-INVASIVENESS OF HUMAN EPITHELIAL CELLS BY Pseudomonas aeruginosa IS INCREASED BY HYPERMUTABILITY.

Los Cocos

Congreso; XVII Congreso Argentino de Microbiología General; 2022

SAMIGE

Pseudomonas aeruginosa is an opportunistic pathogen that chronically infects the airways of patients with cystic fibrosis (CF). The establishment of a chronic infection is a transformative process for the bacterium 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 of adaptive phenotypes of P. aeruginosa providing increased tolerance and resistance. One mechanism that could contribute to the persistence and survival capacity of P. aeruginosa in this environment is the ability to thrive in the intracellular environment of the eukaryotic cell. Despite being considered an extracellular pathogen, numerous studies have shown that P. aeruginosa can internalize in different eukaryotic cell types, including epithelial and endothelial cells. Here we performed a longterm evolution experiment by carrying out successive reinfection assays. Each round of infection consists in an antibiotic exclusion assay in which A549 lung epithelial cells are infected with hypermutator or wt strains of P. aeruginosa. In each round of infection, we recovered intracellular bacterial 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 and persist in the intracellular milieu of eukaryotic cells. Interestingly, we observed that after round 4 of infection, the recovery of intracellular hypermutator but not wt bacterial cells began to increaseuninterruptedly until round 10. Importantly, localization of bacteria inside eukaryotic cells was confirmed by laser scanning confocal microscopy. Moreover, high-content imaging and flow cytometry showed an increase in the invasive capacity as the number of rounds of infection progressed, which was more pronounced in the hypermutator strain compared with the wt. Flow cytometry also allowed the evaluation of the cytotoxicity capacity by using a viability dye that can be used to irreversibly label dead cells. We also characterized the diversity of the evolved population recovered from Round 10, by isolating 10 different clones of the wt and hypermutator strains to measure 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 pathways involved. These results shed light on the progressive adaptive process of P. aeruginosa to the intracellular milieu of eukaryotic cells and suggest that hypermutability plays an important role in this adaptation.