REGULATION OF THE (POLY) SIALIC ACID VIRULENCE FACTOR

这项建议的主要目的是增进我们的理解 病原微生物中细菌多糖合成的研究。这个 K1抗原，或称聚唾液酸胶囊，是α2，8-的均聚物- 连接唾液酸基残基。胶囊的合成依赖于大约15 KPS致病基因座的基因至少被组织成两个 可转换转录的操纵子，其中一个由 温度调节的KPSF启动子。唾液酸类化合物的抗识别作用 酸(抗吞噬作用；抑制抗体非依赖性补体 固定；宿主抗原的分子模拟)使这些独特的九- 碳羧酸是细菌致病力的重要决定因素 不同的人类和动物病原体。大肠杆菌K1是一种领先的 新生儿脑膜炎的病因和尿路的常见病因 儿童感染和成人菌血症。接种疫苗以预防 这些疾病可能并不总是实用的，而常规药物 治疗是有问题的。可能出现替代或改进的治疗方法 从更好的理解胶囊多糖的表达。 应用的目的是了解分子机制。 胶囊体温调节和唾液酸前体生物合成。 此信息对于构建已定义的突变体是必需的 将使我们能够毫不含糊地确定胶囊与 和内毒素O抗原。要实现这些目标 在目标方面，我们将追求三个具体目标。第一，体温调节 将通过体外转录系统进行研究 测定KPSF启动子对温度的内在敏感性。 正向调节器的作用将由隔离和 不再调控KPSF启动子的突变体的特征。 第二，唾液酸生物合成前体N-的来源。 乙酰甘露糖胺，将由遗传和生化决定 接近了。最后，胶囊的作用及其与 O抗原将在动物模型中使用已定义的突变体来确定 大肠杆菌K1脑膜炎和菌血症。在完成这项工作后 研究，我们希望已经清楚地描绘出 细胞表面多糖在发病机制中的作用。我们也 希望已经确定了潜在治疗的特定新靶点 旨在阻断胶囊在体内表达的研究进展。

The broad objective of this proposal is to increase our understanding of bacterial polysaccharide synthesis in pathogenic microorganisms. The K1 antigen, or polysialic acid capsule is a homopolymer of alpha2,8- linked sialyl residues. Synthesis of the capsule depends on about 15 genes of the kps pathogenicity locus that is organized into at least two convergently transcribed operons, one of which is controlled by the thermoregulated kpsF promoter. The anti-recognition functions of sialic acids (anti-phagocytosis; inhibition of antibody-independent complement fixation; molecular mimicry of host antigens) make these unique nine- carbon carboxy sugar acids important determinants of virulence in diverse human and animal pathogens. Escherichia coli K1 is a leading cause of neonatal meningitis and a frequent cause of urinary tract infections in children and bacteremia in adults. Vaccination against these diseases may not always be practical, and conventional drug therapy is problematic. Alternative or improved therapies may emerge from a better understanding of capsular polysaccharide expression. Objectives of the application are to understand the molecular mechanisms of capsule thermoregulation and sialic acid precursor biosynthesis. This information is necessary for construction of defined mutants that will allow us to unambiguously determine the association between capsule and lipopolysaccharide O antigen during disease. To accomplish these objectives, we will pursue three specific aims. First, thermoregulation will be investigated with an in vitro transcription system designed to determine the intrinsic sensitivity of the kpsF promoter to temperature. The role of a positive regulator will be determined by isolation and characterization of mutants that no longer regulate the kpsF promoter. Second, the origin of sialic acid biosynthetic precursor, N- acetylmannosamine, will be determined by genetic and biochemical approaches. Finally, the role of the capsule and its association with O antigen will be determined using defined mutants in an animal model of E. coli K1 meningitis and bacteremia. At the completion of this research, we expect to have clearly delineated the relative contributions of cell surface polysaccharides to pathogenesis. We also expect to have identified specific new targets for potential therapeutic developments aimed at blocking capsule expression in vivo.