Regulation of the Poly Sialic Virulence Factor

描述（由申请方提供）：肠外致病性大肠埃希菌（ExPEC）是主要的病原体，主要发生在非常年轻、年老或免疫功能低下的人群中，每年导致数万人死亡和数十亿美元的医疗费用。例如，主要的ExPEC亚型，E. coli O 18：K1表达O 18体细胞抗原和K1多聚唾液酸荚膜抗原，这两种抗原是导致这些菌株成为新生儿细菌性脑膜炎的主要原因的两个主要毒力因子。利用微生物遗传学和碳水化合物分析的创新方法，我们定义了唾液酸转运、合成、聚合、催化和修饰单体和多聚唾液酸的完整途径。修饰机制与一种新型细菌病毒（CUS-3）有关，该病毒携带可变相乙酰化酶基因，催化唾液酸环外链的随机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靶向它们的方法用于新的治疗开发。我们设计了三个具体目标来检验该假设：1）通过遗传改变乙酰转移酶NeuD和乙酰酯酶NeuA*（NeuA-star）来确定单体唾液酸O-乙酰化对总体聚唾液酸修饰的贡献。该目的集中于在将O-乙酰基酯掺入聚唾液酸之前，参与O-乙酰基酯与单体唾液酸相互加成（NeuD）或相减（NeuA*）的酶。2)使用创新的流式细胞术技术和显微镜方法确定体内荚膜聚唾液酸修饰的动力学，以区分不同宿主隔室中的乙酰化和非乙酰化相。3)通过（i）构建所有区域1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-辛酮糖酸，（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，和（v）确定所述引发复合物的化学结构。

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.