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The DeltaF508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability.

ABSTRACT: Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing DeltaF508-CFTR significantly enhanced P. aeruginosa biofilm formation, and DeltaF508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on DeltaF508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing DeltaF508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The DeltaF508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.

SUBMITTER: Moreau-Marquis S

PROVIDER: S-EPMC2494796 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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The DeltaF508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability.

Moreau-Marquis Sophie S Bomberger Jennifer M JM Anderson Gregory G GG Swiatecka-Urban Agnieszka A Ye Siying S O'Toole George A GA Stanton Bruce A BA

American journal of physiology. Lung cellular and molecular physiology 20080321 1

Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aerug ...[more]

PMID: 18359885

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Project description:The survival of bacteria in nature is greatly enhanced by their ability to grow within surface-associated communities called biofilms. Commonly, biofilms generate proliferations of bacterial cells, called microcolonies, which are highly recalcitrant, 3-dimensional foci of bacterial growth. Microcolony growth is initiated by only a subpopulation of bacteria within biofilms, but processes responsible for this differentiation remain poorly understood. Under conditions of crowding and intense competition between bacteria within biofilms, microevolutionary processes such as mutation selection may be important for growth; however their influence on microcolony-based biofilm growth and architecture have not previously been explored. To study mutation in-situ within biofilms, we transformed Pseudomonas aeruginosa cells with a green fluorescent protein gene containing a +1 frameshift mutation. Transformed P. aeruginosa cells were non-fluorescent until a mutation causing reversion to the wildtype sequence occurs. Fluorescence-inducing mutations were observed in microcolony structures, but not in other biofilm cells, or in planktonic cultures of P. aeruginosa cells. Thus microcolonies may represent important foci for mutation and evolution within biofilms. We calculated that microcolony-specific increases in mutation frequency were at least 100-fold compared with planktonically grown cultures. We also observed that mutator phenotypes can enhance microcolony-based growth of P. aeruginosa cells. For P. aeruginosa strains defective in DNA fidelity and error repair, we found that microcolony initiation and growth was enhanced with increased mutation frequency of the organism. We suggest that microcolony-based growth can involve mutation and subsequent selection of mutants better adapted to grow on surfaces within crowded-cell environments. This model for biofilm growth is analogous to mutation selection that occurs during neoplastic progression and tumor development, and may help to explain why structural and genetic heterogeneity are characteristic features of bacterial biofilm populations.

Project description:The critical role of bacterial biofilms in chronic human infections calls for novel anti-biofilm strategies targeting the regulation of biofilm development. However, the regulation of biofilm development is very complex and can include multiple, highly interconnected signal transduction/response pathways, which are incompletely understood. We demonstrated previously that in the opportunistic, human pathogen P. aeruginosa, the PP2C-like protein phosphatase SiaA and the di-guanylate cyclase SiaD control the formation of macroscopic cellular aggregates, a type of suspended biofilms, in response to surfactant stress. In this study, we demonstrate that the SiaABC proteins represent a signal response pathway that functions through a partner switch mechanism to control biofilm formation. We also demonstrate that SiaABCD functionality is dependent on carbon substrate availability for a variety of substrates, and that upon carbon starvation, SiaB mutants show impaired dispersal, in particular with the primary fermentation product ethanol. This suggests that carbon availability is at least one of the key environmental cues integrated by the SiaABCD system. Further, our biochemical, physiological and crystallographic data reveals that the phosphatase SiaA and its kinase counterpart SiaB balance the phosphorylation status of their target protein SiaC at threonine 68 (T68). Crystallographic analysis of the SiaA-PP2C domain shows that SiaA is present as a dimer. Dynamic modelling of SiaA with SiaC suggested that SiaA interacts strongly with phosphorylated SiaC and dissociates rapidly upon dephosphorylation of SiaC. Further, we show that the known phosphatase inhibitor fumonisin inhibits SiaA mediated phosphatase activity in vitro. In conclusion, the present work improves our understanding of how P. aeuruginosa integrates specific environmental conditions, such as carbon availability and surfactant stress, to regulate cellular aggregation and biofilm formation. With the biochemical and structural characterization of SiaA, initial data on the catalytic inhibition of SiaA, and the interaction between SiaA and SiaC, our study identifies promising targets for the development of biofilm-interference drugs to combat infections of this aggressive opportunistic pathogen.

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Project description:While both iron and nitric oxide (NO) are redox-active environmental signals shown to regulate biofilm development, their interaction and roles in regulating biofilms have not been fully elucidated. In this study, exposure of Pseudomonas aeruginosa biofilms to exogenous NO inhibited the expression of iron acquisition-related genes and the production of the siderophore pyoverdine. Furthermore, supplementation of the culture medium with high levels of iron (100??M) counteracted NO-induced biofilm dispersal by promoting the rapid attachment of planktonic cells. In the presence of iron, biofilms were found to disperse transiently to NO, while the freshly dispersed cells reattached rapidly within 15?min. This effect was not due to the scavenging of NO by free iron but involved a cellular response induced by iron that led to the elevated production of the exopolysaccharide Psl. Interestingly, most Psl remained on the substratum after treatment with NO, suggesting that dispersal involved changes in the interactions between Psl and P. aeruginosa cells. Taken together, our results suggest that iron and NO regulate biofilm development via different pathways, both of which include the regulation of Psl-mediated attachment. Moreover, the addition of an iron chelator worked synergistically with NO in the dispersal of biofilms.IMPORTANCE Nitric oxide (NO), which induces biofilm dispersal, is a promising strategy for biofilm control in both clinical and industrial contexts. However, competing environmental signals may reduce the efficacy of NO. The results presented here suggest that the presence of iron represents one such environmental cue that antagonizes the activity of NO as a biofilm-dispersing agent. Based on this understanding, we developed a strategy to enhance dispersal by combining NO with an iron-scavenging agent. Overall, this study links two important environmental signals, iron and NO, with their roles in biofilm development and suggests new ways for improving the use of NO in biofilm control strategies.

Dataset Information

The DeltaF508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability.

Publications

The DeltaF508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability.

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