Initiation of conjugative transfer of IncC conjugative plasmids and associated genomic islands circulating in Gammaproteobacteria

了解驱动抗生素耐药基因传播的分子机制对于对抗多重耐药细菌的出现至关重要。不相容群C （incompatibility group C, IncC）的共轭质粒是肠杆菌科和弧菌科多种病原菌产生多药耐药的关键因素。在世界范围内，有关IncC质粒与广谱ß-内酰胺酶和碳青霉烯酶相关的报告越来越多，令人不安地出现在从环境、食品、食品生产动物、人类及其宠物中回收的肠道病原体中。IncC质粒还推动具有多重耐药性的基因组岛的繁殖，如沙门氏菌基因组岛1 (SGI1)，它作为IncC质粒的寄生虫。IncC质粒编码一种共轭转移机制，也被称为IV型分泌系统，它将遗传物质（DNA）从供体分泌到受体细胞。目前，介导这种细胞间DNA转移的分子机制尚不清楚。DNA的易位是由属于HD磷酸二酯酶家族的松弛酶TraI在转移起点（oriT）启动的，需要MobI辅助蛋白。由HD磷酸二酯酶引发的共轭转移是不寻常的，这表明一种独特而新颖的机制需要揭示。SGI1可能通过抑制TraI和MobI之间的相互作用来干扰IncC质粒转移的启动。此外，细菌基因组数据库的数据挖掘表明，IncC质粒可以推动与SGI1远亲的一个全新的基因组岛家族在广泛的细菌物种中传播。本研究的主要目的是表征IncC质粒共轭转移启动的机制，以及SGI1和相关基因组岛干扰这一过程的途径。更具体地说，我们的目标是鉴定所有参与oriT DNA加工和DNA分泌到受体细胞中的蛋白质，以及允许通过IncC质粒编码的IV型分泌系统识别和分泌这些蛋白质的蛋白质易位信号。我们还将确定SGI1干扰这些功能的机制，以确保其自身的繁殖和IncC质粒的不稳定。最后，我们将研究IncC质粒在广泛存在于海洋微生物基因组中的假定的sgi1样基因组岛的迁移中的作用。该研究将对IncC质粒的基础生物学产生根本性的见解，并填补其在促进不相关基因组岛转移中的作用方面的重要知识空白。从长远来看，这些研究将为发现新的潜在分子靶点提供坚实的基础，这些靶点可用于开发旨在应对多药耐药进展的工具。

Knowledge of the molecular mechanisms that drive the dissemination of antibiotic resistance genes is essential to fight the emergence of multidrug-resistant bacteria. Conjugative plasmids of the incompatibility group C (IncC) are key contributors to the multidrug resistance of many pathogenic species of Enterobacteriaceae and Vibrionaceae. Reports associating IncC plasmids with extended-spectrum ß-lactamases and carbapenemases are unsettlingly rising worldwide in enteric pathogens recovered from the environment, food products, food-producing animals, humans and their pets. IncC plasmids also drive the propagation of multidrug resistance-conferring genomic islands such as the Salmonella genomic island 1 (SGI1) that acts as a parasite of IncC plasmids. IncC plasmids code for a conjugative transfer machinery, also known as a type IV secretion system, that secretes genetic material (DNA) from a donor into a recipient cell. Currently, the molecular mechanisms mediating this intercellular DNA transfer are not well understood. Translocation of DNA is initiated at the origin of transfer (oriT) by the relaxase TraI that belongs to a family of HD phosphodiesterases and requires a MobI auxiliary protein. Conjugative transfer initiated by an HD phosphodiesterase is unusual and suggests a unique and novel mechanism that needs to be revealed. SGI1 interferes with the initiation of transfer of IncC plasmids likely by inhibiting the interaction between TraI and MobI. Furthermore, data mining of bacterial genome databases suggests that IncC plasmids can drive the dissemination of a completely new family of genomic islands distantly related to SGI1 from and to a broad range of bacterial species. The main goal of this proposal is to characterize the mechanism of conjugative transfer initiation of IncC plasmids, and the pathways used by SGI1 and related genomic islands to interfere with this process. More specifically, our objectives are to identify all the proteins involved in DNA processing at oriT and the secretion of DNA into the recipient cells as well as the protein translocation signals that allow recognition and secretion of these proteins through the type IV secretion system encoded by IncC plasmids. We will also determine the mechanisms by which SGI1 interferes with these functions to ensure its own propagation and the destabilization of the IncC plasmids. Finally, we will investigate the role of IncC plasmids in the mobility of putative SGI1-like genomic islands that are widespread in the genome of marine-dwelling microorganisms. This study will yield fundamental insights about the basic biology IncC plasmids and fill important knowledge gaps in their role in promoting transfer of unrelated genomic islands. On the long term, these studies will provide strong foundations for the discovery of new potential molecular targets that could be used for the development of tools aimed at tackling the progression of multidrug resistance.