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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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(A and B) Biofilm growth prevention under aerobic conditions [~98% Fe(III)] (A) and anaerobic conditions [~10 µM Fe(II) and 10 µM Fe(III)] (B) by conalbumin [a Fe(III) chelator] and ferrozine [a Fe(II) chelator]. (C and D) Biofilm dissolution under aerobic (C) and anaerobic (D) conditions by conalbumin and ferrozine. In all cases, chelator effects are mitigated by the addition of Fe in excess of the chelation capacity [80 µM Fe(III) under oxic conditions; Fe(II) under anoxia]. Asterisks represent significance versus untreated controls. Error bars represent standard errors of the means (n = 12).
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fig4: (A and B) Biofilm growth prevention under aerobic conditions [~98% Fe(III)] (A) and anaerobic conditions [~10 µM Fe(II) and 10 µM Fe(III)] (B) by conalbumin [a Fe(III) chelator] and ferrozine [a Fe(II) chelator]. (C and D) Biofilm dissolution under aerobic (C) and anaerobic (D) conditions by conalbumin and ferrozine. In all cases, chelator effects are mitigated by the addition of Fe in excess of the chelation capacity [80 µM Fe(III) under oxic conditions; Fe(II) under anoxia]. Asterisks represent significance versus untreated controls. Error bars represent standard errors of the means (n = 12).

Mentions: Given that the CF sputum environment contains a mixture of Fe(III) and Fe(II), what implications does this have for treating biofilm infections? Might abundant Fe(II) levels in infected environments compromise the success of Fe(III)-specific chelation therapies targeting P. aeruginosa? This was first suggested in a recent study that tested the efficacy of several iron-binding compounds in the disruption of P. aeruginosa biofilm growth under both oxic and hypoxic conditions (14). While biofilm formation was prevented under most conditions tested, the specific oxidation state of iron was unknown. Motivated by these experiments, we utilized a high-throughput biofilm assay to measure biofilm formation in the presence of Fe(III) and Fe(II) with or without oxidation-state-specific iron chelators. First, we tested whether ferrozine, a Fe(II)-specific chelator, could act synergistically with conalbumin, a Fe(III)-specific chelator, to prevent biofilm development. Consistent with previous studies (10, 14), 100 µM conalbumin prevented biofilm formation by 66% (P < 0.001) under aerobic conditions where all iron (20 µM) was Fe(III) (Fig. 4A). In contrast, 200 µM ferrozine [Fe(II) specific] had no significant effect, nor did the combination of ferrozine and conalbumin treatments relative to conalbumin alone. Conversely, under hypoxic conditions designed to mimic airway microenvironments during late-stage infection, conalbumin was ineffective in preventing biofilm accumulation when ~10 µM Fe(II) and 10 µM Fe(III) were present (Fig. 4B). Here, 200 µM ferrozine significantly reduced biofilm accumulation by 29% (P = 0.012), and more notably, the combination of 100 µM conalbumin and 200 µM ferrozine reduced biofilm accumulation by 54% (P < 0.001), suggesting that targeting both oxidation states of iron in vivo might be more effective than targeting Fe(III) alone in the prevention of biofilm growth. Under both oxic and anoxic conditions, the addition of 80 µM iron (resulting in 100 µM total) in excess of the chelation capacity (conalbumin binds iron in a 2:1 ratio; ferrozine binds in a 3:1 ratio) restored biofilm accumulation, demonstrating that the chelator effect is likely due to iron sequestration rather than nonspecific interactions.


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)

(A and B) Biofilm growth prevention under aerobic conditions [~98% Fe(III)] (A) and anaerobic conditions [~10 µM Fe(II) and 10 µM Fe(III)] (B) by conalbumin [a Fe(III) chelator] and ferrozine [a Fe(II) chelator]. (C and D) Biofilm dissolution under aerobic (C) and anaerobic (D) conditions by conalbumin and ferrozine. In all cases, chelator effects are mitigated by the addition of Fe in excess of the chelation capacity [80 µM Fe(III) under oxic conditions; Fe(II) under anoxia]. Asterisks represent significance versus untreated controls. Error bars represent standard errors of the means (n = 12).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig4: (A and B) Biofilm growth prevention under aerobic conditions [~98% Fe(III)] (A) and anaerobic conditions [~10 µM Fe(II) and 10 µM Fe(III)] (B) by conalbumin [a Fe(III) chelator] and ferrozine [a Fe(II) chelator]. (C and D) Biofilm dissolution under aerobic (C) and anaerobic (D) conditions by conalbumin and ferrozine. In all cases, chelator effects are mitigated by the addition of Fe in excess of the chelation capacity [80 µM Fe(III) under oxic conditions; Fe(II) under anoxia]. Asterisks represent significance versus untreated controls. Error bars represent standard errors of the means (n = 12).
Mentions: Given that the CF sputum environment contains a mixture of Fe(III) and Fe(II), what implications does this have for treating biofilm infections? Might abundant Fe(II) levels in infected environments compromise the success of Fe(III)-specific chelation therapies targeting P. aeruginosa? This was first suggested in a recent study that tested the efficacy of several iron-binding compounds in the disruption of P. aeruginosa biofilm growth under both oxic and hypoxic conditions (14). While biofilm formation was prevented under most conditions tested, the specific oxidation state of iron was unknown. Motivated by these experiments, we utilized a high-throughput biofilm assay to measure biofilm formation in the presence of Fe(III) and Fe(II) with or without oxidation-state-specific iron chelators. First, we tested whether ferrozine, a Fe(II)-specific chelator, could act synergistically with conalbumin, a Fe(III)-specific chelator, to prevent biofilm development. Consistent with previous studies (10, 14), 100 µM conalbumin prevented biofilm formation by 66% (P < 0.001) under aerobic conditions where all iron (20 µM) was Fe(III) (Fig. 4A). In contrast, 200 µM ferrozine [Fe(II) specific] had no significant effect, nor did the combination of ferrozine and conalbumin treatments relative to conalbumin alone. Conversely, under hypoxic conditions designed to mimic airway microenvironments during late-stage infection, conalbumin was ineffective in preventing biofilm accumulation when ~10 µM Fe(II) and 10 µM Fe(III) were present (Fig. 4B). Here, 200 µM ferrozine significantly reduced biofilm accumulation by 29% (P = 0.012), and more notably, the combination of 100 µM conalbumin and 200 µM ferrozine reduced biofilm accumulation by 54% (P < 0.001), suggesting that targeting both oxidation states of iron in vivo might be more effective than targeting Fe(III) alone in the prevention of biofilm growth. Under both oxic and anoxic conditions, the addition of 80 µM iron (resulting in 100 µM total) in excess of the chelation capacity (conalbumin binds iron in a 2:1 ratio; ferrozine binds in a 3:1 ratio) restored biofilm accumulation, demonstrating that the chelator effect is likely due to iron sequestration rather than nonspecific interactions.

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