HIPERMUTABILITY CAN FACILITATE THE ADAPTATION OF Pseudomonas aeruginosa TO THE INTRACELLULAR MILIEU OF EUKARYOTIC CELLS?

LOPEZ VERONICA ALEJANDRA; MOYANO ALEJANDRO

Congreso; LVI SAIB Meeting ?XV SAMIGE Meeting; 2020

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 adaptivephenotypes and causes of the increased tolerance and resistance of P. aeruginosa. Another mechanism through whichpathogens 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, to date there are few studies about the relevance of this mechanismin the ability of P. aeruginosa to persist in CF chronic infections. Here we evaluated a wt and a hypermutator strain of thePAO1 P. aeruginosa strain, on their ability to invade and persist in the intracellular milieu of A549 lung epithelial cells byperforming antibiotic exclusion assays. A549 cells were then lysed to recover intracellular bacterial cells to measureinvasiveness (t0), or left for additional 4, 8, 12 and 24 h post-infection (t4, t8, t12 and t24, respectively) to evaluate bacterialpersistence. As a first result, no differences were observed between the wt or hypermutator strain regarding invasiveness orpersistence in all the time-periods tested. To extend our analysis on the adaptability of both strains to this environment weperformed a long-term evolution experiment by carrying out successive reinfection assays, which consisted in usingintracellular bacterial cells recovered from the A549 as the inoculum for the next round of infection. We chose bacterial cellsrecovered 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 tobe the most resistant, we performed a parallel experiment (T24exp) by using t24 bacterial cells as inoculum for ten successiverounds of infections, always recovering bacteria at t0 and t24. Interestingly, no differences were observed between the wt andhypermutator strain during the first 3 rounds of the t4 experiment (T4exp). However, after round 4 of infection, the recoveryof intracellular hypermutator but not wt bacterial cells begun to increase uninterruptedly until round 10. In fact, round 10 ofT4exp showed a 8.3-fold increase in invasiveness (t0) and a 11.2-fold increase in recovered bacterial cells at t4 respect toround 1. On the other hand, T24exp only showed a successive increase in invasiveness (t0) which reached a 9,2-fold increasein round 10 but only a 3.7-fold increase of recovery at t24. These results suggest that hypermutability enhanced the adaptationof P. aeruginosa to the intracellular milieu of eukaryotic cells. Further experiments will be required to explore the molecularbases of this adaptive process, which might play a role in the evolution of chronic infections in the airways of CF patients.