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 emerging from the environment 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 from this group associated with chronic lung infections that has been extensively studied in this context, but the mechanism 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 isolate 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 exposure 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 response, iron acquisition, biofilm formation and those previously associated with chronic infection. Importantly the observed changes in the proteome were distinct from changes 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 of 13 antibiotics tested; increased biofilm (1.8-4.1-fold (p<0.0001) and decreased pyocyanin production (2.6-fold, p<0.0001). All hypoxia-adapted cultures showed decreased siderophore production (2.9-3.6-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 phenotype and proteome changes that are also observed in P. aeruginosa CF lung chronic infection, possibly opening the path to treatment of chronic infections.
Author Summary
Opportunistic antibiotic-resistant bacterial infections emerging from the natural environment 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 long-term 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 most bacteria and several of them are also observed in isolates that had adapted to chronic infection. Similar adaptive responses in other opportunistic environmental pathogens suggest that this adaptation could be targeted to create universal therapeutic approaches preventing antibiotic overuse and improving patient outcomes.
