Caracterización bioquímica y estructural de la maquinaria conjugativa del plásmido R388 para un diseño racional de inhibidores

• Autores: Tamara Menguiano Vázquez
• Directores de la Tesis: Ignacio Arechaga Iturregui (dir. tes.), María Elena Cabezón Navarro (dir. tes.)
• Lectura: En la Universidad de Cantabria ( España ) en 2025
• Idioma: inglés
• Títulos paralelos:
  • Biochemical and structural characterization of the R388 plasmid conjugative machinery for a rational design of inhibitors
• Tribunal Calificador de la Tesis: Mario Mencía Caballero (presid.), María Jesús Lucas Gay (secret.), Alejandro Peña Ontalvilla (voc.)
• Programa de doctorado: Programa de Doctorado en Biología Molecular y Biomedicina por la Universidad de Cantabria
• Enlaces
  • Tesis en acceso abierto en:  UCrea  UCrea 
• Resumen
  • español

    La conjugación es el principal mecanismo de transferencia horizontal de genes responsable de la diseminación de genes de resistencia a antibióticos. Por tanto, la inhibición de este proceso es fundamental para frenar la propagación de bacterias multirresistentes. En este trabajo describimos nuevos inhibidores de la conjugación bacteriana. Además, presentamos la caracterización bioquímica de varios subcomplejos del relaxosoma conjugativo y sus interacciones con componentes del Sistema de Secreción Tipo IV. Estos nuevos hallazgos ofrecen una comprensión más detallada de los mecanismos que subyacen a la conjugación bacteriana y respaldan el desarrollo de nuevos inhibidores eficaces.

  • English

    Conjugation is the main mechanism of horizontal gene transfer responsible for the dissemination of antibiotic resistance genes. This process requires the coordinated action of three key complexes in the bacterial donor cell: the relaxosome, the Type IV Secretion System (T4SS), and the pilus. The energy required for the assembly of the T4SS and substrate transport is provided by specific ATPases (TrwB, TrwC, TrwD, and TrwK in the R388 plasmid). The R388 relaxosome, a DNA-processing machinery, consists of the relaxase TrwC, the accessory protein TrwA, the integration host factor IHF, and the plasmid origin of transfer (oriT). TrwB, the Type IV Coupling Protein (T4CP) of the R388 plasmid, acts as the connector between the T4SS and the translocated substrate by interacting with both the T4SS and the relaxosome. In this work, we have been able to track the localization of the R388 ATPases that power conjugation, in the presence and absence of recipient cells, using fluorescence microscopy. The structure of the R388 relaxosome, as well as the functional implications of the TrwB-TrwC interaction during conjugation, remain unknown. Here, various subcomplexes of the relaxosome have been purified, and their structural characterization would allow for the modeling of the entire relaxosome. Furthermore, the effect of TrwC on TrwBΔN70 ATPase activity has been analyzed, demonstrating that TrwC inhibits the ATPase activity of TrwBΔN70, possibly as an energy-saving mechanism in the absence of conjugation. The components of the conjugative machinery represent key targets for new inhibition strategies. In this context, two novel inhibitors of conjugation, Benserazide and a Benzenesulfonamide derivative, have been identified, and the established R388 conjugation inhibitor, 2-Bromopalmitic acid, has also been shown to significantly reduce the conjugation of F plasmids, which are of high clinical relevance due to their widespread distribution among pathogenic Enterobacteriaceae. As an alternative inhibitory strategy, two R388 fertility inhibitors, E-Factor and FiwA, have been found to interact in vitro with the T4CP TrwBΔN70. Altogether, these new findings provide deeper insight into the mechanisms underlying bacterial conjugation and support the development of new effective inhibitors, which may be crucial in the fight against multidrug-resistant bacteria.