Long-term adaptation to hypoxia provides insight into mechanisms facilitating the switch of Pseudomonas aeruginosa to chronic lung infections
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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 that causes chronic lung infections, and has been extensively studied in this context, but the mechanism(s) driving its adaptation towards chronic colonisation in the CF lung is 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 examined how an early CF strain adapted to 6% oxygen over 28 days using an experimental evolution approach and showed that two distinct P. aeruginosa small colony variants (SCVs) emerged, one exclusively in hypoxia-adapted cultures. Importantly, SCVs were more prevalent in hypoxia-adapted cultures than in normoxia-adapted cultures (98% vs <35%). Proteomic analysis revealed significant changes in the abundance of >200 proteins within 28 days, including proteins involved in antibiotic resistance, stress responses, iron acquisition, biofilm formation and proteins previously associated with chronic infection. Importantly the observed stable changes in the proteome were distinct from changes previously reported after short-term exposure to hypoxia. Phenotypic characterisation of adapted cultures supported these findings, showing that hypoxia- adapted cultures developed higher resistance to 8 out of 13 antibiotics tested; increased biofilm (1.8 to 4.1-fold (p<0.0001) and decreased pyocyanin production (2.6-fold, p<0.0001). All hypoxia-adapted cultures showed decreased siderophore production (2.9 to 3.6-fold, p<0.02). Two distinct patterns of adaptation to hypoxic conditions were observed, with the phenotypes of one culture being markedly different from the other two. Overall, we demonstrate that long-term hypoxia exposure contributes to multiple changes in phenotype and proteome that were also observed in P. aeruginosa CF lung chronic infection isolates, possibly opening the path to treatment of chronic infections.
Author Summary
Opportunistic 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 of P. aeruginosa exposed to low oxygen conditions (an important environmental pressure in the CF lung), for 28 days, to examine whether it drives the process of persistence. We found that prolonged exposure to hypoxia induced changes in the pathogen’s characteristics, which were 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.
