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Ferrous iron is a significant component of bioavailable iron in cystic fibrosis airways.

Hunter RC, Asfour F, Dingemans J, Osuna BL, Samad T, Malfroot A, Cornelis P, Newman DK - MBio (2013)

Bottom Line: Previous studies have focused on ferric iron [Fe(III)] as a target for antimicrobial therapies; however, here we show that ferrous iron [Fe(II)] is abundant in the CF lung (-39 µM on average for severely sick patients) and significantly correlates with disease severity (ρ = -0.56, P = 0.004), whereas ferric iron does not (ρ = -0.28, P = 0.179).Because limiting Fe(III) acquisition inhibits biofilm formation by P. aeruginosa in various oxic in vitro systems, we also tested whether interfering with Fe(II) acquisition would improve biofilm control under anoxic conditions; concurrent sequestration of both iron oxidation states resulted in a 58% reduction in biofilm accumulation and 28% increase in biofilm dissolution, a significant improvement over Fe(III) chelation treatment alone.Ferric iron chelation therapy has been proposed as a novel therapeutic strategy for CF lung infections, yet until now, the iron oxidation state has not been measured in the host.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, California, USA.

ABSTRACT

Unlabelled: ABSTRACT Chronic, biofilm-like infections by the opportunistic pathogen Pseudomonas aeruginosa are a major cause of mortality in cystic fibrosis (CF) patients. While much is known about P. aeruginosa from laboratory studies, far less is understood about what it experiences in vivo. Iron is an important environmental parameter thought to play a central role in the development and maintenance of P. aeruginosa infections, for both anabolic and signaling purposes. Previous studies have focused on ferric iron [Fe(III)] as a target for antimicrobial therapies; however, here we show that ferrous iron [Fe(II)] is abundant in the CF lung (-39 µM on average for severely sick patients) and significantly correlates with disease severity (ρ = -0.56, P = 0.004), whereas ferric iron does not (ρ = -0.28, P = 0.179). Expression of the P. aeruginosa genes bqsRS, whose transcription is upregulated in response to Fe(II), was high in the majority of patients tested, suggesting that increased Fe(II) is bioavailable to the infectious bacterial population. Because limiting Fe(III) acquisition inhibits biofilm formation by P. aeruginosa in various oxic in vitro systems, we also tested whether interfering with Fe(II) acquisition would improve biofilm control under anoxic conditions; concurrent sequestration of both iron oxidation states resulted in a 58% reduction in biofilm accumulation and 28% increase in biofilm dissolution, a significant improvement over Fe(III) chelation treatment alone. This study demonstrates that the chemistry of infected host environments coevolves with the microbial community as infections progress, which should be considered in the design of effective treatment strategies at different stages of disease.

Importance: Iron is an important environmental parameter that helps pathogens thrive in sites of infection, including those of cystic fibrosis (CF) patients. Ferric iron chelation therapy has been proposed as a novel therapeutic strategy for CF lung infections, yet until now, the iron oxidation state has not been measured in the host. In studying mucus from the infected lungs of multiple CF patients from Europe and the United States, we found that ferric and ferrous iron change in concentration and relative proportion as infections progress; over time, ferrous iron comes to dominate the iron pool. This information is relevant to the design of novel CF therapeutics and, more broadly, to developing accurate models of chronic CF infections.

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Fe(II) percentage of the total iron pool relative to sputum phenazine content. Fe(II) dominates the iron pool at high concentrations of total phenazines (PYO plus PCA) (A) and phenazine-1-carboxylic acid (PCA) (B) but not pyocyanin (PYO) (C). These data likely reflect the higher reactivity of PCA with Fe(III) under anoxic conditions (30).
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fig2: Fe(II) percentage of the total iron pool relative to sputum phenazine content. Fe(II) dominates the iron pool at high concentrations of total phenazines (PYO plus PCA) (A) and phenazine-1-carboxylic acid (PCA) (B) but not pyocyanin (PYO) (C). These data likely reflect the higher reactivity of PCA with Fe(III) under anoxic conditions (30).

Mentions: The alteration of total iron concentrations and the rise in Fe(II) over time likely result from multiple inputs by both host and pathogen (24, 26). For example, iron levels are known to increase due to inflammation (27); loss of intracellular iron by ΔF508 epithelial cells (28); altered production of the iron-related proteins heme, ferritin, and transferrin (26); and their proteolysis (29). In addition, redox-active phenazine metabolites produced by P. aeruginosa are abundant in CF sputum (22), some of which can readily reduce Fe(III) to Fe(II) (30). Iron reduction by phenazines has been demonstrated to circumvent iron chelation in vitro, promoting the formation of biofilms (31). Based on our recent demonstration of a strong correlation between sputum phenazine levels and pulmonary decline (22), we used high-pressure liquid chromatography (HPLC) to assess whether elevated levels of two phenazines, pyocyanin (PYO) and phenazine-1-carboxylic acid (PCA), also correlated with high Fe(II) concentrations. Consistent with our previous findings from an independent adult patient cohort, the majority of sputum samples tested had detectable phenazine concentrations (76 of 97 samples tested contained >10 µM total phenazine; see Table S1 in the supplemental material). In sputum samples with low concentrations of phenazines, the percentage of the total iron pool that was Fe(II) ranged anywhere from 0 to 100%, revealing that phenazines are not required for the presence of ferrous iron (Fig. 2A). Yet, phenazines may facilitate Fe(III) reduction in vivo, as evidenced by the generally high percentage of Fe(II) once phenazine levels rise above ~50 µM in expectorated sputum. Treating each sputum sample independently, we found a strong trend between PCA abundance and Fe(II) % (ρ = 0.185, P = 0.069), yet no correlation between PYO abundance and Fe(II) % (ρ = 0.042, P = 0.67) (Fig. 2B and C). This may reflect that PCA can reduce Fe(III) much faster than can PYO under anoxic conditions (30). It remains to be determined whether the PCA/Fe(II) % trend would pass our test of statistical significance (P < 0.05) with additional sampling. We treated samples independently in this analysis in order to compare phenazine concentrations and Fe(II) % within a particular environment; given the variability of sputum chemistry over time (and likely also space) for individual patients (see Fig. S2 and Table S1), averaging and comparing these values per patient would not have been meaningful.


Ferrous iron is a significant component of bioavailable iron in cystic fibrosis airways.

Hunter RC, Asfour F, Dingemans J, Osuna BL, Samad T, Malfroot A, Cornelis P, Newman DK - MBio (2013)

Fe(II) percentage of the total iron pool relative to sputum phenazine content. Fe(II) dominates the iron pool at high concentrations of total phenazines (PYO plus PCA) (A) and phenazine-1-carboxylic acid (PCA) (B) but not pyocyanin (PYO) (C). These data likely reflect the higher reactivity of PCA with Fe(III) under anoxic conditions (30).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753050&req=5

fig2: Fe(II) percentage of the total iron pool relative to sputum phenazine content. Fe(II) dominates the iron pool at high concentrations of total phenazines (PYO plus PCA) (A) and phenazine-1-carboxylic acid (PCA) (B) but not pyocyanin (PYO) (C). These data likely reflect the higher reactivity of PCA with Fe(III) under anoxic conditions (30).
Mentions: The alteration of total iron concentrations and the rise in Fe(II) over time likely result from multiple inputs by both host and pathogen (24, 26). For example, iron levels are known to increase due to inflammation (27); loss of intracellular iron by ΔF508 epithelial cells (28); altered production of the iron-related proteins heme, ferritin, and transferrin (26); and their proteolysis (29). In addition, redox-active phenazine metabolites produced by P. aeruginosa are abundant in CF sputum (22), some of which can readily reduce Fe(III) to Fe(II) (30). Iron reduction by phenazines has been demonstrated to circumvent iron chelation in vitro, promoting the formation of biofilms (31). Based on our recent demonstration of a strong correlation between sputum phenazine levels and pulmonary decline (22), we used high-pressure liquid chromatography (HPLC) to assess whether elevated levels of two phenazines, pyocyanin (PYO) and phenazine-1-carboxylic acid (PCA), also correlated with high Fe(II) concentrations. Consistent with our previous findings from an independent adult patient cohort, the majority of sputum samples tested had detectable phenazine concentrations (76 of 97 samples tested contained >10 µM total phenazine; see Table S1 in the supplemental material). In sputum samples with low concentrations of phenazines, the percentage of the total iron pool that was Fe(II) ranged anywhere from 0 to 100%, revealing that phenazines are not required for the presence of ferrous iron (Fig. 2A). Yet, phenazines may facilitate Fe(III) reduction in vivo, as evidenced by the generally high percentage of Fe(II) once phenazine levels rise above ~50 µM in expectorated sputum. Treating each sputum sample independently, we found a strong trend between PCA abundance and Fe(II) % (ρ = 0.185, P = 0.069), yet no correlation between PYO abundance and Fe(II) % (ρ = 0.042, P = 0.67) (Fig. 2B and C). This may reflect that PCA can reduce Fe(III) much faster than can PYO under anoxic conditions (30). It remains to be determined whether the PCA/Fe(II) % trend would pass our test of statistical significance (P < 0.05) with additional sampling. We treated samples independently in this analysis in order to compare phenazine concentrations and Fe(II) % within a particular environment; given the variability of sputum chemistry over time (and likely also space) for individual patients (see Fig. S2 and Table S1), averaging and comparing these values per patient would not have been meaningful.

Bottom Line: Previous studies have focused on ferric iron [Fe(III)] as a target for antimicrobial therapies; however, here we show that ferrous iron [Fe(II)] is abundant in the CF lung (-39 µM on average for severely sick patients) and significantly correlates with disease severity (ρ = -0.56, P = 0.004), whereas ferric iron does not (ρ = -0.28, P = 0.179).Because limiting Fe(III) acquisition inhibits biofilm formation by P. aeruginosa in various oxic in vitro systems, we also tested whether interfering with Fe(II) acquisition would improve biofilm control under anoxic conditions; concurrent sequestration of both iron oxidation states resulted in a 58% reduction in biofilm accumulation and 28% increase in biofilm dissolution, a significant improvement over Fe(III) chelation treatment alone.Ferric iron chelation therapy has been proposed as a novel therapeutic strategy for CF lung infections, yet until now, the iron oxidation state has not been measured in the host.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, California, USA.

ABSTRACT

Unlabelled: ABSTRACT Chronic, biofilm-like infections by the opportunistic pathogen Pseudomonas aeruginosa are a major cause of mortality in cystic fibrosis (CF) patients. While much is known about P. aeruginosa from laboratory studies, far less is understood about what it experiences in vivo. Iron is an important environmental parameter thought to play a central role in the development and maintenance of P. aeruginosa infections, for both anabolic and signaling purposes. Previous studies have focused on ferric iron [Fe(III)] as a target for antimicrobial therapies; however, here we show that ferrous iron [Fe(II)] is abundant in the CF lung (-39 µM on average for severely sick patients) and significantly correlates with disease severity (ρ = -0.56, P = 0.004), whereas ferric iron does not (ρ = -0.28, P = 0.179). Expression of the P. aeruginosa genes bqsRS, whose transcription is upregulated in response to Fe(II), was high in the majority of patients tested, suggesting that increased Fe(II) is bioavailable to the infectious bacterial population. Because limiting Fe(III) acquisition inhibits biofilm formation by P. aeruginosa in various oxic in vitro systems, we also tested whether interfering with Fe(II) acquisition would improve biofilm control under anoxic conditions; concurrent sequestration of both iron oxidation states resulted in a 58% reduction in biofilm accumulation and 28% increase in biofilm dissolution, a significant improvement over Fe(III) chelation treatment alone. This study demonstrates that the chemistry of infected host environments coevolves with the microbial community as infections progress, which should be considered in the design of effective treatment strategies at different stages of disease.

Importance: Iron is an important environmental parameter that helps pathogens thrive in sites of infection, including those of cystic fibrosis (CF) patients. Ferric iron chelation therapy has been proposed as a novel therapeutic strategy for CF lung infections, yet until now, the iron oxidation state has not been measured in the host. In studying mucus from the infected lungs of multiple CF patients from Europe and the United States, we found that ferric and ferrous iron change in concentration and relative proportion as infections progress; over time, ferrous iron comes to dominate the iron pool. This information is relevant to the design of novel CF therapeutics and, more broadly, to developing accurate models of chronic CF infections.

Show MeSH
Related in: MedlinePlus