The Effects of B. anthracis Cell Wall in a Small Animal

炭疽芽孢杆菌(B.anthracis)是当今美国的一大生物恐怖主义威胁。在2001年的生物恐怖主义袭击中，所有需要血管加压剂支持的患者的死亡率为100%。这与更常见的细菌性败血症形成对比，在这些类型的细菌性败血症中，需要类似治疗的患者的死亡率通常接近60%。虽然致死性和水肿性毒素(分别为LeTx和ETx)的产生都会导致与炭疽杆菌相关的休克，但仅使用这些毒素并不会导致活体细菌感染所引起的组织损伤的全部范围。这表明，其他毒力因素也导致了对这种有机体的震惊。炭疽杆菌的细胞壁就是这样一个候选者。 革兰氏阳性菌的细胞壁由几个结构大分子组成，包括胃多糖(PG)和磷壁酸(TA)。肽聚糖层是由N-乙酰氨基葡萄糖(GlcNAc)和N-乙酰胞壁酸(MurNAc)两种交替的氨基糖形成的线性链形成的晶格结构。革兰氏阳性细菌的这一层(20至80 nm)比革兰氏阴性细菌(7至8 nm)厚得多，约占前者细菌干重的90%，但仅为后者的10%。磷壁酸是通过磷酸二酯键连接的甘油或核糖醇的聚合物。在革兰氏阴性细菌中没有发现它们。它们可以共价结合到肽聚糖层的N-乙酰胞壁酸，也可以连接到细胞膜中的质膜脂类，形成脂磷壁酸(LTA)。这些成分在细菌细胞壁中起着结构性作用，提供了细胞壁的形状和结构强度，并抵消了细胞质的渗透压力。然而，这些分子除了结构功能外，就像革兰氏阴性细菌的脂多糖一样，也可以作为天然免疫系统的强大刺激剂。一些革兰氏阳性细菌表面的肽聚糖与PG识别蛋白(PGRPs)、Toll样受体2(TLR-2)、核苷酸结合寡聚化结构域(NOD)蛋白(NOD1、NOD2或低温比林)相互作用，导致Caspase-1、NF-kB和MAP激动酶的激活，从而导致多种促炎细胞因子和趋化因子的表达和释放。一些细菌的脂磷壁酸还可以与CD14、CD36、TLR-2和可能的LBP相互作用，诱导细胞因子和趋化因子的释放。这些细胞壁组分激活炎症反应，进而导致败血症和感染性休克相关的血流动力学不稳定和组织损伤。 细胞壁肽聚糖和脂磷壁酸的特殊结构和免疫刺激作用因革兰氏阳性细菌而异。一些金黄色葡萄球菌的细胞壁，如高毒力金黄色葡萄球菌，是一种强烈的炎症刺激物，在体内给药时具有高度毒性。然而，来自其他细菌类型的细胞壁，如无毒力的枯草杆菌，几乎不会引起炎症反应。目前可从体外研究中获得的数据表明，炭疽杆菌细胞壁具有相当大的促炎作用。在一系列实验中，波波夫等人。已证明炭疽杆菌细胞壁强烈刺激外周血单核细胞产生炎性细胞因子肿瘤坏死因子？、白介素10和白介素6(17)。然而，这些刺激效应是否会在体内造成可见的损伤，还有待检验。同样重要的是要注意到，在动物和人类中，炭疽杆菌的致命感染与细菌载量有关，而细菌载量比通常观察到的致命细菌的载菌量要大得多。因此，与其他细菌类型相比，可能对炭疽杆菌造成伤害的细胞壁的数量要大得多。 在一系列实验和研究中，我们确定了高纯度形式的LeTx和ETX单独和联合对大鼠心肺功能、炎性细胞因子和趋化因子的产生、一氧化氮释放和组织学变化的影响。在我们开发的模型中，毒素在24小时内注入，以更好地模拟活细菌感染期间发生的模式。 本研究采用大鼠模型研究炭疽杆菌细胞壁的作用。在到目前为止的实验中，我们已经注意到，尽管细胞壁输注与循环中白细胞和血小板的减少呈剂量依赖关系，与炎症反应一致，但血压并没有下降，尽管使用了相当高的细胞壁剂量，致命性也是最小的。将细胞壁作为丸剂给药对循环细胞和血小板产生类似的影响，但确实会产生一些血液动力学不稳定和更多的致命性。然而，总体而言，这些研究支持炭疽杆菌细胞壁必须大量存在才能在炭疽杆菌感染期间引起休克和致死的可能性。正在编写摘要，描述该议定书的早期工作，其他研究正在进行中。

Bacillus anthracis (B. anthracis) is a major bioterrorism threat in the United States (US) today. In the 2001 bioterrorism attack, the mortality rate in all patients who required vasopressor support was 100%. This contrasts with more commonly encountered types of bacterial sepsis in which mortality rates in patients requiring similar treatment is typically close to 60%. Although production of both lethal and edema toxins (LeTx and ETx respectively) contribute to the shock associated with B. anthracis, administration of these toxins alone do not result in the full spectrum of tissue injury noted with live bacterial infection. This suggests that other virulence factors contribute to shock with this organism. The cell wall of B. anthracis is one such candidate. The cell wall of gram positive bacteria is composed of several structural macromolecules, including peptoglycans (PG) and teichoic acids (TA). The peptidoglycan layer is a crystal lattice structure formed from linear chains of two alternating amino sugars, N-acetyl glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc). This layer is substantially thicker in Gram-positive bacteria (20 to 80 nm) than in Gram-negative bacteria (7 to 8 nm) and comprises around 90% of the dry weight of the former bacteria but only 10% of the latter. Teichoic acids are polymers of glycerol or ribitol linked via phosphodiester bonds. They are not found in gram-negative bacteria. They can be either covalently bonded to N-acetylmuramic acid of the peptidoglycan layer or linked to the plasma membrane lipids found in the cytoplasmic membrane forming lipoteichoic acids (LTA). These components serve a structural role in the bacterial cell wall, giving the wall shape and structural strength, as well as counteracting the osmotic pressure of the cytoplasm. In addition to their structural functions however, these molecules, just like lipopolysaccharide (LPS) from gram-negative bacteria, can also serve as powerful stimulants of the innate immune system. Interaction of peptidoglycan with PG recognition proteins (PGRPs), toll-like receptor 2 (TLR-2), nucleotide-binding oligomerization domain (NOD) proteins (NOD1, NOD2 or cryopyrin) on the surface of some gram-positive bacteria results in the activation of Caspase-1, NF-kB and MAP kinases and the subsequent expression and release of a variety of pro-inflammatory cytokines and chemokines. Lipotechoic acid from some bacteria can also interact with CD14, CD36, TLR-2 and possibly LBP to induce cytokine and chemokine release. Activation of the inflammatory response by these cell wall components can in turn produce the hemodynamic instability and tissue injury associated with sepsis and septic shock. The specific structure and immunostimulatory effects of cell wall peptidoglycan and lipotechoic acid varies among gram-positive bacteria. The cell wall of some, such as highly virulent S. aureus, is a strong inflammatory stimulant and is highly toxic when administered in vivo. The cell wall from other bacteria types however, like nonvirulent B. subtilis, induces little inflammatory response. Data now available from in vitro studies indicates that B. anthracis cell wall has considerable pro-inflammatory effects. In a series of experiments, Popov et al. has shown that B. anthracis cell wall strongly stimulates peripheral blood monocyte production of the inflammatory cytokines TNF??, IL-1?O, and IL-6 (17). It remains to be tested however, whether these stimulatory effects result in observable injury in vivo. It is also important to note that lethal infection with B. anthracis both in animals and man is associated with bacterial loads that are much greater than is typically observed with virulent bacteria. Thus, the amount of cell wall that may contribute to injury with B. anthracis, may be much greater than with other bacteria types. In a series of experiments and studies, we have defined the effects of highly purified forms of LeTx and ETx, both alone and together on cardiopulmonary function, inflammatory cytokine and chemokine production, nitric oxide release and histological changes in rats. In the model we developed, toxin is infused over 24 h to better simulate the pattern that occurs during live bacterial infection. The present study is employing the rat model to study the effects of B. anthracis cell wall. In experiments to date we have noted that although infusion of cell wall is associated with dose dependent reductions in circulating white blood cells and platelet, consistent with an inflammatory response, blood pressure is not decreased and lethality, despite using fairly high cell wall doses, is minimal. Administration of cell wall as a bolus creates similar effects on circulating cells and platelets, but does produce some hemodynamic instability and more lethality. Overall however these studies support the possibility that B. anthracis cell wall must be present in very amounts to contribute to shock and lethality during B. anthracis infections. Abstracts are being prepared describing early work in this protocol and other studies are ongoing.