Universal in vitro culture model as a replacement strategy to study pathogenic treponemes

通用体外培养模型作为研究致病性密螺旋体的替代策略

• 批准号: BB/X010589/1
• 负责人: Linda Grillova
• 金额: $ 51.8万
• 依托单位: Wellcome Sanger Institute
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX010589%2F1
• 关键词: Universal; vitro; culture; model; replacement

Pathogenic treponemes are bacteria causing distinct diseases such as syphilis, yaws, bejel and atypical treponematosis in humans and in some animals. Despite causing severe life-threatening infections, very little is known about their basic biology and pathogenesis. This is mainly because these bacteria are generally considered to be unculturable. Treponemes have a very small genome that lacks multiple metabolic pathways necessary for the de novo biosynthesis of various co-factors, fatty acids, and nucleotides. So, to survive and grow, these bacteria need to obtain their essential nutrients through interaction with their host cells. Therefore, a culture system that provides all of these environmental requirements should, in theory, be able to sustain the growth of these bacteria, in vitro. However, despite rigorous attempts by the scientific community, so far, the optimisation of an axenic culture system has been unsuccessful. Therefore, for decades, large number of rabbits have been used to propagate clinically relevant treponemes. Only recently, after decades of experiments the first successful long-term cultivation of pathogenic treponemes co-cultured with rabbit skin epithelial cells (Sf1Ep) was introduced. Although technically challenging and requiring state-of-the-art technical skills, this system has opened new avenues for the study of the basic biology of pathogenic treponemes. Unfortunately, due to a very high number of treponemal cells needed to initiate the culture, the Sf1Ep co-culture system remains restricted only to rabbit-propagated strains, and cannot be used for the propagation of new strains. Moreover, even when a high number of viable treponemes are available, for unknown reasons, some treponemal strains remain unculturable under these conditions, limiting the scopes of treponemal research to only a few reference strains. The ultimate objective of this proposal is to generate high-quality multi-omics data to systematically obtain the necessary information for the optimisation of a universal culture condition that could support the growth and propagation of multiple different treponemal strains with limited number of viable bacteria to initiate the culture. This will not only replace the use of animals for treponemal expansion and syphilis diagnosis, but will also allow us to study the basic biology of pathogenic treponemes, their pathogenesis, routes of transmission and alternative treatments.I am aiming to using the Sf1Ep co-culture system to track the longitudinal changes in genome-wide expression patterns of treponemes during the adaptation from in vivo into the in vitro conditions. Using these transcriptional data combined with subsequent phenotypic profiling, I will model metabolic pathways and identify the metabolic interaction network of Sf1Ep cells and treponemes. Based on these observations, I will identify the essential nutrients and requirements that are crucial for the axenic culture of pathogenic treponemes. Subsequently, I will evaluate the effect of the medium supplements on the growth and viability of treponemes both in the presence and absence of Sf1Ep cells. Alternatively, I will also explore the potential of improving the co-culture system by either supplementing the Sf1Ep cells with essential nutrients or by replacing the Sf1Ep cells with other mammalian cells which may be more effective at supporting the in vitro growth of treponemes.Our success in this project will not only pave the way for the basic science research of these fastidious bacteria, but will also significantly reduce the number of rabbits that are sacrificed for treponemal propagation and diagnosis. Furthermore, our proposed systematic pipeline could also set precedence for other similar microorganisms, translating into a vast 3Rs impact. Finally, alongside the main objectives, this project will also provide new important insights regarding the biology, physiology and genetics of pathogenic treponemes.

致病性密螺旋体是在人类和一些动物中引起不同疾病如梅毒、雅司病、贝杰尔病和非典型密螺旋体病的细菌。尽管引起严重的危及生命的感染，但对其基本生物学和发病机制知之甚少。这主要是因为这些细菌通常被认为是不可培养的。密螺旋体具有非常小的基因组，其缺乏各种辅因子、脂肪酸和核苷酸的从头生物合成所必需的多种代谢途径。因此，为了生存和生长，这些细菌需要通过与宿主细胞的相互作用获得必需的营养物质。因此，提供所有这些环境要求的培养系统在理论上应该能够在体外维持这些细菌的生长。然而，尽管科学界进行了严格的尝试，但迄今为止，无菌培养系统的优化一直不成功。因此，几十年来，大量的兔子被用于传播临床相关的密螺旋体。直到最近，经过几十年的实验，第一个成功的长期培养致病性密螺旋体与兔皮肤上皮细胞（Sf1Ep）共培养。虽然在技术上具有挑战性，需要最先进的技术技能，该系统已经打开了致病密螺旋体的基础生物学研究的新途径。不幸的是，由于启动培养需要非常高数量的密螺旋体细胞，Sf1Ep共培养系统仍然仅限于兔繁殖的菌株，并且不能用于新菌株的繁殖。此外，即使有大量的可行的密螺旋体，由于未知的原因，一些密螺旋体菌株仍然无法在这些条件下培养，限制了密螺旋体研究的范围，只有少数参考菌株。该提案的最终目标是生成高质量的多组学数据，以系统地获得优化通用培养条件所需的信息，该通用培养条件可以支持多种不同密螺旋体菌株的生长和繁殖，其中有限数量的活菌用于启动培养。这不仅将取代使用动物的密螺旋体扩张和梅毒诊断，但也将使我们能够研究致病性密螺旋体的基本生物学，其发病机制，传播途径和替代treatment.I的目的是使用Sf1Ep共培养系统跟踪纵向变化的全基因组表达模式的密螺旋体在适应从体内到体外条件。使用这些转录数据结合随后的表型分析，我将模拟代谢途径，并确定Sf1Ep细胞和密螺旋体的代谢相互作用网络。基于这些观察，我将确定致病性密螺旋体的纯培养所必需的营养素和要求。随后，我将评估在Sf1Ep细胞存在和不存在的情况下，培养基补充剂对密螺旋体生长和活力的影响。另外，我还将探索通过补充Sf1Ep细胞的必需营养素或用其他哺乳动物细胞替代Sf1Ep细胞来改善共培养系统的潜力，这些细胞可能更有效地支持密螺旋体的体外生长。我们在这个项目中的成功不仅将为这些难养细菌的基础科学研究铺平道路，而且还将显著减少为密螺旋体繁殖和诊断而牺牲的兔子的数量。此外，我们提出的系统管道也可以为其他类似的微生物设定优先级，转化为巨大的3R影响。最后，除了主要目标，该项目还将提供有关致病性密螺旋体的生物学，生理学和遗传学的新的重要见解。