INCREASED INVASIVENESS AND PERSISTANCE OF HYPERMUTATOR PSEUDOMONAS AERUGINOSA WITHIN LUNG EPITHELIAL CELLS

LOPEZ VERONICA ALEJANDRA

Congreso; Congreso SAIB 2022; 2022

Pseudomonas aeruginosa is an opportunistic pathogen that chronically infects the airways of patientswith cystic fibrosis (CF). One mechanism that could contribute to the persistence and survival capacityof P. aeruginosa, in a changing and heterogeneous environment like CF´s lungs, is the ability to thrive inthe intracellular environment of the eukaryotic cell. Despite P. aeruginosa is considered an extracellularpathogen, numerous studies showed that P. aeruginosa can be internalized in different cell types,including epithelial and endothelial cells. Also is important to mention that major traits such as biofilmgrowth mode and hypermutability are considered a source of adaptive phenotypes providing increasedP. aeruginosa tolerance and resistance. Here we performed a long-term evolution experiment withhypermutator and wild-type strains of P. aeruginosa by carrying out successive reinfection assays, whichconsisted in using intracellular bacterial cells, recovered after antibiotic exclusion assays from A549 lungepithelial cells, as the inoculum for the next round of infection. In this way we performed 10 successiveinfection assays to evaluate and compare the ability of P. aeruginosa to invade and persist in theintracellular milieu of eukaryotic cells. Interestingly, we observed that after round 4 of infection, therecovery of intracellular hypermutator but not wild-type bacterial cells began to increase uninterruptedlyuntil round 10.The localization of bacteria inside eukaryotic cells was confirmed by laser scanning confocal microscopy.Furthermore, the use of flow cytometry and high-content imaging analyses showed an increase in theinvasive capacity as we progress in the number of rounds of infection which was more pronounced in thehypermutator strain compared to the wild-type. Flow cytometry also allowed the evaluation ofcytotoxicity by using a viability dye to label dead cells. Finally we characterized 10 different isolated clonesof the hypermutator and wild-type strains to evaluate the diversity of the evolved population recoveredfrom Round 10 by measuring the invasive capacity of each individual isolate. These results suggest thathypermutability plays an important role in this progressive adaptive process of P. aeruginosa to theintracellular milieu of eukaryotic cells. Further experiments such as whole-genome sequencing will berequired to explore the molecular bases of this adaptive process, which might play a role in the evolutionof chronic infections in the airways of CF patients.