REGULATION OF CAPSULE BIOSYNTHESIS IN N MENINGITIDIS

描述（改编自申请人的摘要）：唾液酸是一种 广泛分布的微生物毒力因子，表达于 脑膜炎奈瑟菌、无乳链球菌、埃希氏菌 coli K1和N.淋球菌作为荚膜多糖和/或末端 N-乙酰神经氨酸（NANA）残基与脂寡糖连接。 唾液酸的表达是为了规避人类宿主的防御和调节 生物体与宿主细胞的接触依赖性相互作用。 尽管 唾液酸在微生物致病中的重要作用， 已知这些分子表达的遗传调控， 人类 作为这一过程的范例，调查人员试图确定 如何合成B、C、Y和W-135 N组的唾液酸胶囊。 脑膜炎在人类感染期间受到调节。 研究者 提出转录控制两个操纵子参与胶囊 生物合成和运输是唾液酸的主要调节机制， 酸胶囊N.脑膜炎，以及环境条件， 人宿主和/或人细胞因子影响该调节。 在两 具体目标，研究人员建议探索遗传基础， 脑膜炎球菌对唾液酸的表达和转录调节。 在目标I中，研究者将确定促进者和监管者。 连接趋异操纵子的134 bp基因间区的元件 参与CMP-NANA/唾液酸囊生物合成和囊膜 运输 这将通过转录报告基因来完成 构建体和基因间区域的协调诱变。 TN916 诱变、体外诱变和使用爱马仕质粒，或E.杆菌 然后将使用互补来鉴定转录因子， 调节生物合成和运输操纵子。 在Aim II中， 研究者建议研究以下表达、调节和作用： 在特定事件（鼻咽炎）期间的包膜基因的发病机制 定植、血流侵入）， 感染 研究者将首先研究胶囊的调节 在不同的体外环境生长条件下， 脑膜炎球菌在感染过程中可能遇到的那些， 定殖（pH、铁限制、Ca++、温度和厌氧）。 然后，研究者将这些研究扩展到人类鼻咽 器官培养物和人血清。 直接评估关节囊 通过ELISA和免疫显微镜技术， 基因定义的同基因突变体，转录报告基因 构建体，并定量胶囊基因mRNA。 这些 研究直接应用于新的脑膜炎球菌的开发， 疫苗战略，并应提供有关的重要信息 在其他重要的细菌和生物系统中调节唾液酸。

DESCRIPTION (Adapted from the applicant's abstract): Sialic acid is a widely distributed microbial virulence factor that is expressed on the surface of Neisseria meningitidis, Streptococcus agalactiae, Escherichia coli K1 and N. gonorrhoeae as capsular polysaccharide and/or terminal N-acetylneuraminic acid (NANA) residues attached to lipooligosaccharides. Sialic acids are expressed to circumvent human host defenses and to regulate contact-dependent interactions of the organisms with host cells. Despite the important role of sialic acids in microbial pathogenesis, little is known about the genetic regulation of expression of these molecules in humans. As a paradigm for this process, the investigator seeks to identify how synthesis of the sialic acid capsules of groups B, C, Y and W-135 N. meningitidis are regulated during human infection. The investigator proposes that transcriptional control of two operons involved in capsule biosynthesis and transport is a major regulatory mechanism of the sialic acid capsules of N. meningitidis, and that environmental conditions in the human host and/or human cell factors influence the regulation. In two specific aims, the investigator proposes to explore the genetic basis for expression and transcriptional regulation of sialic acid by meningococci. In Aim I, the investigator will identify the promoters and regulatory elements of the 134bp intergenic region linking the divergent operons involved in CMP-NANA/sialic acid capsule biosynthesis and capsule membrane transport. This will be accomplished by transcriptional reporter gene constructs, and orchestrated mutagenesis of the intergenic region. Tn916 mutagenesis, in vitro mutagenesis and use of the Hermes plasmid, or E. coli complementation will then be used to identify transcriptional factors which regulate the biosynthesis and transport operons. In Aim II, the investigator proposes to investigate the expression, regulation and role in pathogenesis of capsule genes during specific events (nasopharyngeal colonization, bloodstream invasion) observed for meningococci during infection. The investigator will first study the regulation of capsule genes during different in vitro environmental growth conditions that mimic those the meningococcus would likely encounter during infection or colonization (pH, iron limitation, Ca++, temperature, and anaerobiosis). The investigator will then expand these studies to human nasopharyngeal organ cultures and human serum. Direct assessment of capsular polysaccharide by ELISA and immunomicroscopic techniques, genetically-defined isogenic mutants, transcriptional reporter gene constructs, and quantitation of capsule gene mRNA will be used. These studies have direct application to the development of new meningococcal vaccine strategies and should provide important information about the regulation of sialic acid in other important bacterial and biologic systems.