Regulation of the Poly Sialic Virulence Factor

描述（由申请人提供）：肠外致病性大肠杆菌（ExPEC）是大多数非常年轻、老年或免疫功能低下人群的主要病原体，每年导致数万人死亡，并造成数十亿美元的医疗费用。例如，主要的ExPEC亚型，大肠杆菌O18:K1，表达O18体细胞抗原和K1多唾液酸胶囊抗原，作为两种主要毒力因子，使这些菌株成为新生儿细菌性脑膜炎的主要原因。利用微生物遗传学和创新的碳水化合物分析方法，我们确定了唾液酸运输、合成、聚合、分解代谢和单体和聚唾液酸修饰的完整途径。该修饰机制与一种携带可变相位乙酰化酶基因的新型细菌病毒cus3有关，该病毒可催化唾液酸外环链的随机o -乙酰化。我们假设，了解这些细菌抗原的体内功能将有助于针对它们开发新的治疗方法。我们设计了三个具体目标来验证这一假设：1)通过基因改变乙酰转移酶NeuD和乙酰酯酶NeuA* (NeuA-star)，确定单体唾液酸O-乙酰化对整体聚唾液酸修饰的贡献。本研究的重点是在单体唾液酸掺入聚唾液酸之前，O-乙酰酯与单体唾液酸相互加成（NeuD）或相互减法（NeuA*）的酶。2)利用创新的流式细胞技术和显微方法确定囊状聚唾液酸在体内的修饰动力学，以区分不同寄主室中乙酰化和非乙酰化相。3)通过(i)构建所有1区输出基因的框内缺失，（ii）确定生物合成是否需要3-脱氧-d -甘露糖-八氟索酸酯，（iii）鉴定与辅助蛋白KpsC相互作用的聚合酶结构域，（iv）使用量子点技术和K1特异性噬菌体确定胶囊输出位点，(v)确定起始复合物的化学结构，确定偶联多糖合成出口的分子基础。

DESCRIPTION (provided by applicant): Extraintestinal pathogenic Escherichia coli (ExPEC) are major pathogens in mostly the very young, aged or immunocompromised human population resulting in tens of thousands of deaths each year and billions of dollars in healthcare costs. For example, the predominant ExPEC subtype, E. coli O18:K1, expresses O18 somatic and K1 polysialic acid capsule antigens as two major virulence factors responsible for making these strains the leading causes of neonatal bacterial meningitis. Using microbial genetics and innovative methods of carbohydrate analysis, we defined the complete pathways for sialic acid transport, synthesis, polymerization, catabolism and modification of both monomeric and polysialic acids. The modification mechanism was linked to a novel bacterial virus (CUS-3) carrying the phase-variable acetylase gene catalyzing stochastic O-acetylation of the sialic acid exocyclic chain. We hypothesize that understanding the in vivo functions of these bacterial antigens will facilitate approaches targeting them for new therapeutic development. We have designed three specific aims to test this hypothesis: 1) Determine the contribution of monomeric sialic acid O- acetylation to overall polysialic acid modification by genetically altering the acetyltransferase, NeuD, and acetylesterase, NeuA* (NeuA-star). This aim focuses on the enzymes involved in the reciprocal addition (NeuD) or subtraction (NeuA*) of O- acetyl esters to or from monomeric sialic acids prior to their incorporation into polysialic acid. 2) Determine the dynamics of capsular polysialic acid modification in vivo using an innovative flow cytometric technique and microscopic methods to distinguish between acetylated and unacetylated phases in different host compartments. 3) Determine the molecular basis for coupling polysaccharide synthesis to export by (i) constructing in frame deletions of all region 1 export genes, (ii) determining whether 3-deoxy-D-manno- octulosonate is required for biosynthesis, (iii) identifying the polymerase domain(s) interacting with the accessory protein, KpsC, (iv) using Quantum-dot technology and K1- specific phage to determine the site of capsule export, and (v) determining the chemical structure of the initiation complex.