Listeriosis Pathogenesis: Effect of Serogroup-specific Wall Teichoic Acid Changes

描述（由申请人提供）：在了解单核细胞增生李斯特菌的发病机制方面取得了巨大进展，特别是在细胞生物学水平上。取得这一进展的原因之一是广泛使用单一血清1/2 L.单核细胞增生菌株EGD。然而，来自其他更普遍的血清群（包括血清4）的代表性菌株仍未得到充分研究。从人类和其他动物中分离出来的与疾病相关的分离株主要为血清4组菌株。李氏血清群是由壁磷壁酸（WTA）结构定义的。所有的李氏菌分离株都具有两种根本不同的WTA结构之一——以1/2血清群结构为代表的结构和以4血清群结构为代表的结构。最近的独立研究表明，血清4组WTA组成的某些变化正在显著减弱。在本提案中，我们研究了小鼠口服毒性血清4组菌株的发病机制。我们有一个特定的目标：1)构建和表征改变血清4组壁壁壁酸（WTA）组成的突变体。我们将系统地改变血清4组WTA的组成，并将组成变化与体内和体外致病性的变化联系起来。这一目的有三个目标：(a)突变体的构建和生化表征；等位基因交换将被用于将框内缺失突变引入基因中，这些基因的产物是产生和适当替换血清组4 WTA重复单元所必需的。(b)体外突变体表征；细胞内生长和细胞间扩散将在培养的小鼠肠细胞和巨噬细胞单层中进行评估。(c)体内和体外突变体特征；每个突变体的口腔毒力将在感染指数实验中确定，该实验评估突变体从肠腔的易位程度。此外，将对每个WTA突变体的炎症细胞浸润的引发、PMN和单核吞噬细胞的吞噬杀伤以及TNF的产生水平进行检查。总的来说，我们预计我们的结果将扩展我们在一个高度相关的血清组的李斯特菌发病机制的知识。此外，我们的结果有可能影响几个领域的国家卫生政策和程序。例如：(i)风险评估——了解血清组4 WTA组成如何影响毒力，可促进基于凝集素的简单分析方法的发展，以评估在出现菌体污染的情况下是否需要召回产品。（ii）诊断——了解WTA成分如何影响致病潜力和疾病表现，可以促进诊断，提高诊断分析的特异性，并提高流行病学研究的能力。（iii）治疗——特定的治疗策略可以扩展治疗方式（例如，专门用于破坏WTA生物合成中的特定步骤）。

DESCRIPTION (provided by applicant): Great strides have been made in understanding Listeria monocytogenes pathogenesis, especially at the cell biological level. One reason for this progress has been the extensive use of a single serogroup 1/2 L. monocytogenes strain, EGD. However, representative strains from other more prevalent serogroups, including serogroup 4, have remained understudied. Serogroup 4 strains constitute the preponderance of disease associated isolates from humans and other animals. Listerial serogroups are defined by their wall teichoic acid (WTA) structure. All listerial isolates have one of two fundamentally different WTA structures--those represented by the 1/2 serogroup structure and those represented by serogroup 4 structure. Recent independent studies indicate that certain alterations to serogroup 4 WTA composition are profoundly attenuating. In this proposal we examine the pathogenesis of a mouse oral virulent serogroup 4 strain. We have one specific aim: 1) Construct and characterize mutants that have altered serogroup 4 wall teichoic acid (WTA) composition. We will systematically alter the serogroup 4 WTA composition and correlate compositional changes to alterations in pathogenicity in vivo and in vitro. This aim has three objectives: (a) Mutant construction and biochemical characterization; allelic exchange will be employed to introduce in-frame deletion mutations into genes whose products are required for the generation of, and the proper substitution of the serogroup 4 WTA repeat unit. (b) Mutant characterization in vitro; intracellular growth and cell-to-cell spread will be assessed in cultured mouse enterocyte and macrophage monolayers. (c) Mutant characterization in vivo and ex vivo; the oral virulence of each mutant will be determined in infective index experiments that assess the degree of translocation of the mutants from the intestinal lumen. Additionally, the elicitation of inflammatory cell infiltrates, phagocytic killing by PMN and mononuclear phagocytes, and level of TNF production will be examined for each WTA mutant. Overall, we anticipate that our results will extend our knowledge of listerial pathogenesis in a highly relevant serogroup. Additionally, our results have the potential to impact national health policies and procedures in several areas. For example: (i) Risk assessment-a knowledge of how serogroup 4 WTA composition influences virulence could facilitate the development of simple lectin based assays to assess the need for product recalls in cases of listerial contamination. (ii) Diagnostics-a knowledge how WTA composition influences pathogenic potential and disease presentation could facilitate diagnosis, improve the specificity of diagnostic assays, and improve the power of epidemiological studies. (iii) Therapies--specific therapeutic strategies could expand treatment modalities (e.g., ones tailored to disrupt specific steps in WTA biosynthesis).