The Role of LPS and Toll-like Receptors in Plague

革兰氏阴性菌鼠疫耶尔森氏菌是鼠疫的病原体。鼠疫耶尔森氏菌和其他一些病原体能够改变其脂多糖/脂A的酰基链，以减少Toll样受体4(TLR4)的信号转导。因此，鼠疫耶尔森氏菌在37℃时产生一种TLR4激活能力较差的四酰基脂多糖，而在26℃时合成一种有效的六酰基脂类化合物。我们已经证明，通过产生一种修饰的细菌株来合成同样在37℃下激活内毒素的六酰化TLR4，从而在37℃下产生的先天免疫反应对于鼠疫耶尔森菌的毒力是必要的。改造后的菌株含有LpxL，这是一种来自大肠杆菌的脂类A生物合成酶，在鼠疫杆菌中不存在，其毒力降低了100多万倍。我们的主要假设是，躲避LPS-TLR4信号对于鼠疫杆菌的毒力是必不可少的，而对脂类A结构的严格调控是发生这种逃避的必要条件。我们建议使用产生修饰内毒素的鼠疫杆菌菌株来研究鼠疫杆菌对天然免疫的逃避和激活。我们的模型系统似乎非常适合描述对抗鼠疫杆菌的有效天然免疫机制，我们的长期目标是定义这种机制和细菌对策。1)鼠疫耶尔森氏菌表达LpxP，这是一种脂类A生物合成基因，可能是在较低温度下产生六酰TLR4激活内毒素所必需的。我们建议研究LpxP在37℃下的表达调节，因为调节似乎是毒力所必需的。2)我们还希望研究LPS-TLR4信号的回避在鼠疫杆菌进化为其最接近的祖先假结核杆菌(Y.ptb)的高毒力病原体中的作用，该菌只可能导致轻微的胃肠炎。有趣的是，Y.PTB含有LpxL基因。我们的建议建议研究Y.PtB LpxL的功能，这将包括Y.PtB LpxL在鼠疫耶尔森菌中的表达，以及研究内毒素的活性和结构。3)初步结果表明，IL-1的释放和信号转导在清除鼠疫菌LpxL感染方面比肿瘤坏死因子和I型干扰素更有效。我们将在体外和体内分析鼠疫杆菌诱导和控制IL-1的机制。

The gram-negative bacterium Yersinia pestis is the causative agent of plague. Y. pestis and some other pathogens have the ability to modify the acyl chains in their lipopolysaccharide (LPS) /lipid A in order to minimize Toll-like receptor 4 (TLR4) signaling. Consequently, Y. pestis produces a tetra-acyl LPS with poor TLR4-activating ability at 37C, while synthesizing a potent hexa-acyl lipid at 26C. We have shown that the resulting evasion of innate immune responses at 37C is necessary for Y. pestis virulence via the peripheral route, by generating a modified bacterial strain synthesizing a hexa-acylated TLR4-activating LPS also at 37C. The modified strain contained LpxL, a lipid A biosynthesis enzyme from E. coli, that is absent in Y. pestis, and has more than a million-fold reduced virulence. Our main hypothesis is that evasion of LPS-TLR4 signaling is essential for the virulence of Y. pestis, and that a tight regulation of lipid A structure is necessary for this evasion to occur. We propose to use Y. pestis strains that generate modified LPS to study evasion and activation of innate immunity by the plague bacillus. Our model system appears well suited to describe efficient innate immune mechanisms against Y. pestis, and our long-term goal is to define such mechanisms and bacterial countermeasures. 1) Y. pestis expresses LpxP, a lipid A biosynthesis gene that likely is necessary for the production of a hexa-acyl TLR4-activating LPS at lower temperatures. We propose to study regulation of LpxP expression at 37C, as regulation appears necessary for virulence. 2) We also wish to study the role of evasion of LPS-TLR4 signaling in the evolution of Y. pestis to a highly virulent pathogen from its closest ancestor, Y. pseudotuberculosis (Y. ptb), which only may cause a mild gastroenteritis. Interestingly, Y. ptb harbors an LpxL gene. Our proposal suggests studies of Y. ptb LpxL function, this will include expression of Y. ptb LpxL in Y. pestis, and study LPS activity and structures. 3) Preliminary results indicate that interleukin-1 (IL-1) release and signaling is effective in clearing infection with Y. pestis-LpxL, more so than TNF and type I IFN. We will analyze mechanisms by which Y. pestis induces and is controlled by IL-1, in vitro and in vivo.