Systematic evaluation of the host range and host cell requirements for Gram-negative conjugative plasmids with biotechnological potential

当前抗生素耐药性的时代需要新的策略来对抗细菌感染。结合是微生物交换遗传物质的一种自然机制。重新利用偶联过程可以帮助开发旨在对抗抗生素耐药性的新策略。我们的团队之前已经描述了一种共轭质粒TP114，在小鼠肠道内显示了令人印象深刻的体内转移率。这种质粒在体内高速转移的能力为对抗细菌感染和开发微生物组编辑工具提供了新的途径。然而，关于宿主范围的共轭质粒及其与供体和受体细胞的相互作用，仍存在一些问题。我们建议开发一种创新的高通量偶联实验来表征TP114的宿主范围，并研究供体或受体细菌的遗传需求。在我们的高通量实验中，我们将系统地将TP114转移到大量菌株中，包括来自人类和动物微生物群的病原体和共生菌，以及环境菌株。我们将进一步将TP114转移到Keio集合中，这是一个包含所有可能的单基因缺失的大肠杆菌突变文库，以评估每个必要基因对偶联过程的影响。为了进一步鉴定其他与TP114性质相似的质粒，我们将研究属于革兰氏阴性菌中最普遍的不相容群的一组具有代表性的共轭质粒的宿主范围和所需的遗传决定因素。最后，我们将测试共轭质粒作为微生物组编辑工具的功效。为了做到这一点，我们将使用含有表达CRISPR/Cas9功能的修饰TP114的供体细菌来消耗克罗恩病小鼠模型中的粘附-侵袭性大肠杆菌（AIEC）。我们的研究有望为具有巨大生物技术潜力的共轭质粒提供第一张宿主范围的综合地图。此外，我们期望确定宿主的生理和代谢对偶联过程的贡献。因此，我们的研究结果将有助于设计新的微生物工程工具，这些工具可以用于创新的方法来操纵微生物组。我们的结果也可能导致新的策略，以防止水平基因转移和抗生素耐药基因的传播。此外，该项目将提供一个高度激励和竞争的培训机会，受训者将使用最先进的设备和技术进行前沿研究。该项目将建立并显著提高华盛顿大学在国际公认的细菌偶联方面的专业知识。它将使UdeS成为细菌偶联、水平基因转移和微生物组编辑领域的领导者。

The current age of antibiotic resistance calls for new strategies to combat bacterial infections. Conjugation is a natural mechanism by which microorganisms exchange genetic material. Repurposing the conjugation process could help the development of novel strategies aimed at fighting antibiotic resistance. Our group has previously described a conjugative plasmid, TP114, showing impressive transfer rates in vivo in the mouse intestinal tract. The ability of such plasmids to transfer in vivo at high rates provides new avenues to fight bacterial infections and develop microbiome editing tools. However, several questions remain regarding the host range of conjugative plasmids and their interactions with donor and recipient cells. We propose to develop an innovative high-throughput conjugation assay to characterize the host range of TP114 and investigate the genetic requirements of donor or recipient bacteria. In our high-throughput assay, we will systematically transfer TP114 into a vast collection of strains that includes pathogens and commensals from the human and animal microbiota, as well as environmental strains. We will further transfer TP114 into the Keio collection, an Escherichia coli mutant library comprising all possible single gene deletions, to assess the influence of every dispensable gene on the conjugation process. To further identify other plasmids with properties similar to TP114, we will investigate the host range and required genetic determinants of a representative set of conjugative plasmids belonging to the most prevalent incompatibility groups in Gram-negative bacteria. Finally, we will test the efficacy of conjugative plasmids as a microbiome editing tool. To do this, we will use a donor bacterium containing a modified TP114 expressing CRISPR/Cas9 functions to deplete Adherent-Invasive Escherichia coli (AIEC) in a Crohn's disease mouse model.Our study is expected to provide the first comprehensive map of host ranges for conjugative plasmids with great biotechnological potential. In addition, we anticipate identifying the contribution of the host's physiology and metabolism to the conjugation process. Hence our results will be helpful to design novel microbial engineering tools that can be used in innovative approaches to manipulate microbiomes. Our results could also lead to new strategies to prevent horizontal gene transfer and the dissemination of antibiotic resistance genes. In addition, this project will provide a highly stimulating and competitive training opportunity, in which trainees will use state-of-the-art equipment and technologies to perform cutting-edge research. This project will build on and significantly improve the expertise at UdeS on bacterial conjugation that is already recognized internationally. It will position UdeS as a leader in bacterial conjugation, horizontal gene transfer and microbiome editing.