Long-term adaptation to hypoxia provides insight into mechanisms facilitating the switch of Pseudomonas aeruginosa to chronic lung infections

Opportunistic bacterial infections are an increasing threat, especially for immunocompromised individuals such as people with cystic fibrosis (CF), driving morbidity and mortality. Pseudomonas aeruginosa is a key pathogen associated with chronic lung infections, that has been extensively studied in this context, but the mechanism(s) driving its adaptation towards chronic colonisation in the lung are not fully understood. This work focuses on the adaptations of P. aeruginosa to long-term hypoxia, one of the important environmental pressures present in the CF lung, to investigate whether it drives the development of persistence in CF patients. We used an experimental evolution approach to investigate how an early CF strain adapted to 6% oxygen over 28 days. We focussed on the impact of long-term hypoxia on the proteome and investigated the emergence of stable changes in phenotype. Changes in the abundance of >140 proteins were observed compared to the ancestral strain, including proteins involved in antibiotic resistance, stress response, iron homeostasis, biofilm formation and those previously associated with chronic infection. Significant changes in the abundance of proteins regulating cellular c-di-GMP levels were also observed. We show that two distinct P. aeruginosa small colony variants (SCVs) emerged, one exclusively in hypoxia exposed cultures. Hypoxia-adapted cultures developed resistance to 8 out of 13 antibiotics tested; increased biofilm and exopolysaccharide production; and decreased pyocyanin production, consistent with the changes in the proteome. All hypoxia-adapted cultures showed decreased siderophore production. Overall, we demonstrate that long-term hypoxia exposure contributes to multiple changes in phenotype and proteome that are frequently observed in P. aeruginosa CF lung chronic infection isolates. This suggests that hypoxia is driving these adaptations, at least in part, and opens a new path to treatment. Author SummaryOpportunistic antibiotic-resistant bacterial infections are an increasing threat, particularly for immunocompromised individuals such as people with cystic fibrosis (CF). These infections increase disease severity, the number of deaths and healthcare costs. Pseudomonas aeruginosa is a major pathogen in this context, causing persistent infections, yet the mechanisms driving its adaptation towards chronic colonisation in the CF lung are not understood. We investigated the adaptations that P. aeruginosa exposed to low oxygen conditions (an important environmental pressure in the CF lung) for 28 days, to examine whether it drives adaptations that lead to chronic infection. We found that prolonged exposure to hypoxia induced stable changes in P. aeruginosa, which are distinct from those reported after short term exposure. We observed multiple adaptations in hypoxia adapted cultures, many of which were associated with important signalling networks linked to lifestyle shifts in bacteria and several were also observed in isolates that had adapted to chronic infection. Similar adaptive responses in other opportunistic environmental pathogens suggest that this process of adaptation could be targeted to create universal therapeutic approaches preventing antibiotic overuse and improving patient outcomes.