Abstract
Chronic pulmonary infections with Pseudomonas aeruginosa are a debilitating component of the genetic disease cystic fibrosis (CF). The persistence of these infections is partly due to iron uptake as this contributes to bacterial growth and the formation of resilient biofilms. In conditions of restricted availability, iron can be sequestered by the endogenous siderophores pyoverdine and pyochelin and may also be acquired as haem through the Has and Phu pathways.
This research primarily sought to ascertain the use and relative importance of the pyoverdine, pyochelin, Has and Phu systems in iron acquisition in CF. Further objectives were to elucidate how the lung environment affects iron uptake, to characterise regulatory components of the Has system and to ascertain the effect of a synthetic iron chelator (ICL670) on P. aeruginosa growth.
A quantitative reverse transcriptase-PCR (qRT-PCR) approach was developed to measure the expression of P. aeruginosa iron uptake systems. In vitro studies verified that expression of all four iron acquisition systems was significantly reduced in the presence of iron (Fe3+) and that auto-regulation of the pyoverdine and pyochelin systems occured. This qRT-PCR approach was therefore used to measure system activity in 56 sputum samples from 23 CF patients. Here, the pyoverdine system was active in most samples, while the pyochelin system demonstrated a lower level of activity and the Has and Phu systems showed high levels of expression in some samples. Siderophore assays of CF sputum were consistent with the preferential use of pyoverdine for iron uptake however the high levels of haem system expression also suggested active acquisition of haem.
A global reduction in iron uptake system expression was observed during acute and post acute phases of infection, illustrating that the lung environment affects iron uptake. Expression of haem uptake systems remained comparatively high and it was postulated that this was a response to elevated haem levels during pulmonary exacerbations. The strain of P. aeruginosa resident in the CF airway also affected iron acquisition, as the ability to produce pyoverdine or pyochelin was lost from some clinical strains. ICL670 was only able to delay growth of pyoverdine-deficient bacteria and therefore has limited potential to treat P. aeruginosa infections.
In vitro studies characterising the Has system determined that P. aeruginosa has a higher affinity for haemoglobin than for haem, and that the haem-sequestering protein HasAp promotes haem uptake. Furthermore, the has system is regulated by HasI, a sigma-factor which is expressed from the unique hasIS operon in response to iron availability. HasI promoted expression of genes encoding HasAp and the outer membrane receptor HasR however further members of the HasI regulon could not be identified in microarray studies.
Overall, the P. aeruginosa requirement for iron in CF appears to be met primarily through pyoverdine and haem assimilation. This iron uptake is critical for the resilience of P. aeruginosa biofilms which contribute to the persistence of infection. Medical interventions that restrict these iron uptake pathways may therefore be therapeutic for CF patients.