title: Positive signature-tagged mutagenesis in Pseudomonas aeruginosa: tracking patho-adaptive mutations promoting airways chronic infection.
abstract: The opportunistic pathogen Pseudomonas aeruginosa can establish life-long chronic infections in the airways of cystic fibrosis (CF) patients. Persistent lifestyle is established with P. aeruginosa patho-adaptive variants, which are clonal with the initially-acquired strains. Several reports indicated that P. aeruginosa adapts by loss-of-function mutations which enhance fitness in CF airways and sustain its clonal expansion during chronic infection. To validate this model of P. aeruginosa adaptation to CF airways and to identify novel genes involved in this microevolution, 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, indicating that P. aeruginosa is prone to revise the interaction with its host during persistent lifestyle. Finally, 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.
authors.affiliation: San Raffaele Scientific Institute, Division of Immunology, Transplantation and Infectious Diseases, Milano, Italy.
authors.forename: Irene
authors.initials: I
authors.lastname: Bianconi
authors.forename: Andrea
authors.initials: A
authors.lastname: Milani
authors.forename: Cristina
authors.initials: C
authors.lastname: Cigana
authors.forename: Moira
authors.initials: M
authors.lastname: Paroni
authors.forename: Roger C
authors.initials: RC
authors.lastname: Levesque
authors.forename: Giovanni
authors.initials: G
authors.lastname: Bertoni
authors.forename: Alessandra
authors.initials: A
authors.lastname: Bragonzi
mesh_terms: D000220:Adaptation, Biological / Q000235:genetics*; D000465:Algorithms; D000818:Animals; D002478:Cells, Cultured; D002908:Chronic Disease; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016296:Mutagenesis / Q000502:physiology*; D009154:Mutation / Q000502:physiology; D011552:Pseudomonas Infections / Q000150:complications / Q000235:genetics / Q000382:microbiology* / Q000473:pathology; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity*; D012141:Respiratory Tract Infections / Q000209:etiology / Q000382:microbiology* / Q000473:pathology; D014774:Virulence / Q000235:genetics; D037521:Virulence Factors / Q000235:genetics
chemical_list: D037521:Virulence Factors