¿DISSECTION OF THE ROLE OF P. AERUGINOSA VIRULENCE FACTORS AND HOST GENETIC BACKGROUND DURING RESPIRATORY INFECTION '

Pseudomonas aeruginosa is the fourth most commonly isolated hospital pathogen, multidrug resistant and potentially deadly causes of pneumonia. Patients at risk of acquiring P. aeruginosa are immune-compromised , due to immunosuppressive therapies or underlying diseases such as cancer, AIDS or the hereditary disease cystic fibrosis (CF). P. aeruginosa-host interaction is driven by several factors including the bacterial phenotype and the host genetic make- up. From the bacterial side, several virulence factors have been attributed to P. aeruginosa pathogenicity while many others remain to be established. One third of the P. aeruginosa genome has no attributed homology to any previously reported sequences with only 6.7% of its genes having a function verified experimentally. To identify novel virulence genes, a genome-wide multi-host in vitro and in vivo screening was carried out. A library of 57,360 P. aeruginosa Tn-5 mutants was screened for phenotypic virulence traits (e.g. swarming, pyocyanin, and protease). A total of 404 Tn5-mutants showing pleiotropic phenotypes were tested in the C. elegans and D. melanogaster infection models and further reduced for validation in cell culture and animal models. In particular, 28 selected Tn5-mutants were tested for invasion and IL-8 production in immortalized respiratory A549 cells. Mortality, inflammation and lung pathology of eight selected mutants were scored in C57BL/6 mice and five out of eight mutants were significantly attenuated in inducing murine mortality. The Tn5 gene insertion sites were mapped and their conservation established across seven P. aeruginosa genomes by comparative sequence analysis. The Tn-5 inserted genes encoded proteins from several functional classes including hypothethical, catabolism regulation, iron uptake and quorum sensing. This screening generated a list of virulence-related genes relevant for pathogenicity in multi-hosts and favour much needed insight into the aspects of P. aeruginosa/host interaction. From the host side, the clinical outcome of P. aeruginosa infections may be extremely variable among individuals at risk including CF patients. However, risk factors for P. aeruginosa infection remain largely unknown. To characterize how the genetic background influences P. aeruginosa lung infections, nine different inbred mouse strains were infected with P. aeruginosa clinical isolate and monitored for body weight change and mortality up to seven days. Next, one of the most susceptible and resistant mouse strains were further characterized by scoring bacterial count, cell-mediated immunity, cytokines and chemokines profile and lung pathology in an early time course. Susceptible mice showed a significantly higher bacterial burden, higher cytokines and chemokines levels but lower leukocyte recruitment, and a lower inflammatory severity damage when compared to resistant ones. Our results indicated that host genetic make-up may have a role in the modulation of cell-mediated immunity playing a critical role in the control of P. aeruginosa infection