Factors Affecting longterm in vitro culture of Treponema pallidum

摘要 梅毒螺旋体和密切相关的细菌是梅毒，雅司病，贝杰尔（地方性）的病原体 梅毒）和品他。据估计，仅梅毒一项，全球就有1800万例患病，2016年， 美国报告的梅毒病例总数是1993年以来的最高记录。之一 研究T.梅毒螺旋体不能在体外连续培养螺旋体;它是一种 几种主要的细菌病原体（沿着麻风分枝杆菌）具有这种区别。上对初级 本项目的数据，我们报告了一致的，长期的乘法T。苍白球亚种苍白球 梅毒的病原体，在组织培养系统中。该培养体系由T.螺旋体 用兔上皮细胞在含有1.5% 氧气和5%的二氧化碳。T的指数乘法。苍白球已获得超过100天的持续 实验，具有良好的生存力（通过运动性测量）和感染性（通过 实验感染兔子）。T.苍白球发生与继代培养在6至7天的时间间隔 和周期性的介质交换，这被认为是保持接近稳态的条件， 培养物进入生长的稳定期。在这个项目中，我们建议继续和扩大 对影响T. pallidum增殖，并探讨其在T.螺旋体 研究，例如突变分析。具体目标是：1）确定最佳条件 为T.组织培养体系中的pallidum亚种和菌株; 2）利用组织 培养体系，明确营养需求，实现T.苍白球下 无菌条件（即没有哺乳动物细胞）;和3）检查体外遗传分析的可行性 培养T.通过化学诱变，用寡核苷酸转化，穿梭载体和 自杀质粒和转座子诱变。进一步证实和发展了T.苍白球 体外培养系统将有助于T.苍白球研究，包括生理学研究， 结构、抗菌药物敏感性、发病机制和宿主免疫。

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.