Caracterización funcional y estructural de LMM-PBP4 y análisis de su papel moderador en la integridad molecular del peptidoglicano para el modelo bacteriano "Pseudomonas aeroginosa"

Tesis doctoral inédita leída en la Universidad Aautónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 23-05-2015Nosocomial and community-acquired infections by Pseudomonas aeruginosa continue to represent a major therapeutic challenge where the choice of an appropriate antibiotic is essential. Resistance of this pathogen against β-lactam antibiotics is mainly determined by the production of inactivating enzymes (β-lactamases) AmpC type whose regulatory mechanism is much more complex than the existing in members of the Enterobacteriaceae family, by including the protein AmpR responsible for regulation of additional genes in regards to ampC, such as ampE and creD as well as dacB, involved in the overproduction of this cephalosporinase. The connection between the replacement and recycling processes of peptidoglycan and its regulatory pathways for constitutive overproduction of chromosomal AmpC β-lactamase, particularly the mechanism associated with inactivation of PBP4, raises a need for detailed structural and functional research of this LMM-PBP class C subclass C1 in Pseudomonas aeruginosa that allows to establish similarities and differences with other low molecular mass penicillin-binding proteins in this bacterial model and between functional orthologs from related species, also evaluating its participation in the metabolism of cell wall and the biology of resistance to β-lactam antibiotics. The methodological approach designed and implemented to address this Thesis demonstrated the lack of β-lactamase activity for the LMM-PBP4 native form, the presence of DD-carboxypeptidase and DD-endopeptidase activities in this protein (this last was defined as the predominant activity) and its absence at the site of cell division discarding its association with septal and bacterial divisome proteins. Its subcellular localization in the inner membrane and periplasmic space strengthens its proposal as a potential hydrolase-autolysin associated with the maturationrecycling of the peptidoglycan. The high affinity demonstrated between the β-lactam antibiotics cefoxitin and imipenem with LMM-PBP4 (purified and bound to membrane) correlates with the binding activity for these proteins and their probable inactivation and its role on constitutive overproduction of AmpC β-lactamase. The structural model for the monomeric unit of LMM-PBP4 confirms the presence of domains II and III which define it as a LMM-PBP class C subclass C1 allowing to estimate the dimension of the cavity in the active site that favors the access of large substrates and recognizes the existence of a threonine amino acid at position 386 (T386) which would accommodate the peptide chain of a second strand of peptidoglycan. These structural precedents support the development of a DD-endopeptidase activity in this protein. Analysis of the cell wall in relation to its muropeptides composition from the reference strain and mutants in regulatory proteins for AmpC production (DacB, AmpE, AmpDh2) under natural conditions, supplementationoverexpression and antibiotic inactivation of LMM-PBP4, exhibits no changes to propose an activation system for ampC via an effector of pentapeptide nature but confirms in vivo the DD-peptidase bifunctionality mentioned above. Moreover, it was achieved the identification of new muropeptides related to structure and functionality still not described in Pseudomonas aeruginos