ANAEROBIOSIS, CMP-NANA AND GONOCOCCAL PATHOGENESIS

厌氧、CMP-NANA 和淋球菌发病机制

• 批准号: 6031869
• 负责人: RICHARD REST
• 金额: $ 10.01万
• 依托单位: MCP HAHNEMANN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-06-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6031869
• 关键词: N acetylneuraminate; Neisseria gonorrhoeae; anaerobiosis; antibacterial antibody; bacterial cytopathogenic effect; bacterial proteins; binding proteins; bioassay; cytosine nucleotides; enzyme activity; enzyme induction /repression; enzyme mechanism; gene expression; genetic library; gonorrhea; laboratory mouse; laboratory rabbit; lipopolysaccharides; method development; molecular cloning; nucleic acid sequence; nucleic acid structure; protein purification; sialyltransferases; site directed mutagenesis

Neisseria gonorrhoeae (the gonococcus, gonococci, gc) infects about 2 million civilians a year in the USA, and several millions more in the rest of the world. In women, untreated cervical disease (gonorrhea) often ascends to the endometrium and fallopian tubes to cause pelvic inflammatory disease (PID), which in turn can lead to fallopian tube scarring, sterility and ectopic pregnancy. Each year in the USA, treating gonorrhea, PID and their sequelae costs hundreds of millions of dollars. In addition to helping prevent and treat gonococcal infections and disease and their sequelae, studying gonococcal pathogenesis is important to help us better understand other mucosal pathogens, including those causing serious respiratory and meningeal infections (e.g., meningitis caused by N. meningitidis), and to help us better understand other sexually transmitted diseases. Gonococci grown in vitro interact with host defenses, including serum, epithelial cells and phagocytes, quite differently than do gc grown in vivo. The goal of the research presented in this proposal is to investigate the mechanisms by which gc modulate their lipooligosaccharide (LOS) under conditions thought to exist in vivo, i.e. in the presence of cytidine monophosphate N-acetylneuraminic acid (CMP-NANA) and under decreased oxygen tension (anaerobiosis). We have observed that anaerobiosis acts synergistically with low concentrations of CMP-NANA to induce high level serum resistance in gc. Serum resistance is due (at least in part) to the facile sialylation of LOS by gonococcal sialyltransferase (STase) using host CMP-NANA. We observed that anaerobic gc, compared to aerobic gc, express increased STase activity and altered LOS. To our knowledge, this is the first observation of the regulation of gonococcal virulence factors by conditions thought to exist in vivo (other than iron). To began to explain these observations, I propose the following SPECIFIC AIMS: 1. Develop a rapid, quantitative assay to measure gonococcal sialyltransferase (STase) activity. 2. Identify, clone, sequence and analyze the gonococcal STase gene(s), or genes associated with the expression of STase activity. 3. Purify and biochemically characterize the gonococcal protein(s) responsible for STase activity. 4. Begin investigations to determine the degree, level (transcriptional, translational, or post translational) and mechanism(s) of regulation of gonococcal STase activity or expression by anaerobiosis.

淋病奈瑟菌（淋球菌、淋球菌、gc）感染约 2 美国每年有数百万平民，其他地区还有数百万平民 世界的。在女性中，未经治疗的宫颈疾病（淋病）常常 上升至子宫内膜和输卵管引起盆腔 炎症性疾病 (PID)，进而导致输卵管病变 疤痕、不育和宫外孕。每年在美国，治疗 淋病、盆腔炎及其后遗症花费数亿美元。 除了帮助预防和治疗淋球菌感染和疾病 及其后遗症，研究淋球菌发病机制对于帮助 我们更好地了解其他粘膜病原体，包括那些引起 严重的呼吸道和脑膜感染（例如，由以下原因引起的脑膜炎） 脑膜炎奈瑟球菌），并帮助我们更好地了解其他性行为 传播疾病。 体外生长的淋球菌与宿主防御相互作用，包括血清、 上皮细胞和吞噬细胞，与生长的 gc 完全不同 体内。本提案中提出的研究目标是 研究GC调节脂寡糖的机制 （LOS）在被认为存在体内的条件下，即存在 胞苷一磷酸 N-乙酰神经氨酸 (CMP-NANA) 及以下 氧张力降低（厌氧）。我们观察到 厌氧与低浓度 CMP-NANA 协同作用 在GC中诱导高水平的血清抵抗。血清抵抗是由于（在 至少部分）淋球菌对 LOS 的轻松唾液酸化 使用宿主 CMP-NANA 的唾液酸转移酶 (STase)。我们观察到厌氧 与有氧 gc 相比，gc 表达增加的 STase 活性并改变 视距。据我们所知，这是监管部门首次观察到 淋球菌毒力因素被认为存在于体内的条件（其他 比铁）。 为了开始解释这些观察结果，我提出以下具体建议 目的： 1. 开发一种快速、定量的检测方法来测量淋球菌 唾液酸转移酶（STase）活性。 2. 鉴定、克隆、测序和 分析淋球菌 STase 基因或与淋球菌 STase 相关的基因 STase 活性的表达。 3. 纯化并进行生化表征 负责 STase 活性的淋球菌蛋白。 4. 开始 调查以确定程度、水平（转录、 翻译或翻译后）和监管机制 淋球菌 STase 活性或厌氧表达。