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

目前抗生素耐药性的时代要求采取新的战略来抗击细菌感染。接合是微生物交换遗传物质的一种自然机制。改变接合过程的目的可能有助于开发旨在对抗抗生素耐药性的新策略。我们的小组之前已经描述了一种接合质粒TP114，它在小鼠肠道中显示出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抗击细菌感染和开发微生物组编辑工具提供了新的途径。然而，关于结合质粒的宿主范围以及它们与供体细胞和受体细胞的相互作用，仍有几个问题。我们建议开发一种创新的高通量结合试验来表征TP114的宿主范围，并调查供体或受体细菌的遗传需求。在我们的高通量分析中，我们将系统地将TP114转移到大量菌株中，其中包括来自人类和动物微生物区系的病原体和共生菌，以及环境菌株。我们将进一步将TP114转移到KEIO文库中，这是一个包含所有可能的单基因缺失的大肠杆菌突变体库，以评估每个可选基因对接合过程的影响。为了进一步鉴定具有类似TP114特性的其他质粒，我们将调查一组具有代表性的接合质粒的宿主范围和所需的遗传决定因素，这些接合质粒属于革兰氏阴性细菌中最普遍的不相容组。最后，我们将测试结合质粒作为微生物组编辑工具的有效性。为此，我们将在克罗恩病小鼠模型中使用含有表达CRISPR/Cas9功能的修饰TP114的供体细菌来耗尽粘附性侵袭性大肠杆菌(AIEC)。我们的研究有望提供第一张具有巨大生物技术潜力的接合质粒宿主范围的全面图谱。此外，我们期望确定寄主的生理和新陈代谢对接合过程的贡献。因此，我们的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。我们的结果还可能导致防止水平基因转移和抗生素耐药基因传播的新策略。此外，该项目将提供一个极具刺激性和竞争性的培训机会，学员将使用最先进的设备和技术进行尖端研究。该项目将在UDES已经得到国际认可的细菌接合方面的专业知识的基础上，并显著提高其水平。它将使UdeS在细菌接合、水平基因转移和微生物组编辑方面处于领先地位。

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.