ADAPTATION OF Pseudomonas aeruginosa TO THE INTRACELLULAR MILIEU OF EUKARYOTIC CELLS

MARTINO, ROMÁN A.; MOYANO, ALEJANDRO J.; LOPEZ, VERONICA; SAKA, HA; SMANIA, ANDREA M.

MODALIDAD VIRTUAL

Congreso; LVII Reunión Anual de Sociedad Argentina de Investigaciones Bioquímicas y Biología Molecular (SAIB) y el XVI Congreso Anual de la Asociación Civil de Microbiología General (SAMIGE); 2021

SAIB & SAMIGE

Pseudomonas aeruginosa is an opportunistic pathogen that chronically infects the airways of cystic fibrosis (CF) patients. Major traits such as a biofilm mode of growth and hypermutability, are considered to constitute a source for adaptive phenotypes and causes of the increased tolerance and resistance of P. aeruginosa. Another mechanism through which pathogens are capable of evading the immune response, as well as exposure to some antibiotics, is the ability to thrive in theintracellular environment of the eukaryotic cell. However, the relevance of this mechanism in the ability of P. aeruginosa to persist in CF chronic infections is poorly explored. Here we performed a long-term evolution experiment with hypermutator and wt strains of P. aeruginosa by carrying out successive reinfection assays, which consisted in using intracellular bacterial cells, recovered after antibiotic exclusion assays from A549 lung epithelial cells, as the inoculum for the next round ofinfection. A549 cells were lysed to recover intracellular bacterial cells in each infection assay to measure invasiveness (t0), or left for additional 4 and 24 h post-infection (t4 and t24, respectively) to evaluate bacterial persistence. We chose bacterial cells recovered from t4, which showed the best recovery values, and repeated this for ten further successive rounds of infections,always lysing A549 cells and recovering bacteria at t0 and t4 (T4exp). In addition, since bacteria recovered from t24 are expected to be the most resistant, we performed a parallel experiment (T24exp) by using t24 bacterial cells as inoculum for ten successive rounds of infections, always recovering bacteria at t0 and t24. Interestingly, we observed that after round 4 of infection, the recovery of intracellular hypermutator but not wt bacterial cells begun to increase uninterruptedly until round 10. Flow cytometry analyses showed an increase in the invasive capacity as we progress in the number of rounds of infection which was more pronounced in the hypermutator strain compared to the wt. High-content imaging confirmed these results and showed an uneven infection of A549 cells, whereas some remained uninfected, others were infected by several bacterial cells.We also characterized the bacterial cells of hypermutator and wt strains recovered from rounds 1, 4, 7 and 10 by evaluating their capacity to form biofilms and swarming motility. We observed an increase in biofilm formation capacity as we progressed through the rounds, which was higher in the hypermutator strain. This was consistent with a reduced swarming motility showed by intracellular-recovered bacteria compared to the parental strains. These results shed light on the progressive adaptive process of P. aeruginosa to the intracellular milieu of eukaryotic cells. Further experiments will be required to explore the molecular bases of this adaptive process, which might play a role in the evolution of chronic infections in the airways of CF patients.