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，表达在 脑膜炎奈瑟菌、无乳链球菌、大肠杆菌表面 ColiK1和淋病奈瑟菌作为衣壳多糖和/或末端 N-乙酰神经氨酸(NANA)残基附着在脂寡糖上。 唾液酸的表达是为了绕过人类宿主的防御并调节 生物体与宿主细胞的接触依赖的相互作用。尽管 唾液酸在微生物发病机制中的重要作用 已知这些分子在体内表达的遗传调节 人类。作为这一过程的范例，调查者试图确定 B、C、Y和W-135N组唾液酸胶囊的合成。 脑膜炎在人类感染期间受到调节。调查员 提出囊膜中两个操纵子的转录调控 唾液酸的生物合成和转运是一种主要的调节机制 脑膜炎奈瑟氏菌的酸性胶囊，以及环境条件 人类宿主和/或人类细胞因素影响这一调节。分成两份 为了达到特定的目的，研究人员建议探索其遗传基础 脑膜炎双球菌对唾液酸的表达和转录调控。 在AIM I中，调查员将确定发起人和监管机构 连接发散操纵子的134bp基因间隔区的元件 参与CMP-NaNA/唾液酸胶囊的生物合成和胶囊膜 运输。这将通过转录报告基因来完成 构建，并对基因间隔区进行精心策划的突变。Tn916 Hermes质粒的诱变、体外诱变和使用 然后，互补将被用来识别转录因子，这些转录因子 调节生物合成和运输操纵子。在AIM II中， 研究人员建议调查该基因的表达、调控和作用 特定事件(鼻咽部)中被膜基因的发病机制 定植、血流侵袭)观察脑膜炎双球菌 感染。调查者将首先研究胶囊的调节 基因在不同的体外环境生长条件下模拟 脑膜炎双球菌在感染期间或 定植(pH、铁限制、Ca++、温度和厌氧)。 然后研究人员将把这些研究扩展到人类鼻咽部 器官培养和人血清。对包膜的直接评估 用酶联免疫吸附试验和免疫显微镜技术检测多糖， 基因定义的等基因突变体，转录报告基因 构建，并定量的胶囊基因的mRNA将被使用。这些 研究对开发新的脑膜炎双球菌有直接应用 疫苗策略，并应提供有关 唾液酸在其他重要细菌和生物系统中的调节。

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.