Mechanisms of propagation of antibiotic resistance-conferring mobile genetic elements in Enterobacteriaceae and Vibrionaceae

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Knowledge of the genetic determinants and molecular mechanisms that drive the mobility of antibiotic resistance genes is essential to fight the emergence and spread of multidrug resistant bacteria. The integrative and conjugative elements of the SXT/R391 family (SRIs) and the conjugative plasmids of the A/C incompatibility group (ACPs), are major vectors of multidrug resistance genes in pathogenic Enterobacteriaceae and Vibrionaceae. SRIs is responsible for antibiotic resistance of the seventh pandemic lineage of Vibrio cholerae, the etiologic agent of cholera, which remains a major cause of mortality and morbidity on a global scale. Meanwhile, reports associating ACPs with extended-spectrum ß-lactamases and carbapenemases are unsettlingly rising worldwide in enteric pathogens recovered from the environment, food-producing animals, food products and humans. ACPs also facilitate the propagation of the multidrug resistance-conferring Salmonella Genomic Island 1 (SGI1). Because ACPs and SRIs derive from a common ancestor, they code for highly similar conjugative transfer machineries, which mediate the translocation of genetic material from donor to recipient cells. Currently, the molecular mechanisms mediating these transfers are not well understood. Translocation of ACP and SRI DNA is initiated at the origin of transfer (oriT) by the relaxase TraI and the auxiliary protein MobI, both encoded by ACPs and SRIs. TraI belongs to an atypical family of relaxases, which are predicted HD phosphodiesterases and seems to require a MobI-like auxiliary protein to function, suggesting a completely unique and novel mechanism of conjugative transfer initiation. We anticipate that TraI and MobI are key factors involved in the ACP-mediated transfer of genomic islands such as SGI1. The main goal of this proposal is to characterize the mechanism of conjugative transfer initiation of ACPs and SRIs, and the molecular interactions between the conjugative machinery of ACPs and genomic islands that they mobilize. More specifically, we propose to identify all the partner proteins that are involved in DNA processing at oriT. Moreover, we aim at discovering the protein translocation signals that allow recognition and secretion of proteins through the type IV secretion system encoded by ACPs and SRIs. We will also functionally map with single-nucleotide accuracy the oriT to determine what specific sequence features promote assembly of a high-order complex at this locus. Finally, we will investigate the interactions between the ACP-encoded relaxosome and genomic islands that are mobilized by ACPs. Our results will yield fundamental insights about the basic biology ACPs and SRIs, thereby providing the scientific community with strong foundations and new potential molecular targets that could be used for the development of tools aimed at tackling the progression of multidrug resistance.