Factors Affecting longterm in vitro culture of Treponema pallidum

梅毒螺旋体长期体外培养的影响因素

• 批准号: 10000844
• 负责人: Diane G Edmondson
• 金额: $ 46.13万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-17 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10000844
• 关键词: Affect; Antimicrobial susceptibility; Atmosphere; Bacteria; Bejels; Carbon Dioxide; Cell division; Cells; Cellular Structures; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Cryoelectron Microscopy; DNA biosynthesis; Data; Dependence; Development; Disease; Dose; Environment; Epithelial Cells; Expression Profiling; Feasibility Studies; Funding; Gene Expression; Generations; Genes; Genetic; Genome; Globus Pallidus; Goals; Growth; Hares; Hour; Immunity; In Vitro; Infection; Inflammatory; Laboratories; Lesion; Maintenance; Mammalian Cell; Measures; Modification; Mutagenesis; Mutation; Mutation Analysis; Mycobacterium leprae; Nutrient; Nutritional Requirements; Oligonucleotides; Order Spirochaetales; Organism; Oryctolagus cuniculus; Oxygen; Parasites; Pathogenesis; Peptidoglycan; Periodicity; Phase; Physiology; Pinta; Plasmids; Prevalence; Procedures; Property; Reaction; Reporting; Research; Shuttle Vectors; Structure; Suicide; Syphilis; System; Testing; Testis; Time; Tissues; Treponema pallidum; Tube; United States; Writing; Yaws; axenic culture; cell motility; experimental study; feeding; genetic analysis; human pathogen; logarithm; member; pathogen; pathogenic bacteria; preservation; protein profiling; success; tissue culture

ABSTRACT Treponema pallidum and closely related bacteria are the causative agents of syphilis, yaws, bejel (endemic syphilis), and pinta. Syphilis alone has an estimated prevalence of 18 million cases worldwide, and in 2016 the total number of reported syphilis cases in the United States was the highest recorded since 1993. One of the barriers to research on T. pallidum has been the inability to culture the spirochete continuously in vitro; it is one of the few major bacterial pathogens (along with Mycobacterium leprae) with this distinction. In the preliminary data for this project, we report the consistent, long-term multiplication of T. pallidum subsp. pallidum, the causative agent of syphilis, in a tissue culture system. The culture system consists of co-incubation of T. pallidum with rabbit epithelial cells in a modified tissue culture medium under a microaerobic atmosphere containing 1.5% O2 and 5% CO2. Exponential multiplication of T. pallidum has been obtained for over 100 days in ongoing experiments, with excellent retention of viability (as measured by motility) and infectivity (examined by experimental infection of rabbits). Optimal replication of T. pallidum occurs with subculture at 6 to 7 day intervals and periodic medium exchange, which are thought to maintain near-homeostatic conditions and to minimize entrance of the cultures into the stationary phase of growth. In this project we propose to continue and expand studies on the factors affecting T. pallidum multiplication in this system and to explore its utility in T. pallidum research, as exemplified by mutational analysis. The specific aims are to 1) determine the optimal conditions for continuous growth of T. pallidum subspecies and strains in the tissue culture system; 2) utilize the tissue culture system to define the nutritional requirements with the purpose of achieving culture of T. pallidum under axenic conditions (i.e. without mammalian cells); and 3) examine the feasibility of genetic analysis of in vitro cultured T. pallidum through chemical mutagenesis, transformation with oligonucleotides, shuttle vectors and suicide plasmids, and transposon mutagenesis. The further confirmation and development of the T. pallidum in vitro culture system will likely facilitate many aspects of T. pallidum research, including studies of physiology, structure, antimicrobial susceptibility, pathogenesis, and host immunity.

抽象的 treponema pallidum和密切相关的细菌是梅毒，偏航，bejel的病因（流行 梅毒）和pinta。仅梅毒就在全球范围内估计有1800万例，2016年 美国报告的梅毒病例总数是自1993年以来的最高记录。 对pallidum的研究的障碍是无法在体外持续培养螺旋体。这是一个 在少数主要的细菌病原体（以及麻风分枝杆菌）中，有这种区别。在初步 该项目的数据，我们报告了T. pallidum subsp的一致的长期乘法。 Pallidum， 在组织培养系统中的梅毒的病因。培养系统包括pallidum t. t. t. 在含有1.5％ O2和5％CO2。在正在进行中，已获得超过100天的pallidum的指数乘法 实验，具​​有极好的生存能力（通过运动性衡量）和感染性（通过 兔子的实验感染）。在6至7天的间隔中，pallidum的最佳复制在亚文化中发生 和周期性的媒介交换，这些交换被认为维持近彻底的条件并最小化 培养物进入生长的固定阶段。在这个项目中，我们建议继续并扩展 研究影响该系统中粒子繁殖的因素，并探索其在T. pallidum中的效用 研究，例如突变分析的例证。具体目的是1）确定最佳条件 为了持续生长组织培养系统中的粒子亚种和菌株； 2）利用组织 培养系统来定义营养需求，目的是实现T. pallidum的培养 轴原条件（即没有哺乳动物细胞）； 3）检查体外遗传分析的可行性 通过化学诱变，寡核苷酸的转化，穿梭载体和 自杀质粒和转座子诱变。在 体外培养系统可能会促进T. pallidum研究的许多方面，包括生理学研究， 结构，抗菌敏感性，发病机理和宿主免疫。