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

Abstract

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.

Figures

FIG 1

Direct detection of iron abundance and oxidation state within CF sputum. Total iron [Fe(III) plus Fe(II)] (A), Fe(II) (B), and Fe(II) % (D) all increase as pulmonary function (FEV1%) declines. There is no significant increase in Fe(III) (C). Each point represents the average of measurements on multiple sputum samples from a single CF patient.

FIG 2

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).

FIG 3

(A) Fe(II)-relevant gene expression in CF sputum. bqsS is upregulated in planktonic cultures of P. aeruginosa in response to 50 µM Fe(II) (black) relative to 50 µM Fe(III) (white) or no treatment (light gray). A similar result is seen with bqsR. Points represent average CT values from three independent experiments; bars represent the standard deviations. By comparison, expression levels of these Fe(II)-sensitive genes in CF sputum (dark gray) vary over 5 orders of magnitude. Points represent relative gene expression calculated from CT values from triplicate measurements of an individual sputum sample. Transcriptional activity is shown relative to the endogenous housekeeping gene oprI. (B) Expression of diverse iron uptake pathways within CF sputum. feoA and feoB encode proteins that transport Fe(II), while fptA, pvdA, and hasAp encode proteins that are involved in Fe(III) acquisition. Expression levels are shown compared to those in laboratory cultures treated with Fe(II), Fe(III), and no iron as described above.

FIG 4

(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).