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

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。<br><br>革兰氏阳性菌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。已表明炭疽杆菌细胞壁强烈刺激外周血单核细胞产生炎症细胞因子肿瘤坏死因子Q、白介素1受体和白介素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，可能导致炭疽杆菌损伤的细胞壁的数量可能比其他细菌类型大得多。<br><br>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。<br><br>本研究采用大鼠模型研究炭疽杆菌细胞壁的作用。在迄今为止的实验中，我们已经注意到，细胞壁的输注与循环中白细胞和血小板的剂量依赖性减少以及细胞因子和一氧化氮水平的增加有关，这与炎症反应是一致的。这些变化与氧合异常有关，反映为肺泡至动脉血氧分压升高(AaO2)。与非致死量的炭疽细胞壁相比，致死量的AaO2的增加更大，而且与金黄色葡萄球菌致病株的细胞壁产生的AaO2非常相似。基于这些发现，我们假设炭疽细胞壁会恶化注射LeTx的结果。然而，在一种非常意想不到的模式下，给动物注射非致死剂量的炭疽细胞壁实际上保护了动物免受致死剂量的LeTx的伤害。进一步的研究正在进行中，以了解这种效应的基础。

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. <br><br>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 Q, IL-1 R, 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.<br><br>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. <br><br>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 infusion of cell wall is associated with dose dependent reductions in circulating white blood cells and platelet and increases in cytokine and nitric oxide levels, consistent with an inflammatory response. These changes are associated with abnormalities of oxygenation as reflected by increased alveolar to arterial oxygen gradients (AaO2). These increases in AaO2 are greater with lethal compared to nonlethal doses of anthrax cell wall and are very similar to ones produced by cell wall from a pathogenic strain of S. aureus. Based on these findings we hypothesized that anthrax cell wall would worsen outcome with LeTx infusion. However, in a highly unexpected pattern, administration of nonlethal doses of anthrax cell wall actually protected animals from lethal doses of LeTx. Further studies are underway to undertstand the basis for this effect.