The role of calcium-DPA in the virulence of Bacillus anthracis spores

描述(申请人提供)：2001年10月的炭疽病袭击造成5人死亡和17人患病，扰乱了商业和政府活动，并引起了广泛的焦虑。炭疽杆菌的感染形式是孢子，这是一种在胁迫期间形成的休眠和抵抗结构。通过吸入途径，炭疽杆菌的孢子被肺泡巨噬细胞吞噬并在那里萌发。有趣的是，炭疽杆菌不会干扰吞噬小体的成熟，并能够在吞噬小体中茁壮成长。在延迟一段时间后，新萌发的细胞产生一种在宿主巨噬细胞中生存所必需的三方毒素。与对炭疽杆菌孢子的脆弱性不同，巨噬细胞很容易杀死产生毒素的营养炭疽杆菌细胞。这些结果是违反直觉的：新萌发的细胞不会很早产生毒素，应该比营养细胞更容易受到巨噬细胞的攻击。为了解释这一悖论，我们提出炭疽芽胞特有的因素可以解释萌发细胞在巨噬细胞吞噬小体中存活的能力。一个独特的候选者是炭疽杆菌孢子萌发时释放的与二吡啶甲酸(DPA)络合的大量钙离子。当炭疽杆菌孢子在宿主内萌发时，Ca-DPA储存库将定位于巨噬细胞内。哺乳动物细胞中的钙水平受到严格控制，细胞内钙的干扰可能会导致细胞凋亡。或者，DPA的释放可以缓冲吞噬酶体的酸化。作为原理的证明，我们已经证明了含有K+而不是Ca+2的炭疽芽胞显示出较低的细胞毒性(图2)。根据这些结果，我们推测，钙和/或DPA的释放保护萌发的炭疽细胞免受巨噬细胞的作用，是炭疽毒力的决定因素。为了验证我们的假设，我们将研究两个相关的问题：(1)炭疽杆菌孢子的细胞毒性依赖于钙和/或DPA浓度吗？(2)钙和DPA在感染的巨噬细胞中的命运是什么？

DESCRIPTION (provided by applicant): The anthrax attacks of October 2001 caused five fatalities and 17 illnesses, disrupted business and government activities, and caused widespread anxiety. The infective form of B. anthracis is the spore, a dormant and resistant structure formed during periods of stress. Following the inhalation route, B. anthracis spores are phagocytized by alveolar macrophages where they germinate. Interestingly, B. anthracis does not interfere with phagosome maturation and is able to thrive in the phagolysosome. After a delay, the newly germinated cells produce a tripartite toxin that is necessary for survival in the host macrophage. In contrast to their vulnerability to B. anthracis spores, macrophages kill toxin-producing vegetative B. anthracis cells with ease. These results are counterintuitive: Newly germinated cells do not produce toxins early and should be more vulnerable than vegetative cells to macrophage attack. To account for this paradox, we propose that factors unique to Bacillus anthracis spores can account for the ability of germinated cells to survive the macrophage's phagosome. One distinctive candidate is the large concentrations of calcium ions complexed with dipicolinic acid (DPA) that is released upon B. anthracis spore germination. The Ca-DPA depot will be localized inside macrophages when B. anthracis spores germinate in the host. Calcium levels are strictly controlled in mammalian cells and disruptions can lead to apoptosis. Alternatively, DPA release could buffer the acidification of the phagolysosome. As proof-of principle, we have shown that B. anthracis spores containing K+ instead of Ca+2 exhibit reduced cytotoxicity (Fig. 2). Based on these results, we hypothesize that the release of calcium and/or DPA protects germinated B. anthracis cells from macrophage action and is a determining factor in anthrax virulence. To test our hypothesis, we will look into two related issues: (1) is te cytotoxicity of B. anthracis spores dependent on calcium and/or DPA concentrations and (2) what is the fate of calcium and DPA in infected macrophages?