Accessory Genome Accountability in Pseudomonas aeruginosa Persistent Infections

Descripción del proyecto

Unos genes accesorios bacterianos favorecen la resistencia a antibióticos

La fibrosis quística (FQ) es una enfermedad hereditaria provocada por mutaciones en el gen regulador de la conductancia transmembrana de la fibrosis quística (RTFQ). Los pacientes no logran excretar el moco de los pulmones, lo que conduce a infecciones persistentes y disminuye su calidad de vida. El proyecto financiado con fondos europeos AGAPE se centra en «Pseudomonas aeruginosa», un patógeno oportunista que puede ser resistente al tratamiento y está presente en la mayoría de los pacientes con FQ. Sus investigadores pretenden comprender el papel del genoma accesorio de «P. aeruginosa» en el desarrollo de las infecciones persistentes. Dado el aumento de la resistencia a antibióticos, los resultados del proyecto arrojarán luz sobre el mecanismo de adaptación de los patógenos en distintos entornos.

Objetivo

Bacterial survival to antibiotic treatment poses an urgent problem to humanity, causing 25.000 deaths per year in the European Union alone. The inability to fully eradicate pathogens with antibiotics leads to the establishment of persistent infections (PIs). Pseudomonas aeruginosa, an opportunistic pathogen, is found chronically in the airways of 70% of Cystic Fibrosis (CF) patients. In CF, the anomalous production of mucus in the lung promotes the establishment of bacteria, with infections decreasing the life quality and expectancy of patients. The mechanisms by which P. aeruginosa is able to escape treatment and generate PIs within the CF lung have not been fully elucidated. Mostly, the success of P. aeruginosa relies on its adaptability, which derives from its large genome carrying genes for survival in a wide spectrum of environments. Most of these genes are not conserved among strains, composing the accessory genome (AG), which is built up through horizontal gene transfer (HGT). Previous studies have identified components of the AG involved in virulence and resistance, yet the extent of the AG involvement in PIs is unknown. Through AGAPE I seek to elucidate the relevance of the AG in the establishment of PIs in CF lungs. I will do so by studying both the conformation and function of the AG, through novel CRISPR-Cas tools in P. aeruginosa. First, I will determine the identity and quantity of HGT during infection by recording transfer events with a CRISPR-Cas recorder system. Second, I will generate individual and combinatorial knockdowns of every accessory gene through CRISPR interference (CRISPRi), to screen the effects of the repression on fitness during infection. I will follow the infections of the generated strains on lung organoids derived from the corresponding hosts. Overall, this bona fide action will allow to uncover novel mechanisms behind PIs relying on accessory genes, and to identify novel markers and targets to improve patient treatment.