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

了解推动抗生素耐药基因传播的分子机制对于抗击多重耐药细菌的出现至关重要。不相容C组结合质粒(IncC)是许多肠杆菌科和弧菌科致病菌种多重耐药的关键因素。在全球范围内，从环境、食品、食品类动物、人类及其宠物中回收的肠道病原体中，将IncC质粒与超广谱β-内酰胺酶和碳青霉烯酶联系在一起的报告正在令人不安地上升。IncC质粒还驱动与多重耐药相关的基因组岛的繁殖，如沙门氏菌基因组岛1(SGI1)，它充当IncC质粒的寄生虫。IncC质粒编码为接合转移机制，也称为IV型分泌系统，将供体的遗传物质(DNA)分泌到受体细胞中。目前，这种细胞间DNA转移的分子机制还不是很清楚。DNA的转位是由松弛酶TRAI启动的，它属于HD磷酸二酯酶家族，需要MOBI辅助蛋白。由HD磷酸二酯酶启动的接合转移是不寻常的，提示了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，对细菌基因组数据库的数据挖掘表明，IncC质粒可以推动一个全新的基因组岛家族的传播，这些基因组岛与SGI1有遥远的亲缘关系，来自或到达广泛的细菌物种。这一建议的主要目的是描述IncC质粒的接合转移起始机制，以及SGI1和相关基因组岛干扰这一过程的途径。更具体地说，我们的目标是确定所有参与ORIT的DNA加工和DNA分泌到受体细胞的蛋白质，以及允许这些蛋白质通过IncC质粒编码的IV型分泌系统识别和分泌的蛋白质易位信号。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质粒在假设的SGI1样基因组岛移动中的作用，这些基因组岛广泛存在于海洋微生物的基因组中。这项研究将产生关于基本生物学IncC质粒的基本见解，并填补它们在促进无关基因组岛转移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.