EARLY ESTABLISHMENT STAGES OF ANTHRAX INFECTION

该提案调查炭疽感染的最初几个小时；在体内萌发的炭疽芽孢，巨噬细胞的生存，生长和逃逸的营养杆菌。在宿主外，内生孢子保持代谢休眠，即使暴露在恶劣的环境条件下也能保持毒力。内孢子是炭疽传染病，进入体内后被局部巨噬细胞吞噬。内生孢子“感知”新的环境，发芽并长成植物状态。我们的初步数据定义了在这些步骤中被阻断的离散突变体。逃离后，大量细菌感染，毒血症和死亡随之而来。我们的数据还表明，炭疽内生孢子具有独特的体内感觉和信号机制来触发萌发。在培养的巨噬细胞中萌发迅速。非致病性芽孢杆菌内生孢子在巨噬细胞中萌发未增加。炭疽杆菌转座子介导的诱变系统允许选择单个孢子内突变体，这些突变体不能在巨噬细胞中萌发，但完全能够在细菌培养基中萌发和生长。几种独特的突变体被鉴定出来。其中一个这样的基因座被命名为gerP（萌发质粒），位于毒力（毒素）质粒pXO1上。因此，gerP的突变消除了寄主特有的萌发，而不是一般的萌发反应。本研究的目的是：a.定义和表征炭疽芽孢杆菌的萌发基因和寄主化学信号，以确定它们在寄主特异性萌发反应中的作用；B.确定确定的细胞内事件和营养杆菌使用的细菌基因，使其能够从巨噬细胞中存活和逃脱；c.了解在小鼠模型中，炭疽芽孢杆菌宿主特异性萌发系统和早期细胞内事件在发病机制方面的相关性。了解炭疽的这些关键“建立”阶段可能为接触炭疽内生孢子后的早期干预提供目标。了解这种从内孢子的绝对代谢休眠到生长的毒性杆菌的快速而戏剧性的转变，可以使炭疽成为研究细菌感染周期早期阶段的有效模型。

This proposal investigates the first hours of anthrax infections; in vivo germination of the Bacillus anthracis endospore, macrophage survival, growth and escape of the vegetative bacilli. Outside the host, endospores remain metabolically-dormant, preserving virulence even when exposed to harsh environmental conditions. Endospores are the anthrax contagion, entering the body where they are phagocytosed by regional macrophages. Endospores "sense" the new locale, germinate and outgrow to a vegetative state. Our preliminary data defined discrete mutants blocked at each of these steps. After escape, massive bactermia, toxemia and death ensues. Our data also indicate that anthrax endospores have unique in vivo sensory and signaling mechanisms for triggering germination. Germination occurs rapidly in cultured macrophages. Non-pathogenic Bacillus sp. endospores show no increased germination in macrophages. A B. anthracis transposon-mediated mutagenesis system allowed selection of individual endospore mutants incapable of germination in macrophages but fully capable of germination and outgrowth in bacterial media. Several unique classes of mutants were characterized. One such loci, named gerP (germination Plasmid), is located on the virulence (toxin) plasmid pXO1. Thus mutations in gerP eliminate host-specific germination but not general germination responses. The aims of this proposal are to: a. define and characterize the germination genes of B. anthracis and host chemical signals to determine their roles in the host- specific germination response; b. determine defined intracellular events and bacterial genes used by the vegetative bacilli allowing for survival and escape from the macrophage, and; c. understand the relevance of B. anthracis host-specific germination systems and early intracellular events in terms of pathogenesis in the murine model. Knowledge of these critical "establishment" stages of anthrax may provide targets for early intervention after exposure to anthrax endospores. Understanding this rapid and dramatic switch, from absolute metabolic dormancy of the endospore to growing virulent bacilli allows anthrax to be exploited as an effectual model for examining the earliest stages of bacterial infectious cycles.