Estudio comparativo de los dominios transportadores de los sistemas de secreción tipo V de protobacterias y su aplicación en la presentación de anticuerpos en la superficie de E. coli

• Autores: Elvira Marín Muñoz
• Directores de la Tesis: Luis Ángel Fernández Herrero (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2009
• Idioma: español
• Tribunal Calificador de la Tesis: Marta Izquierdo Rojo (presid.), Maria Graciela Pucciarelli (secret.), José Manuel Palacios Alberti (voc.), Kim R. Hardie (voc.), Hilde De Reuse (voc.), Carmen del Rosario Beuzón López (voc.), Antonio Tormo Garrido (voc.)
• Materias:
  • Ciencias de la vida
    • Biología molecular
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Type V secretion system (T5SS) are the largest group of secreted proteins in Gramnegative bacteria and include adhesins, proteases and cytotoxins of important pathogens. The distinct feature of these secreted proteins is their ability to ¿selftranslocate¿ across the bacterial cell envelope in the absence of dedicated protein machineries. T5SS include the secreted proteins referred to as autotransporters (AT) and the Intimin/Invasins.

    This thesis investigates the secretion mechanism of T5SS through a comparative study of the structural and functional properties of the transport domains from different ATs and Intimin and of their interactions with the bacterial machinery involved in the folding and insertion of outer membrane proteins (OMPs). The predicted transport domain of selected ATs from ¿- ß- ¿- and ¿-proteobacteria and Intimin were expressed in E. coli wild type and mutant strains in the major periplasmic chaperones (SurA, DegP, Skp, FkpA) and the essential BamA (YaeT/Omp85) outer membrane protein (OMP) involved in the biogenesis of OMPs with ß¿barrel structure. It is shown that ATs from ¿- ß- and ¿-groups are able to fold and transport peptides to E. coli surface, whereas AT from ¿-proteobacteria could not be expressed in a functional form. All transport domains from ATs require an N-terminal ¿-helix for stability and transport function and form monomers and/or dimers in vitro and in vivo, except for the IgA protease from Neisseria gonorrhoeae that forms a larger oligomer. Intimin transport domain forms a stable dimer. It is also shown that SurA play a role the periplasmic transit and folding of most ATs studied, although the alternative Skp/DegP pathway is used in some cases. Importantly, insertion into the OM of all members of the T5SS is blocked when both periplasmic pathways are inactivated in conditional double mutants. Outer membrane insertion of members of the T5SS is inhibited when BamA is depleted from bacteria, indicating the involvement of the Bam complex in this process. In addition, it is shown the ability of the transport domains of T5SS for the display in the surface of E. coli of single-domain antibodies known as Nanobodies in functional form able to bind their cognate antigen. Transport domains showing optimal display levels were use in magnetic cell sorting (MACS) to select bacteria displaying Nanobodies with high affinity and specificity for a given antigen.