Regulation of the (poly) sialic virulence factor

描述（由申请人提供）：细菌性败血症和脑膜炎是美国感染性病原体导致死亡和住院的主要原因。仅脓毒症一项，每年就造成50万例危及生命的感染，20多万人死亡，每年造成的经济损失大大超过10亿美元。肠道外致病性大肠杆菌（ExPEC）是这些感染的主要病原体，有一种血清型，即大肠杆菌O18:K1:H7，是革兰氏阴性新生儿脑膜炎的主要原因，也是女性无并发症膀胱炎中最常见的分离物，因此使其成为一般ExPEC感染的有用模型。尽管唾液酸修饰细胞表面是一个重要的决定因素，但目前尚不清楚ExPEC如何破坏宿主粘膜并达到引发败血症和脑膜炎所需的全身浓度（菌血症）。在这一继续申请中，我们建议完成我们的研究，以确定唾液酸和多唾液酸毒力因子是如何合成的，然后使用功能基因组方法确定全系统疾病所需的其他基因产物。Specific Aim 1将完成唾液酸生物合成和唾液酸代谢调控的分析。初步数据表明，唾液酸盐生物合成的第一步n -乙酰甘露糖胺（ManNAc）的合成不涉及ManNAc 6-磷酸。x射线晶体学和核磁共振波谱学将补充这一目的的目标。在Specific Aim 2中，我们将通过分析嵌合多唾液基转移酶（polySTs）来确定PSA生物合成的结构基础，并使用位点定向诱变来了解催化机制。我们的初步数据表明，这些重要的酶在结构和功能上与哺乳动物的polyST不同，这表明特异性polyST抑制剂具有广泛的治疗潜力。Specific Aim 3将应用签名标记诱变来识别与PSA不同的疾病特征或与该胶囊协同作用以确定exic毒力。我们的目的是建立最小的系统性ExPEC“病理类型”。提出的研究通过确定非抗生素药物或疫苗设计的新靶点，直接应用于开发新的治疗方法。迫切需要新的方法来预防或治疗快速老龄化的美国人口，非常年轻和越来越多的免疫功能低下患者的exp感染。

DESCRIPTION (provided by applicant): Bacterial sepsis and meningitis are the leading causes of mortality and hospitalization by infectious agents in the United States. Sepsis alone accounts for >700,000 life-threatening infections each year and over 200,000 deaths, with economic losses greatly exceeding one billion annually. Extraintestinal pathogenic Escherichia coli (ExPEC) are the predominant agents of these infections, with one serotype, E. coli O18:K1:H7, emerging as the leading cause of gram-negative neonatal meningitis and the most prevalent isolate from uncomplicated cystitis in women, thus making it a useful model of ExPEC infections in general. How ExPEC breach the host's mucosa and reach the systemic concentration (bacteremia) necessary for triggering sepsis and meningitis is not understood, although cell-surface modification with sialic acid is an important determinant. In this continuation application, we propose to complete our studies to determine how the sialic and polysialic acid virulence factors are synthesized, and then to identify other gene products required for systemic disease using a functional genomic approach. Specific Aim 1 will complete the analysis of sialic acid biosynthesis and the regulation of sialate metabolism. Preliminary data indicate that the synthesis of N-acetylmannosamine (ManNAc), the first committed step in sialate biosynthesis, does not involve ManNAc 6-phosphate. X-ray crystallography and NMR spectroscopy will complement the objectives of this aim. In Specific Aim 2, the structural basis for PSA biosynthesis will be determined by analyzing chimeric polysialyltransferases (polySTs) and using site-directed mutagenesis to understand catalytic mechanism. Our preliminary data indicate that these important enzymes are structurally and functionally distinct from mammalian polySTs, suggesting wide therapeutic potential for specific polyST inhibitors. Specific Aim 3 will apply signature-tagged mutagenesis to identify disease traits that are either distinct from PSA or function in concert with this capsule to define ExPEC virulence. Our objective in this aim is to establish the minimal systemic ExPEC "pathotype". The proposed studies have direct application to the development of new therapeutic approaches by identifying novel targets for non-antibiotic drug or vaccine design. New approaches are urgently needed to prevent or treat ExPEC infections in the rapidly aging US population, the very young and the increasingly large number of immunocompromised patients.