Efecto de la sobre-expresión de los sistemas de bombeo múltiple de drogas, MexAB-OprM y MexCD-OprJ, sobre el sistema de señalización por quorum-sensing de Pseudomonas aeruginosa

• Autores: Manuel Alcalde Rico
• Directores de la Tesis: José Luis Martínez Menéndez (dir. tes.), Miguel Remacha Moreno (dir. tes.), Jorge Andrés Olivares Pacheco (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2017
• Idioma: español
• Tribunal Calificador de la Tesis: José Berenguer Carlos (presid.), Marta Martín Basanta (secret.), Antonio Oliver Palomo (voc.), Jose Antonio Ainsa Claver (voc.), Jesús Blázquez Gómez (voc.)
• Programa de doctorado: Programa Oficial de Doctorado en Bioquímica, Biología Molecular, Biomedicina y Biotecnología (Biociencias Moleculares)
• Materias:
  • Química
    • Bioquímica
  • Ciencias de la vida
    • Microbiología
    • Biología molecular
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Pseudomonas aeruginosa is among the most relevant opportunistic pathogens involved in infections at hospitals, as well as in immunocompromised and cystic fibrosis patients. In addition, this bacterial species displays a characteristic low susceptibility to antibiotics and is able to acquire increased levels of resistance upon selection with antibiotics during treatment. All these circumstances currently make P. aeruginosa infections of special concern. One of the most important causes of resistance in this bacteria is the expression of multidrug efflux pumps, able to extrude disinfectants and antibiotics among others. In this PhD Thesis we analyse the physiological effects, with a special focus on the quorum sensing (QS) regulation network, of the acquisition of high level of resistance due to mutations in the mexR or nfxB genes, which leads to the overexpression of the MexAB-OprM or MexCD-OprJ efflux pumps respectively. In addition, as an independent objective, we have analysed, using experimental evolution and whole genome sequencing tools, the potential mechanisms of resistance of P. aeruginosa against the new antibiotic MDN-57.

    In this work, we demonstrate using transcriptomic analyses, that both the over- expression and the deletion of the MexAB-OprM or MexCD-OprJ efflux systems produce a high impact over the expression of a large number of genes, evidencing the importance of an appropriate expression of these RND systems for cellular homeostasis. We also show that NfxB, the negative regulator of MexCD-OprJ efflux pump, has a potential role as global regulator controlling the expression of a set of genes in a direct or indirect way, beyond the mexCD-oprJ operon. Further, we specially focus in the comparison between the transcriptomes of the mexR* and nfxB* mutants, which overproduce the MexAB-OprM and MexCD-OprJ efflux system respectively in order to decipher the differences and similarities observed in gene expression between these two strains. In this sense, we have found that the main similarity observed when each one of the mexR* and nfxB* transcriptomes are compared with that of the wild-type parental strain is the alteration in the expression of a large set QS-regulated genes. These results could be initially considered as a consequence of an unspecific burden associated to the hyperactivity of either of these two RND systems. However, we show that these changes are dependent on the specific efflux system overexpressed, being different the underlying mechanism by which mexR* and nfxB* mutants present an impaired QS signalling network.

    In contrast with previous claims, we demonstrate that defective QS response associated with the overexpression of MexAB-OprM, rather than being a consequence of an excessive 3- oxo-C12-HSL extrusion, is due to an impaired production of both PQS and HHQ, probably caused by a decreased production of its immediate precursor, octanoic acid. On the other hand, we show that the impaired QS-response observed in the nfxB* strain is mainly caused by an excessive non-physiological extrusion of the autoinducer signal HHQ, which produces a decrease in its own production and in the synthesis of PQS. Despite we also demonstrate that MexCD- OprJ is able to extrude kynurenine, another precursor of AQs, our results indicate that this extrusion is not the main cause of the low AQs production observed in the nfxB* mutant. Altogether, these results highlight the relevance of these two RND efflux systems in the modulation of the QS-response.

    Finally, we have showed that prolonged exposure of P. aeruginosa cultures to the new antibiotic MDN-57 leads to selection of resistant mutants containing mutations in hemA and/or hemD genes. Since these two genes are implicated in heme biosynthesis, it is possible that this pathway would be the target of MDN-57. In addition, we also show that the MexAB-OprM, MexCD-OprJ and MexEF-OprN efflux pumps contribute to the intrinsic and the acquired (when overexpressed) resistance to MDN-57.