mBio-2013-Hunter.pdf

Ferrous Iron Is a Significant Component of Bioavailable Iron in Cystic

Fibrosis Airways

Ryan C. Hunter

a,c

Fadi Asfour

Jozef Dingemans

e,f

Brenda L. Osuna

Tahoura Samad

Anne Malfroot

Pierre Cornelis

Dianne K. Newman

a,b,c

Division of Biology,

Division of Geological and Planetary Sciences,

and Howard Hughes Medical Institute,

California Institute of Technology, Pasadena, California, USA;

Department of Pediatric Pulmonology, Children’s Hospital of Los Angeles, Los Angeles, California, USA

; Department of Bioengineering Sciences, Research Group

Microbiology, Flanders Interuniversity Institute of Biotechnology (VIB), Vrije Universiteit Brussel, Brussels, Belgium

; Unit of Microbiology, Expert Group Molecular and

Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK CEN), Mol, Belgium

; Statistical Consulting, Information Technology

Services, University of Southern California, Los Angeles, California, USA

; Cystic Fibrosis Clinic, Universitair Ziekenhuis Brussel (UZB), Brussels, Belgium

ABSTRACT

Chronic, biofilm-like infections by the opportunistic pathogen

Pseudomonas aeruginosa

are a major cause of mortal-

ity 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 develop-

ment 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

M on average for severely sick patients) and significantly correlates with disease severity (

0.56,

0.004),

whereas ferric iron does not (

0.28,

0.179). Expression of the

P. aeruginosa

genes

bqsRS

, whose transcription is upregu-

lated in response to Fe(II), was high in the majority of patients tested, suggesting that increased Fe(II) is bioavailable to the infec-

tious bacterial population. Because limiting Fe(III) acquisition inhibits biofilm formation by

P. aeruginosa

in various oxic

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 chemis-

try 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 infec-

tions, 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 rela-

tive 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.

Received

19 July 2013

Accepted

23 July 2013

Published

20 August 2013

Citation

Hunter RC, Asfour F, Dingemans J, Osuna BL, Samad T, Malfroot A, Cornelis P, Newman DK. 2013. Ferrous iron is a significant component of bioavailable iron in cystic

fibrosis airways. mBio 4(4):e00557-13. doi:10.1128/mBio.00557-13.

Editor

Arturo Casadevall, Albert Einstein College of Medicine

Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported

license

, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

Address correspondence to Dianne K. Newman, dkn@caltech.edu.

oth culture-dependent and molecular identification methods

suggest that

Pseudomonas aeruginosa

is a dominant bacterial

pathogen of patients with cystic fibrosis (CF) (1). As

P. aeruginosa

adapts to the host environment, it adopts a biofilm-like lifestyle

associated with enhanced antibiotic resistance, persistent infec-

tions, and poor pulmonary prognosis (2). Numerous studies have

documented how

P. aeruginosa

responds to specific environmen-

tal cues, including hypoxia, that are thought to be relevant for

biofilm formation and chronic colonization

in vivo

(3, 4); how-

ever, only a few studies have directly measured chemical parame-

ters within the host, and usually only at a single time point (5, 6).

This knowledge gap is an important one to fill because pathogens

are known to coevolve with host environmental chemistry with

respect to their metabolic programs and growth phenotypes, in-

cluding biofilm formation (2, 7). Toward this end, we sought to

characterize the oxidation state of iron within CF sputum and the

biological response of

P. aeruginosa

at different disease states for a

cross section of CF patients.

The competition for iron between pathogens and the human

host has been extensively studied due to its critical importance in

pathogenesis (8, 9). In particular, iron has been identified as an

important parameter that plays a central role in the development

and maintenance of

P. aeruginosa

biofilm infections within the

lung (10–12). On this basis, iron uptake and acquisition pathways

have been identified as potential antimicrobial targets (10–15).

While microbial ferrous iron [Fe(II)] acquisition pathways are

known (16), therapeutic strategies designed to iron-limit patho-

gens have focused on blocking ferric iron [Fe(III)] acquisition

RESEARCH ARTICLE

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because it is commonly assumed to be the dominant physiologi-

cally relevant form. For example, Fe(III) chelation by the host

immune protein lactoferrin and its analog, conalbumin, has been

shown to dramatically reduce biofilm formation under oxic con-

ditions (11–13). EDTA and FDA-approved iron chelation com-

pounds are similarly effective at mitigating biofilm growth while

concomitantly increasing the efficacy of conventional antibiotics

(13, 15). In addition, the transition metal gallium (also FDA ap-

proved) has been shown to disrupt biofilm formation by virtue of

its chemical similarity to iron, disrupting Fe(III) uptake and in-

terfering with Fe signaling (17, 18).

Despite the success of these Fe sequestration strategies in lab-

oratory models, for many of them to be effective

in vivo

, iron

would need to exist in the ferric form. However, in late-stage dis-

ease, oxygen tension is reduced in the lung (5) and neutrophil-

mediated superoxides are generated that can reduce organically

complexed Fe(III) to Fe(II) (19, 20). In addition, dense, biofilm-

like microcolonies form within sputum that can create hypoxic

microenvironments that can maintain iron in its reduced state

(21). Furthermore,

P. aeruginosa

produces a variety of redox-

active metabolites in CF sputum (e.g., phenazines) (22) that can

reduce Fe(III) to Fe(II), even when iron is bound to host chelation

proteins (23). While the CF lung environment is likely to be chem-

ically heterogeneous, with oxic, hypoxic, and anoxic zones, the

potential for locally anoxic and reducing conditions caused us to

suspect that Fe(II) might be abundant in the airways. This report

describes how we tested this hypothesis and explored its conse-

quences for the development of iron-specific therapeutic strate-

gies.

RESULTS

Total and ferrous iron concentrations increase within the lung

environment as infections progress.

Although the total concen-

tration of iron has been measured in the airways (24, 25) and is

known to accumulate in the lavage and explanted lungs of CF

patients (26), its oxidation state has not been defined. We there-

fore set out to measure Fe(II) abundance in CF sputum at differ-

ent stages of disease progression. Accurately measuring the iron

oxidation state is complicated by the rapid oxidation of Fe(II) to

Fe(III) once expectorated sputum is exposed to ambient oxygen.

With this in mind, we designed a sputum collection and process-

ing approach to better preserve and measure iron in its

in vivo

oxidation state. Twenty-four pediatric patients from across the

spectrum of disease severity provided 115 sputum samples that

were rapidly flash frozen upon expectoration. Samples were then

moved to an anaerobic chamber to impede oxidation and me-

chanically homogenized by syringe, and ratios of free Fe(II)/

Fe(III) concentrations were then determined using the ferrozine

assay. As controls, total iron levels were also assayed using induc-

tively coupled plasma mass spectrometry (ICP-MS) and un-

treated samples stored under argon were compared to flash-

frozen samples to test whether the iron oxidation state was

faithfully preserved during cryostorage (see Fig. S1 in the supple-

mental material).

Due to the temporal variability of sputum iron concentrations

and differences in the number of samples that we were able to

collect for a given patient (see Table S1 in the supplemental ma-

terial), we grouped data over the entire period of the study and

treated each patient’s iron measurements as an average, rather

than as independent observations, in order to test for correlations

with patient disease state (Table 1). Using this data set, clustered

by patient (

24), Spearman rank analysis revealed a significant

negative correlation (

0.48,

0.018) between total iron

and declining lung function (measured by forced expiratory vol-

ume [FEV1%]) (Fig. 1A; Table 2). Elevated iron levels (62

M for severely infected patients) (Table 1) were consistent

with previous studies that quantified total iron levels and iron-

related proteins in the CF airways (24–26). Consistent with our

hypothesis, a considerable amount of this iron was found in its

ferrous form, as sputum from severely infected patients had 39

M Fe(II). Here, we found a highly significant negative corre-

lation between absolute Fe(II) concentrations and disease status

(

0.56,

0.004) (Fig. 1B), though a similar relationship

was not found for Fe(III) (

0.28,

0.179) (Fig. 1C). The

percentage of the total iron pool that was present as Fe(II) was also

higher (though not significantly:

0.36,

0.083) in pa-

tients with advanced disease states; in patients with severe lung

obstruction (FEV1%,

40), Fe(II) constituted 56%

15% of the

total iron pool (Fig. 1D). These data reveal that the chemical en-

vironment of the lung is dynamic and evolves with respect to its

iron redox chemistry as CF disease progresses.

Increased Fe(II) correlates with elevated phenazine concen-

trations.

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

F508 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

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 ele-

vated 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

M total phenazine; see Table S1 in the

supplemental material). In sputum samples with low concentra-

tions 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

TABLE 1

Summary of average Fe concentrations grouped by disease

severity

Disease severity FEV1%

Total Fe (

M) Fe(II) (

M) Fe(II) %

Normal to mild

7 18

841

Moderate

40–69 12 48

Severe

5 62

Reported values are mean concentrations

1 standard deviation of iron detected in

sputum samples collected over the study period. Values are conservative estimates

based on ferrozine and ICP-MS measurements (see Fig. S1 in the supplemental

material).

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rise above ~50

M in expectorated sputum. Treating each sputum

sample independently, we found a strong trend between PCA

abundance and Fe(II) % (

0.185,

0.069), yet no correlation

between PYO abundance and Fe(II) % (

0.042,

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 re-

mains to be determined whether the PCA/Fe(II) % trend would

pass our test of statistical significance (

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.

Fe(II)-responsive genes and multiple iron uptake pathways

are expressed by

P. aeruginosa

within CF sputum.

Given the

abundance of Fe(II)

in vitro

, we predicted that extracellular Fe(II)

would be bioavailable to

P. aeruginosa

within the airways. To test

this, we used a quantitative real-time PCR

(qRT-PCR) approach to measure the ex-

pression patterns of two Fe(II)-

responsive genes within expectorated

sputum relative to their expression pat-

terns under controlled conditions. The

P. aeruginosa

genes

bqsR

and

bqsS

encode

a putative response regulator and sensor

kinase, respectively, of a two-component

system that was previously shown to be

specifically upregulated in response to ex-

tracellular Fe(II) (32). We mined the

NCBI Gene Expression Omnibus (GEO)

database (

http://www.ncbi.nlm.nih.gov

/geo/

) for microarray data generated for

P. aeruginosa

grown under conditions

relevant to the CF lung environment.

These data sets revealed no differential

expression of

bqsRS

in response to multi-

ple environmental stimuli, including low

oxygen, pH, phosphate starvation, oxida-

tive stress, biofilm formation, and various

antibiotic treatments (see Table S2 in the

supplemental material). Thus,

bqsRS

ex-

pression levels (relative to the constitu-

tively expressed gene

oprI

) serve as a reli-

able proxy for the bioavailability of Fe(II)

in the lung.

As previously observed, anaerobically

grown laboratory cultures of

P. aerugi-

nosa

upregulated

bqsS

(

90-fold) in re-

sponse to 50

M Fe(II) relative to no

treatment or treatment with 50

M Fe(III) (Fig. 3A). Likewise,

bqsR

was highly expressed (

70-fold) in response to Fe(II) rela-

tive to other treatments. We then utilized these gene expression

patterns in controlled cultures to gain insight into the iron oxida-

tion state perceived by

P. aeruginosa

in sputum. Consistent with

our direct iron analyses, when

bqsR

and

bqsS

transcripts from

sputum samples were quantified, expression was detected within

the majority of patients (Fig. 3A). Transcriptional activity varied

between sputum samples; however, 8 of 16 patients harbored rel-

ative

bqsS

expression patterns comparable to those for Fe(II)-

treated laboratory cultures.

bqsR

transcripts were also detected in

the majority of sputum samples that we tested, and many had

relative expression levels comparable to those for Fe(II)-treated

planktonic cultures. Unfortunately, technical limitations pre-

vented us from measuring gene expression and iron content in the

same sputum sample (see Materials and Methods). Despite poten-

tial differences in degradation rates for each transcript (see Fig. S3

in the supplemental material), given the high direct measure-

ments of Fe(II) in sputum, previous microarray data, and our

control experiments showing specific upregulation of

bqsRS

response to Fe(II), we favor the interpretation that the iron pool

within the CF airways is of a mixed oxidation state and that the

infected lung environment frequently includes a ferrous portion

that is sensed by

P. aeruginosa

In a majority (75%) of patients, the prevalences of

bqsR

and

bqsS

gene expression were comparable within patients (see Fig. S4

in the supplemental material); however, there was not a significant

correlation between

bqsRS

transcriptional levels and disease sever-

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.

TABLE 2

Summary of statistical relationships between iron

concentrations and disease severity (FEV1%)

Spearman rank

coefficient

Sig.

(two-tailed)

Total iron

0.48

0.018

Fe(III)

0.21

0.316

Fe(II)

0.56

0.004

Fe(II) %

0.36

0.083

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ity (

bqsR

0.13;

bqsS

0.01) (see Fig. S5). This is not

surprising because the relative expression of each gene was previ-

ously shown to be upregulated in response to Fe(II) levels greater

than 10

M. In our patient cohort, on average, Fe(II) was fre-

quently detected at levels above 10

M, even in patients in the

early stages of disease. Thus, one would expect expression of these

Fe(II)-sensitive genes across the spectrum of disease severity in

response to the mixed-oxidation state of the iron pool.

A mixed-oxidation state of lung iron is further supported by

our detection of

P. aeruginosa

gene transcripts encoding diverse

Fe(II)- and Fe(III)-specific uptake proteins (Fig. 3B). We targeted

feoA

(encoding a ferrous iron transporter),

fptA

(ferripyochelin

receptor),

pvdA

(pyoverdine biosynthetic protein), and

hasAp

(heme uptake protein) and quantified their relative expression

levels in each sputum sample relative to

oprI

. Transcripts for each

gene were detected in the majority of sputum samples analyzed

(

feoA

, 15/16 patients;

feoB

, 11/16;

fptA

, 13/16;

pvdA

, 11/16;

hasAp

14/16), though relative expression levels of each gene varied over 5

orders of magnitude between patients. Uptake systems for the two

iron oxidation states were simultaneously expressed within sev-

eral individual patients (see Fig. S4 in the supplemental material),

consistent with a recent study that investigated the expression of

these genes in an independent patient cohort (33). Yet, the expres-

sion of Fe(II) uptake pathways did not correlate with the suppres-

sion of uptake pathways specific for Fe(III), or vice versa. Further-

more, because the regulation of these iron uptake pathways is

complex (34, 35) and some (

pvdA

fptA

, and

hasAp

) appear to be

independent from the oxidation state under anoxic conditions

(Fig. 3B), these expression patterns alone are not predictive of the

iron oxidation state

in vivo

. Rather, the expression of multiple iron

uptake systems is supportive of our interpretation that

P. aerugi-

nosa

utilizes a mixed-oxidation pool of iron within the CF sputum

environment.

Interfering with bioavailable Fe(II) limits biofilm formation

under anoxic conditions.

Given that the

CF sputum environment contains a mix-

ture of Fe(III) and Fe(II), what implica-

tions 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

aeruginosa

biofilm growth under both

oxic and hypoxic conditions (14). While

biofilm formation was prevented under

most conditions tested, the specific oxi-

dation state of iron was unknown. Moti-

vated by these experiments, we utilized a

high-throughput biofilm assay to mea-

sure 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 syner-

gistically with conalbumin, a Fe(III)-

specific chelator, to prevent biofilm de-

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

M Fe(II) (black) relative to 50

M Fe(III) (white) or no treatment (light

gray). A similar result is seen with

bqsR

. Points represent average

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

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.

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velopment. Consistent with previous studies (10, 14), 100

conalbumin prevented biofilm formation by 66% (

0.001)

under aerobic conditions where all iron (20

M) was Fe(III)

(Fig. 4A). In contrast, 200

M ferrozine [Fe(II) specific] had no

significant effect, nor did the combination of ferrozine and conal-

bumin treatments relative to conalbumin alone. Conversely, un-

der hypoxic conditions designed to mimic airway microenviron-

ments during late-stage infection, conalbumin was ineffective in

preventing biofilm accumulation when ~10

M Fe(II) and 10

Fe(III) were present (Fig. 4B). Here, 200

M ferrozine signifi-

cantly reduced biofilm accumulation by 29% (

0.012), and

more notably, the combination of 100

M conalbumin and

200

M ferrozine reduced biofilm accumulation by 54% (

0.001), suggesting that targeting both oxidation states of iron

vivo

might be more effective than targeting Fe(III) alone in the

prevention of biofilm growth. Under both oxic and anoxic condi-

tions, the addition of 80

M iron (resulting in 100

M total) in

excess of the chelation capacity (conalbumin binds iron in a 2:1

ratio; ferrozine binds in a 3:1 ratio) restored biofilm accumula-

tion, demonstrating that the chelator effect is likely due to iron

sequestration rather than nonspecific interactions.

Combined Fe(III)/Fe(II) chelation promotes biofilm disso-

lution under anoxic conditions.

In addition to signaling biofilm

formation, iron is essential for maintenance of established biofilm

communities (12). We therefore performed similar mixed Fe(II)/

Fe(III) chelation experiments targeting mature biofilms to test the

ability of conalbumin and ferrozine to dissolve bacterial biofilms

that have already formed. Under aerobic

conditions (Fig. 4C), the application of

either conalbumin (100

M) or ferrozine

(200

M) in molar excess of iron in the

growth medium showed minimal effect

on biofilm dissolution. We hypothesized

that this is due to the presence of both

Fe(III) and Fe(II) in the hypoxic interior

of aerobically grown biofilms. Consistent

with this prediction, the combined appli-

cation of both Fe(III) and Fe(II) chelators

revealed a synergistic dissolution effect,

resulting in a 33% reduction (

0.01) of

biomass in the presence of oxygen. The

addition of excess iron restored the un-

treated phenotype, corroborating an

iron-specific mechanism of chelator-

induced dispersal. Similarly, 100

conalbumin did not significantly reduce

established biofilm growth under anoxic

conditions (Fig. 4D). However, signifi-

cant biofilm dissolution (20%;

0.001)

was observed in the presence of 200

ferrozine, indicating that

P. aeruginosa

biofilms can reduce Fe(III) present in the

growth medium. More notably, when ap-

plied together with conalbumin, ferro-

zine promoted further dissolution of es-

tablished biofilms at levels comparable to

those under oxic conditions (28%;

0.001), supporting the case for targeting

both Fe(III) and Fe(II) to disrupt

P. aeruginosa

biofilm growth in the CF

airways. In contrast to previous experiments performed under

oxic conditions (15), dual exposure to iron chelators and tobra-

mycin did not exhibit a synergistic effect under anoxic conditions

(see Fig. S6 in the supplemental material).

DISCUSSION

The dependence of bacteria on iron acquisition for biofilm forma-

tion has led to its identification as a novel therapeutic to eliminate

P. aeruginosa

infections within the host, particularly for CF pa-

tients. However, as recently pointed out (14), there is a gap in our

understanding of the

in vivo

chemical environments under which

these treatments might be administered, which might significantly

impact their efficacy. Therefore, the goal of this study was to gain

a better understanding of iron chemistry within the lungs of CF

patients and determine how the

in vivo

environment might impact

the success of iron-specific therapies. Using a unique sputum sam-

pling and processing approach, we determined that Fe(II) com-

prises a significant component of the airway iron pool. We then

used quantitative PCR analysis to confirm that elevated Fe(II)

levels were freely available for the infecting bacterial population.

Finally, we found that interfering with a mixed-oxidation state

iron pool can limit biofilm formation and promote biofilm disso-

lution.

On average, each patient had 42

M total iron (range, 3.7 to

118

M) present in his or her sputum, which was highly depen-

dent on the stage of disease (Table 1). These values are consistent

with a range of studies reporting elevated iron levels in CF sputum

FIG 4

(A and B) Biofilm growth prevention under aerobic conditions [~98% Fe(III)] (A) and anaer-

obic conditions [~10

M Fe(II) and 10

M Fe(III)] (B) by conalbumin [a Fe(III) chelator] and ferro-

zine [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

M Fe(III) under oxic conditions; Fe(II) under anoxia]. Asterisks represent

significance versus untreated controls. Error bars represent standard errors of the means (

12).

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and bronchoalveolar lavage. For example, Reid et al. (24) and

Stites et al. (25) both determined that free iron and ferritin nega-

tively correlate with pulmonary function in clinically stable pa-

tients. Similarly, Gifford et al. (36) reported elevated sputum iron

levels in CF patients and yet found no association between Fe

concentrations and lung function in a mixed cohort of stable and

acutely ill subjects. Although we found a correlation between total

iron and FEV1%, this derives from Fe(II), not Fe(III).

Despite the wealth of data on total iron abundance in the CF

airways, to our knowledge, direct measurements of the iron oxi-

dation state have not been made previously. It has been suggested

that the abnormal acidification of the CF airways (37) and the

development of anoxic microenvironments in sputum (5) favor

the maintenance of a bioavailable Fe(II) pool; however, the rapid

oxidation of Fe(II) upon exposure to ambient oxygen has made

this hypothesis difficult to test. Our novel sputum sampling and

processing approach was able to circumvent this problem, and it is

clear that the percentage of the total iron pool that is Fe(II) is

substantial, particularly in severely ill patients (56%

15%).

While our data support the aforementioned low-pH/anoxic hy-

pothesis for iron reduction, it also seems likely that the accumu-

lation of redox-active phenazines over time contributes to a highly

reducing airway environment that can foster Fe(III) reduction to

Fe(II) (Fig. 2). Based on our laboratory observations that PCA-

mediated iron reduction facilitates biofilm formation (31) and a

correlation between concentrations of these metabolites and pul-

monary decline (22), we suggest that PCA-mediated iron reduc-

tion facilitates disease progression in CF patients.

We recognized a need to distinguish between detectable Fe(II)

levels (by ferrozine) and those which are bioavailable for

P. aerugi-

nosa in vivo

. For example, the total soluble iron measurements

determined by our analytical approach likely include a portion

that is bound to complexing ions or ligands that keep it in solu-

tion, though it might not be bioavailable. Moreover, ferrozine

does not react with heme-associated iron (38), which may repre-

sent an important iron source within the lung (4, 9). However,

because we could detect a high level of Fe(II)-specific

bqsRS

ex-

pression (relative to

oprI

) in a majority of sputum samples (com-

pared to tightly controlled laboratory cultures), we can conclude

that some portion of the Fe(II) pool is sensed by

P. aeruginosa in

vivo

. Consistent with previous

in vitro

studies demonstrating that

P. aeruginosa

grown in the presence of a sputum-derived medium

expresses diverse iron-acquisition-related genes (4), we found

genes involved in pyoverdine, pyochelin, and heme uptake to be

expressed in sputum, similar to another recent study (33). In ad-

dition, direct measurements have also confirmed the presence of

the siderophore pyoverdine in a high percentage of CF patients,

but not all, indicating that

P. aeruginosa

uses multiple mecha-

nisms for iron acquisition within the host (52). Intriguingly, our

data indicate that multiple iron uptake pathways are expressed

simultaneously in several patients and that several (e.g.,

pvdA

fptA

, and

hasAp

) do not appear to be iron responsive under anoxic

conditions (Fig. 3B). This apparent loss of Fur regulation may also

reflect mutations that accrue as infections progress, as has been

documented elsewhere (39). A more thorough understanding of

iron-relevant gene regulation

in vivo

will be necessary to deter-

mine how expression patterns of iron uptake machinery may in-

form us about the chemical environment of the airways.

Because bioavailable iron serves as a signal for biofilm forma-

tion (12) and as an integral cation for biofilm stability (40), it is

thought to be required for both the establishment of

P. aeruginosa

biofilms and their chronicity in CF patients (14, 24). It has also

been established that an optimal concentration of iron is required

for the formation of

P. aeruginosa

biofilms (10, 41). In a biofilm

mode of growth, it is generally accepted that bacterial cells are

inherently more resistant to antibiotics and components of the

host immune system, and only once they revert to their planktonic

state are they readily cleared (42). In our studies, the mixed-

oxidation approach (conalbumin/ferrozine) to iron chelation ap-

pears to be promising in preventing biofilm formation, and we

predict that under oxic conditions, it will further sensitize biofilm

cells to conventional antimicrobial treatments, as has previously

been shown (13, 15). However, mixed Fe(III)/Fe(II) chelation

may be most significant under anoxic conditions (thought to be

prevalent throughout the CF airways [5]), by preventing or dis-

rupting biofilms. While cells reverting to a planktonic lifestyle

would likely remain tolerant to conventional antibiotics due to

slow anaerobic growth and physiological changes (43), they would

no longer be as protected from the host immune response, and

possibly more readily cleared from the host environment. Testing

this hypothesis in infection models is a logical next step. A chal-

lenge will be to ensure that these infection models accurately

mimic the environment of the human host for chronic infections.

Collectively, these studies underscore the importance of a dia-

lectic between laboratory and environmental studies of pathogens

such as

P. aeruginosa

. To complement mechanistic studies at the

bench, characterization of the microbial community

in vivo

must

also include an analysis of the chemical conditions under which it

lives. Such combined efforts will provide insight into how infected

environments coevolve with the composition and activities of the

constituent microbiome at different stages in disease progression.

As suggested here, this integrated approach has the potential to

inform effective design and application of novel therapeutic strat-

egies for

P. aeruginosa

biofilm control.

MATERIALS AND METHODS

Study design and sample collection.

Twenty-five participants (aged 7 to

20 years) and eight participants (aged 16 to 38 years) were recruited from

Children’s Hospital Los Angeles (CHLA) and the Academic Hospital UZ

Brussel, respectively. Inclusion criteria were a positive diagnosis of CF,

ability to expectorate sputum, and informed consent. Sputum was flash

frozen in liquid nitrogen shortly after expectoration to minimize oxida-

tion and/or mRNA degradation and stored at

80°C until processing.

Disease severity was determined by FEV1% scores, and patients were clus-

tered using published guidelines (44, 45). This study was approved by the

ethical commissions of the California Institute of Technology, Children’s

Hospital Los Angeles, and the Academic Hospital UZ Brussel.

Sputum processing.

Frozen samples were thawed in an anaerobic

chamber. Sputum was disrupted using a 16-gauge needle and was homog-

enized by vortexing in an equal volume of anoxic 50 mM HEPES buffer.

Sputum was centrifuged at 8,000

for 10 min, and supernatants were

filtered through 0.22-

m-pore-size columns for 20 min at 10,000

Filtrates were analyzed for iron content. When sufficient sputum material

was obtained, 200

l of filtrate was stored at

80°C for inductively cou-

pled plasma mass spectrometry (ICP-MS) analysis.

Iron quantification.

Iron levels were quantified using the ferrozine

assay (46). Briefly, 50

l of sputum filtrate was carefully added (to avoid

introducing bubbles) to 50

l of 1 M HCl to quantify Fe(II). For total iron,

l was treated with 50

l of 10% hydroxylamine hydrochloride in 1 M

HCl to reduce Fe(III) to Fe(II). Samples were added to 100

l of ferrozine

(0.1% [wt/vol] in 50% ammonium acetate) and incubated for 15 min, and

absorbance was measured at 562 nm. Ferrous ammonium sulfate was

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used as the iron standard. Samples were also analyzed by inductively cou-

pled plasma mass spectrometry (ICP-MS). Briefly, 50

l of filtrate was

digested in 100

l 8 N nitric acid and brought to a total of 1.5 ml in 5%

nitric acid-indium standard. Samples were analyzed on an Agilent 7500cx

analyzer equipped with a reaction cell, using He (2 ml/min) and H

(2.5 ml/min) as reaction gases. Fe concentrations were calculated using

Fe and

Fe signal intensities.

Sputum mRNA extraction and quantitative real-time PCR.

Sputum

samples were collected, frozen, and homogenized as described above. Un-

der anoxic conditions, homogenate was added to 1 vol of 0.1-mm zirconia

beads and 3 volumes of Trizol LS, and mRNA was extracted as described

by Lim et al. (47). Purity and degradation were assessed using NanoDrop

spectrophotometry, agarose gel electrophoresis, and an Agilent 2100 Bio-

analyzer. cDNA was reverse transcribed from 1

g of total RNA with the

first-strand cDNA synthesis kit (Amersham Biosciences) or iScript (Bio-

Rad) according to the manufacturer’s protocols. cDNA was then used as a

template for quantitative PCR (RealTime 7500 PCR machine; Applied

Biosystems) using SYBR green with the ROX detection system (Bio-Rad).

Triplicate measurements were made on each sputum sample. As controls,

anaerobically grown

P. aeruginosa

PA14 treated with 50

M Fe(II), 50

Fe(III), or water (no iron) was assayed as previously described (32). For all

primer sets (see Table S3 in the supplemental material), the following

cycling parameters were used: 94°C for 3 min followed by 40 cycles of 94°C

for 60 s, 55°C for 45 s, and 72°C for 60 s, followed by 72°C for 7 min.

oprI

and

clpX

were used to normalize levels of gene expression (48, 49) (see

Fig. S3). Primer efficiencies were determined using iQ5 optical system

software (Bio-Rad), and standard curves were constructed based on four

different known quantities of genomic DNA of

P. aeruginosa

PAO1

(100 ng, 50 ng, 10 ng, and 5 ng) (see Table S3). The threshold cycle (

)

values of each gene were used to calculate relative gene expression using

the 2

method (50). The mRNA extraction protocol precluded a con

current Fe(II) measurement because the coloration of the Trizol reagent

interferes with the ferrozine assay.

HPLC quantification of phenazines.

Phenazine extraction and quan-

tification were performed anaerobically as previously described (22).

Ninety-seven out of 115 samples contained sufficient sputum material for

phenazine analysis (3).

MBEC assay for biofilm prevention and dissolution.

We used a high-

throughput biofilm assay (MBEC physiology and genetics assay) consist-

ing of a 96-well plate and 96-peg lid. Inoculum was prepared by diluting

(30-fold) a 10

-cell/ml suspension of

P. aeruginosa

PA14 in Trypticase soy

broth (TSB). One hundred fifty microliters was dispensed into each of the

60 inner wells, while 200

l of sterile TSB was placed in each perimeter

well. For dissolution experiments, plates were incubated at 37°C for 24 h,

and lids were transferred to a fresh 96-well TSB plate for 24 h at 37°C or to

an anaerobic chamber for 24 h at 37°C in anaerobic TSB containing

50 mM KNO

. Biofilms were exposed to 100

M conalbumin and/or

200

M ferrozine for 24 h (concentrations were selected such that they

were in molar excess of medium iron concentrations). The dual chelator

treatment was also complemented with 8

g/ml tobramycin or 80

ferrous ammonium sulfate where indicated. After treatment, lids were

rinsed once in 50 mM HEPES, air dried for 10 min, and quantified by

crystal violet staining (51). For biofilm growth prevention assays, both

aerobic and anaerobic inocula were amended with 100

M conalbumin

and/or 200

M ferrozine. For aerobic experiments, biofilms developed

for 24 h. For anaerobic growth, the medium was replaced every 24 h by

transferring the lid to a sterile plate containing TSB with or without treat-

ments, and biofilms were developed for 168 h.

Statistical analysis.

Spearman rank analysis (

) was performed on

iron and phenazine concentrations versus lung function (Fig. 1 and

2).Two-tailed Student

tests were used for pairwise comparisons between

chelator treatments and controls (Fig. 4). In all cases,

0.05 was con-

sidered statistically significant.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at

http://mbio.asm.org

/lookup/suppl/doi:10.1128/mBio.00557-13/-/DCSupplemental

Text S1, DOCX file, 0 MB.

Figure S1, JPG file, 0.3 MB.

Figure S2, JPG file, 0.3 MB.

Figure S3, JPG file, 0.5 MB.

Figure S4, JPG file, 1.2 MB.

Figure S5, JPG file, 0.1 MB.

Figure S6, JPG file, 0.1 MB.

Table S1, DOCX file, 0.1 MB.

Table S2, DOCX file, 0.1 MB.

Table S3, DOCX file, 0.1 MB.

ACKNOWLEDGMENTS

This work was supported by the Caltech-UCLA Joint Center for Transla-

tional Medicine, the Webb Foundation, the Howard Hughes Medical In-

stitute, and the National Heart, Lung, and Blood Institute of the National

Institutes of Health under award number R01HL117328. R.C.H. is sup-

ported by the NHLBI under award number 1K99HL114862. J.D. is sup-

ported by a fellowship from the Agentschap voor Innovatie door Weten-

schap en Technologie (IWT). D.K.N. is an HHMI Investigator.

We thank staff at CHLA and UZ Brussel for assistance. We also thank

Y. Lim and F. Rohwer (SDSU) for guidance on mRNA preparation from

sputum and members of the Newman lab for constructive feedback.

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