FUNCTIONAL GENOMICS TO ESTABLISH THE RULE OF PSEUDOMONAS AERUGINOSA ADAPTATION TO THE PROGRESSION TO AIRWAYS CHRONIC INFECTION

Pseudomonas aeruginosa chronic infections cause persistent respiratory symptoms and decline of the lung functions in patients with cystic fibrosis (CF). After a period of intermittent and acute infections, persistent lifestyle is established with P. aeruginosa patho-adaptive variants, which are clonal with the initially-acquired strains. P. aeruginosa adapts itself by loss-of-function mutations which enhance fitness in CF airways and sustain its clonal expansion during chronic infection. Currently, much is known about the bacterial factors needed for acute infections while the mechanisms involved in the colonization and persistence in chronic airways infection remain mostly unknown. In addition antibiotic resistance characterizes P. aeruginosa chronic lung infections in CF patients, highlighting the need of new therapeutic options. Vaccination strategies to prevent or limit P. aeruginosa infection represent a rational approach to positively impact the clinical status of CF patients. The purposes of this thesis were to develop novel approaches of genomics-based method high-throughput screening to directly identify bacterial functions whose inactivation promotes airways long-term chronic infection and to identify novel P. aeruginosa surface exposed antigens with high immunogenic potential to design a new immunotherapy for patients with CF. To identify novel genes involved in this microevolution of P. aeruginosa adaptation in CF airways, we designed a novel approach of positive-selection screening by PCR-based signature-tagged mutagenesis (Pos-STM) in a murine model of chronic airways infection. A systematic positive-selection scheme using sequential rounds of in vivo screenings for bacterial maintenance, as opposed to elimination, generated a list of genes whose inactivation increased the colonization and persistence in chronic airways infection. The phenotypes associated to these Pos-STM mutations reflect alterations in diverse aspects of P. aeruginosa biology which include lack of swimming and twitching motility, lack of production of the virulence factors such as pyocyanin, biofilm formation and metabolic functions. In addition, Pos-STM mutants showed altered invasion and stimulation of immune response when tested in human respiratory epithelial cells. Sequence analysis of Pos-STM genes in longitudinally P. aeruginosa isolates from CF patients identified signs of patho-adaptive mutations within the genome. This novel Pos-STM approach identified bacterial functions that can have important clinical implications for the persistent lifestyle and disease progression of the airway chronic infection. Next to generate a new genome-derived P. aeruginosa antigens database; we identified novel vaccine candidates on the basis of their putative localization on the cell surface by in silico analysis of the genome of P. aeruginosa PAO1 strain and by a combination of advanced genomic approaches, such as microarray technology and negative-STM approach screening. All targets have been validated by a comparative genome analysis with six P. aeruginosa sequenced strains deposited on the web including clinical strains, we excluded from further analysis antigens not conserved in all the genomes. Targets with homology with human and mouse genome ware excluded due the high probability to behave as self−antigens and poorly immunogenic. Selected antigens will be produce as recombinant proteins. To evaluate the immune-protection of conserved vaccine candidates against infection caused by P. aeruginosa we established vaccination protocols in murine models of P. aeruginosa acute and chronic infection. In acute infection, mice were injected with a lethal dose of P. aeruginosa after the last dose of immunization and monitored for survival, whereas to evaluate the protection from chronic infection, bacterial load in the lungs of immunized and control mice was compared after 14 days from P. aeruginosa challenge. The established murine models will be used in the future work to test novel P. aeruginosa genome derived vaccine candidates. These new vaccine candidates may contribute to settle the basis of a novel immunotherapy to contrast P. aeruginosa lung infections in CF patients.