INVASIVENESS OF HUMAN EPITHELIAL CELLS BY PSEUDOMONAS AERUGINOSA IS INCREASED BY HYPERMUTABILITY

LOPEZ VERONICA

Congreso; XVII Congreso Argentino de Microbiología General; 2022

Pseudomonas aeruginosa is an opportunistic pathogen that chronically infects the airways of patientswith cystic fibrosis (CF). The establishment of a chronic infection is a transformative process for thebacterium itself, as it must adjust to the changing and heterogeneous conditions that prevail in the CFlung. Major traits such as biofilm growth mode and hypermutability are considered a source ofadaptive phenotypes of P. aeruginosa providing increased tolerance and resistance. One mechanismthat could contribute to the persistence and survival capacity of P. aeruginosa in this environment isthe ability to thrive in the intracellular environment of the eukaryotic cell. Despite being consideredan extracellular pathogen, numerous studies have shown that P. aeruginosa can internalize indifferent eukaryotic cell types, including epithelial and endothelial cells. Here we performed a longterm evolution experiment by carrying out successive reinfection assays. Each round of infectionconsists in an antibiotic exclusion assay in which A549 lung epithelial cells are infected withhypermutator or wt strains of P. aeruginosa. In each round of infection, we recovered intracellularbacterial cells and used them as inoculum for the next round of infection. In this way, we performed10 successive infection assays to evaluate and compare the ability of P. aeruginosa to invade andpersist in the intracellular milieu of eukaryotic cells. Interestingly, we observed that after round 4 ofinfection, the recovery of intracellular hypermutator but not wt bacterial cells began to increaseuninterruptedly until round 10. Importantly, localization of bacteria inside eukaryotic cells wasconfirmed by laser scanning confocal microscopy. Moreover, high-content imaging and flowcytometry showed an increase in the invasive capacity as the number of rounds of infectionprogressed, which was more pronounced in the hypermutator strain compared with the wt. Flowcytometry also allowed the evaluation of the cytotoxicity capacity by using a viability dye that can beused to irreversibly label dead cells. We also characterized the diversity of the evolved populationrecovered from Round 10, by isolating 10 different clones of the wt and hypermutator strains tomeasure invasive capacity of each individual clone. We are currently analyzing the molecular bases ofthis adaptive process by whole-genome sequencing, in order to identify the genetic pathwaysinvolved. These results shed light on the progressive adaptive process of P. aeruginosa to theintracellular milieu of eukaryotic cells and suggest that hypermutability plays an important role in thisadaptation.