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

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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.