Hipermutability can facilitate the adaptation of Pseudomonas aeruginosa to the intracellular milieu of eukaryotic cells

PANZZETTA, MARÍA E.; MOYANO, ALEJANDRO J.; SMANIA, ANDREA M.; LÓPEZ, VERÓNICA A.; SAKA, HÉCTOR A.

Congreso; LVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular - XV Congreso Argentino de Microbiología General; 2020

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular - Asociación Civil de Microbiología General

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 the intracellular environment of the eukaryotic cell. However, to date there are few studies about the relevance of this mechanism in the ability of P. aeruginosa to persist in CF chronic infections. Here we evaluated of a wt and a hypermutator strain of the PAO1 P. aeruginosa strain, on their ability to invade and persist in the intracellular milieu of A549 lung epithelial cells by performing antibiotic exclusion assays. A549 cells were then lysed to recover intracellular bacterial cells to measure invasiveness (t0), or left for additional 4, 8, 12 and 24 h post-infection (t4, t8, t12 and t24, respectively) to evaluate bacterial persistence. As a first result, no differences were observed between the wt or hypermutator strain regarding invasiveness or persistence in all the time-periods tested. To extend our analysis on the adaptability of both strains to this environment we performed a long-term evolution experiment by carrying out successive reinfection assays, which consisted in using intracellular bacterial cells recovered from the A549 as the inoculum for the next round of infection. 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. 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, no differences were observed between the wt and hypermutator strain during the first 3 rounds of the t4 experiment (T4exp). However, after round 4 of infection, the recovery of intracellular hypermutator but not wt bacterial cells begun to increase uninterruptedly until round 10. In fact, round 10 of T4exp showed a 8.3-fold increase in invasiveness (t0) and a 11.2-fold increase in recovered bacterial cells at t4 respect to round 1. On the other hand, T24exp only showed a successive increase in invasiveness (t0) which reached a 9,2-fold increase in round 10 but only a 3,7-fold increase of recovery at t24. These results suggest that hypermutability enhanced the adaptation 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.