this information to development of therapeutics. The primary goal of this project is to investigate mechanisms of resistance to LT in macrophages. To address this goal, three specific aims are proposed: (1) Investigation of alterations in cellular trafficking and localization using immunofluorescence/confocal microscopy to examine localization of LF in sensitive versus resistant macrophages, (2) Investigation of the role of potential LF intracellular substrates in resistant macrophages, and (3) Analysis of differential microarray analyses to evaluate gene expression in LT-sensitive versus resistant macrophages with a focus on evaluating cytokine expression, MAPK kinase effector molecule expression, and expression of Kif1C, a recently described murine gene product associated with resistance to LT. Successful completion of these studies will yield a fundamental understanding of mechanisms of macrophage resistance to LT.