JB Accepted Manuscript Posted Online 27 April 2015 J. Bacteriol. doi:10.1128/JB.00072-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Iron depletion enhances production of antimicrobials by Pseudomonas

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aeruginosa.

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Angela T. Nguyen1, Jace W. Jones1, Max A. Ruge1, Maureen A. Kane1, and Amanda G.

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Oglesby-Sherrouse1,2*

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University of Maryland 1School of Pharmacy, Department of Pharmaceutical Sciences

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and 2School of Medicine, Department of Microbiology and Immunology, Baltimore,

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Maryland, 21201

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Keywords: Pseudomonas aeruginosa, iron regulation, PQS, Staphylococcus aureus,

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PrrF

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Running Title: Iron regulates P. aeruginosa antimicrobial activity

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*To whom correspondence should be sent: [email protected]

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ABSTRACT

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Cystic fibrosis (CF) is a heritable disease characterized by chronic, polymicrobial

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lung infections. While Staphylococcus aureus is the dominant lung pathogen in young

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CF patients, Pseudomonas aeruginosa becomes predominant by adulthood. P.

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aeruginosa produces a variety of antimicrobials that likely contribute to this shift in

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microbial populations. In particular, secretion of 2-alkyl-4(1H)-quinolones (AQs)

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contributes to lysis of S. aureus in co-culture, providing an iron source to P. aeruginosa

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both in vitro and in vivo. We previously showed that production of one such AQ, the

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Pseudomonas quinolone signal (PQS), is enhanced by iron depletion, and that this

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induction is dependent upon the iron-responsive PrrF sRNAs. Here we demonstrate that

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antimicrobial activity against S. aureus during co-culture is also enhanced by iron

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depletion, and we provide evidence that multiple AQs contribute to this activity.

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Strikingly, a P. aeruginosa ∆prrF mutant, which produces very little PQS in mono-

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culture, was capable of mediating iron-regulated growth suppression of S. aureus. We

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show that the presence of S. aureus suppresses the ∆prrF1,2 mutant’s defect in iron-

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regulated PQS production, indicating a PrrF-independent iron regulatory pathway

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mediates AQ production in co-culture. We further demonstrate that iron-regulated

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antimicrobial production is conserved in multiple P. aeruginosa strains, including clinical

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isolates from CF patients. These results demonstrate that iron plays a central role in

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modulating interactions of P. aeruginosa with S. aureus. Moreover, our studies suggest

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that established iron regulatory pathways of these pathogens are significantly altered

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during polymicrobial infections.

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IMPORTANCE Chronic polymicrobial infections involving P. aeruginosa and S. aureus are a

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significant cause of morbidity and mortality, as the interplay between these two

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organisms exacerbates infection. This is in part due to enhanced production of

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antimicrobial metabolites by P. aeruginosa when these two species are co-cultured.

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Using both established and newly developed co-culture techniques, this report

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demonstrates that iron depletion increases P. aeruginosa’s ability to suppress growth of

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S. aureus. These findings present a novel role for iron in modulating microbial

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interaction, and provide the basis for understanding how essential nutrients drive

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polymicrobial infections.

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INTRODUCTION Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes a

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variety of life-threatening infections. Many of these infections are polymicrobial,

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including those in diabetic foot ulcers (1, 2) and chronic lung infections in cystic fibrosis

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patients (3, 4). Several studies have noted that polymicrobial infections with P.

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aeruginosa are exacerbated as compared to mono-infection (2, 5-10). Thus, defining

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the mechanisms that mediate these microbial interactions is paramount to

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understanding the pathogenesis of polymicrobial infections.

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P. aeruginosa requires iron for growth and virulence (11-14). However, iron is a

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limiting nutrient in the environment and in the host (15, 16). To acquire this valuable

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nutrient, P. aeruginosa encodes several iron uptake systems. Amongst these is the

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production and secretion of siderophores, such as pyoverdine and pyochelin (17), which

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chelate ferric iron at high affinities and deliver it to the cell. Additionally, P. aeruginosa

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encodes a ferrous iron uptake system (Feo) to allow for import of soluble ferrous iron in

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oxygen-depleted environments (18). Finally, P. aeruginosa encodes two heme uptake

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systems, Has and Phu, which transport and degrade heme, releasing iron for use inside

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the cell (19).

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Iron is also toxic at high concentrations, thus P. aeruginosa regulates the expression

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of iron uptake systems in response to intracellular iron concentrations. In the presence

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of iron, genes for iron acquisition are repressed via the ferric uptake regulator (Fur)

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protein, which is essential in P. aeruginosa (20-22). The Fur protein also represses the

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transcription of the PrrF small RNAs (sRNAs), which regulate the expression of many

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iron containing and iron storage proteins (23), and are required for virulence in an acute

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murine lung infection (24). Included in the PrrF regulon are genes encoding anthranilate

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degradation enzymes (AntA and CatBCA), which allow catabolism of this metabolite by

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the TCA cycle (25). Anthranilate also serves as a precursor for the Pseudomonas

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quinolone signal (PQS), a quorum sensing molecule and virulence trait (26, 27). Thus,

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regulation by the PrrF sRNAs promotes production of PQS (25, 28).

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PQS production is initiated by PqsA, a coenzyme ligase that converts anthranilate to

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anthraniloyl-CoA (29). PqsA was previously reported to be required for lysis of S.

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aureus, resulting in increased iron availability to P. aeruginosa (30). The specific

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mechanism for this activity remains unclear and is likely to be multifactorial, as

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anthraniloyl CoA can serve as the precursor for at least fifty-five unique alkylquinolone

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(AQ) molecules (29, 31). One of these, 2-heptyl-4-hydroxyquinoline (HQNO), is an N-

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oxide ubiquinone that inhibits growth of Gram-positive bacteria, including S. aureus (32-

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36). Additionally, PQS stimulates the production of redox active phenazines, which may

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contribute to iron acquisition due to: i) their ability to lyse bacterial and mammalian cells

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(37), thus liberating intracellular iron stores, and ii) redox cycling, which reduces

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insoluble ferric iron to its more bioavailable, ferrous form (38). PQS has also been

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shown to possess iron chelating activity, which may provide a competitive advantage in

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complex microbial environments (39, 40). However, PQS is not a siderophore, in that it

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cannot deliver iron to the cytosol of P. aeruginosa (39, 40). Instead, previous studies

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have hypothesized that PQS serves as an iron trap at the surface of the cell (39). The

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precursor to PQS, HHQ, shares many of the same signaling activities as PQS, including

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the induction of phenazine production (36, 39). However, HHQ does not possess the 3-

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hydroxyl group of PQS and is therefore incapable of chelating ferric iron (40).

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While several of the studies cited above provide a link between iron homeostasis

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and antimicrobial activity in P. aeruginosa, no studies have yet investigated the impact

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of iron availability on polymicrobial interactions involving P. aeruginosa. In this study, we

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demonstrate that iron depletion enhances antimicrobial activity of P. aeruginosa when

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grown in co-culture with S. aureus. While this phenomenon is dependent upon PqsA,

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we found this activity is independent of the PrrF sRNAs, indicating the presence of a

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distinct iron regulatory pathway that controls PQS production. We further show that this

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activity is conserved in multiple strains of P. aeruginosa, including two cystic fibrosis

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isolates. These studies demonstrate that iron availability impacts polymicrobial

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interactions of P. aeruginosa with S. aureus, presenting additional implications for the

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role of AQs in iron acquisition and pathogenesis.

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METHODS

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Bacterial strains and growth conditions. Bacterial strains used in this work are

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listed in Table 1. The pqs mutants were generated in our laboratory’s PAO1 strain by

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allelic exchange (41) using the previously-described ∆pqsE (42) and ∆pqsL (43)

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deletion constructs. Escherichia coli strains were routinely grown in L broth or on L agar

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plates, and P. aeruginosa strains were maintained in brain-heart infusion (BHI) broth or

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on BHI agar plates. For iron-depleted medium, chelex-treated, dialyzed trypticase soy

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broth (DTSB) was prepared as previously described (44). Ferric chloride (FeCl3) was

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added to DTSB a final concentration of 100 µM for iron-replete conditions.

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Co-culture assays. Antimicrobial activity against S. aureus was assayed as

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previously described (30) with some modifications. The indicated P. aeruginosa strains

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were grown in DTSB for 18 hours at 37°C supplemented with or without 100 μM of

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FeCl3. S. aureus strain MRSA-M2, grown overnight on BHI and diluted to an A600 of 0.1,

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was spread well with a cotton swab onto BHI or DTSB plate and allowed to dry. Five μL

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of the indicated P. aeruginosa DTSB culture was spotted onto the S. aureus lawn, and

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plates were allowed to dry at room temperature. Plates were incubated overnight at 37

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°C, and visualized the next day for S. aureus growth.

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To quantify antimicrobial activity against S. aureus, a liquid co-culture system using

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transwell cell culture inserts (Corning Costar®, NY, USA) was developed. Strains were

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grown overnight in DTSB for 18 hours at 37˚C. S. aureus cultures were diluted to an

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OD600 of 0.05 in DTSB supplemented with or without 100 µM FeCl3, and 600 µL of the

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resulting cell suspension was inoculated into the bottom of the transwell plate. The

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transwell insert with a 0.4 µM membrane was then placed onto the plate, and 100 µL of

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P. aeruginosa cultures, diluted to an OD600 of 0.05, were inoculated on top of the

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membrane. The transwell plates were incubated at 37˚C for 18 hours under static

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growth conditions, and S. aureus cell density (OD630) was measured spectroscopically

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in a BioTek® Synergy™ HT plate reader.

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Detection of PQS. Bacteria were grown in DTSB for 18 hours at 37°C, with and

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without 100 μM FeCl3 supplementation as indicated. Each culture was harvested and

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extracted with acidified ethyl acetate as described by Collier, et.al. (45). One half of the

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resulting organic extract was transferred to a clean tube and evaporated to dryness.

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Samples were resuspended in 1:1 acidified ethyl acetate:acetonitrile and analyzed by

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thin-layer chromatography (TLC) (46).

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Ultra performance liquid chromatography (UPLC) tandem mass

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spectrometry (MS/MS). Culture supernatants prepared as described above were

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suspended in water:acetonitrile (1:1) with 0.1 % formic acid and analyzed as is. UPLC

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was performed on a Waters ACQUITY UPLC system (Milford, MA). The separation was

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achieved using a C18 CSH (1.7 µm; 2.1 x 100 mm) column (Waters, Milford, MA).

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Mobile phase A was water with 0.1 % formic acid and mobile phase B was acetonitrile

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with 0.1 % formic acid. The gradient was 30% B for 0.5 minutes, ramped to 95 % B over

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9.5 minutes, held for 3 minutes, ramped to 30% B in 0.75 minutes, and equilibrated for

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2.25 minutes for a total run time of 16 minutes. The flow rate was 0.3 mL/min. The

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column was maintained at 40 °C and the auto-sampler was kept at 5°C. A 5 µL injection

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was used for all samples. The tandem mass spectrometry experiments were performed

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on a Waters SYNAPT G2-S quadrupole time-of-flight (QTOF) mass spectrometer

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(Milford, MA). The instrument was operated in positive ion mode electrospray. The

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capillary voltage was 2.5 kV and sampling cone voltage was 30 V. Nitrogen at a flow of

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650 L/h was used as the desolvation gas with a constant desolvation temperature of

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400°C. The source temperature was set at 125°C. Data were acquired over the m/z

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range of 100-1200. The mass spectrometer was operated in MSE mode with alternating

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low- and high-collision energies. The first scan was set at low-collision energy (4 eV)

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and used to collect precursor ion spectra. The second scan was set at high-collision

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energy and ramped from 25-40 eV which was used for generation of product ion

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spectra. Argon gas was used for collision-induced dissociation (CID). Leucine

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Enkephalin was used as the lock-mass to ensure high mass accuracy data acquisition.

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Data were acquired and analyzed with Waters MassLynx v4.1 software.

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Real time PCR. Real time PCR (qPCR) analysis of prrF, pqsA, antA, and antR gene

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expression in broth cultures was carried out using primers and probes as previously

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described (24, 25, 28), Expression of pqsH was detected using the following primers

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and probe: PqsH.for (TCG AGT TCA TCA GGA AGC AAT C), PqsH.rev (CGA GGG

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TAT TCC TCA GCC AGA), and PqsH.probe (CTT GGT CAG TGG GAA TCG CCC

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TCC).Samples were analyzed using the Applied Biosystems StepOne Plus Real Time

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PCR System (Life Technologies). Relative amounts of cDNA were determined by the

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∆∆CT method or by use of a standard curve generated from cDNA acquired from serial

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dilutions of PAO1 RNA samples. Expression was normalized to oprF cDNA detected in

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each sample.

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RESULTS

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Iron regulates pqsA-mediated growth suppression of S. aureus. We previously

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showed that production of certain AQs is regulated by iron in a PrrF-dependent manner

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(28). Since AQ production is required for antimicrobial activity against S. aureus, we

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tested whether iron depletion would also affect the ability of P. aeruginosa to suppress

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growth of S. aureus. S. aureus was exposed to overnight low iron cultures of either wild

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type (PAO1) or an isogenic ∆pqsA mutant of P. aeruginosa on agar plates containing

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rich, complex medium (BHI). As was previously shown for P. aeruginosa strain PA14

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(30), PAO1, but not the isogenic ∆pqsA mutant, induced a halo of S. aureus growth

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inhibition after overnight incubation (Figure 1A). This effect was also observed when we

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repeated the experiment with low iron (DTSB) agar plates (Figure 1A). However,

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supplementation of this medium with 100 µM FeCl3 significantly reduced the size of the

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growth inhibition halo induced by PAO1 (Figure 1A), suggesting that P. aeruginosa

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antimicrobial activity against S. aureus is linked to iron availability.

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We next developed a transwell growth system to quantify the antimicrobial effects of

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P. aeruginosa co-culture on S. aureus when the strains were separated by a 0.4 µM

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membrane. S. aureus was inoculated into low or high iron DTSB broth below the

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transwell membrane, and the indicated P. aeruginosa strain was inoculated on the top

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side of the membrane (see diagram in Figure 1B). The transwell co-cultures were

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grown without shaking overnight, and S. aureus culture densities were determined

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spectroscopically. Notably, mono-cultures of S. aureus grew just as well in low iron

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versus high iron medium under these conditions (Figure 1B). In contrast, iron depletion

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had a significant impact on S. aureus growth when co-cultured with PAO1 (Figure 1B).

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Moreover, this effect was eliminated in the ∆pqsA mutant (Figure 1B), demonstrating

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growth suppression of S. aureus in this system is due to AQ production. Addition of P.

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aeruginosa culture supernatants to S. aureus low and high iron cultures was not

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sufficient to produce this effect (Figure S1), indicating co-culture of these two strains is

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required for AQ-mediated growth suppression. Together, these data demonstrate that

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AQ-mediated antimicrobial activity against S. aureus is enhanced by iron depletion.

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We next determined if iron-regulated antimicrobial activity was conserved amongst

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different P. aeruginosa strains using our transwell co-culture assay. The wild type P.

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aeruginosa strains PAO1, PAK, and PA14 all show iron-regulated growth suppression

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of S. aureus (Figure 2). Variable degrees of iron regulated growth suppression between

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PAO1 and PA14 were noted during these assays. However, culture densities of S.

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aureus in low iron were not significantly changed when comparing amongst the P.

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aeruginosa strains that were included in the co-culture. Thus, our data indicate that iron-

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regulated antimicrobial activity against S. aureus is conserved amongst commonly used

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laboratory strains of P. aeruginosa.

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PqsA contributes to antimicrobial activity against both Gram-negative and

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Gram-positive bacterial species. To determine if AQ-mediated antimicrobial activity

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was uniquely active against S. aureus, we analyzed the ability of PAO1 and the ∆pqsA

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mutant to suppress growth of two other bacterial pathogens that are often found in the

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sputum from CF patients (47): Haemophilus influenzae, a Gram-negative bacterium and

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normal flora of the upper respiratory tract (48), and Streptococcus mutans, a Gram-

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positive dental pathogen (49). PAO1 inhibited growth of each of these species on iron-

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depleted medium, and this inhibition was substantially diminished in iron-replete

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medium (Figure 3A-B), suggesting that iron-regulated antimicrobial activity is not

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restricted to S. aureus, or even to just Gram-positive bacteria.

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As might be expected, the extent to which P. aeruginosa was capable of inhibiting

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growth of each of these species was variable - the zone of growth inhibition for S.

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mutans was well-defined, whereas a gradient of clearance was observed for H.

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influenzae (Figure 3A-B). Neither of these species were inhibited by the PAO1∆pqsA

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mutant when plated on high iron medium, and the growth suppression halos of both

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species were smaller when exposed to the ∆pqsA mutant on low iron medium (Figure

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3A-B). Thus, it appears that iron-regulated antimicrobial activity of these species is also

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dependent upon AQ production.

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Unfortunately, attempts at quantifying this phenotype in our transwell co-culture

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system were unsuccessful. H. influenzae demonstrated significantly reduced growth in

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low versus high iron medium when grown in mono-culture (Figure 3C), making it

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impossible to assign a specific role for P. aeruginosa in suppressing growth of this

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species in co-culture. In contrast, no significant differences in S. mutans growth were

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observed in low versus high iron medium either in mono-culture or co-culture by this

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assay (Figure 3D). We reasoned that the lack of iron-regulated growth suppression of

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S. mutans could be due to reduced PQS production during static growth in the transwell

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plates, as PQS production is known to be dependent upon oxygen (50). However, iron-

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regulated AQ production was not only retained under these conditions, but was

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significantly enhanced during static versus shaking growth (Figure S2). Thus, while

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PqsA appears to mediate antimicrobial activity against each of these organisms, we are

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not able at this time to make any further conclusions about the impact of iron on these

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interspecies relationships.

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The ∆prrF1,2 mutant retains the ability to mediate iron-regulated growth

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suppression of S. aureus. The PrrF sRNAs were previously shown to be required for

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optimal PQS production by P. aeruginosa grown in low iron conditions (25). Since PQS

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is one of many AQs that promotes growth suppression of S. aureus, we hypothesized

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that the ∆prrF1,2 mutant would also be defective for suppressing S. aureus growth.

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Surprisingly, growth suppression of S. aureus by the ∆prrF1,2 mutant was

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indistinguishable from PAO1, both on BHI and on low iron DTSB media (Figure 1A).

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Moreover, antimicrobial activity of the ∆prrF1,2 mutant was reduced by supplementing

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DTSB medium with 100 µM FeCl3, again in a manner similar to PAO1. This finding was

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further corroborated by our transwell co-culture assay (Figure 1B). These data suggest

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an alternate, PrrF-independent iron regulatory pathway mediates iron regulation of AQ

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production in the presence of S. aureus.

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Based on previously published data that co-culture with S. aureus can induce AQ

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production by P. aeruginosa (6), we reasoned that AQ production might similarly be

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restored to the ∆prrF1,2 mutant when grown in the presence of S. aureus. To test this

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idea, we analyzed PQS production in culture supernatants of PAO1 and the ∆prrF1,2

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mutant grown in high or low iron media, either alone or in co-culture with S. aureus. As

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previously shown by thin layer chromatography (TLC) (28), production of PQS, as well

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as a distinct, fluorescent metabolite, was substantially reduced in the ∆prrF1,2 mutant

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as compared to wild type when grown in low iron mono-cultures (Figure 1C). Like PQS,

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production of this metabolite was dependent upon pqsA, indicating it is likely to be an

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AQ. In contrast to what was observed in mono-culture, production of this metabolite by

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the ∆prrF1,2 mutant was substantially increased by co-culture with S. aureus (Figure

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1C). Moreover, iron-regulated production of this metabolite was restored to the ∆prrF1,2

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mutant when co-cultured with S. aureus (Figure 1C). Thus, our data demonstrate that

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the AQ-deficiency phenotype of the ∆prrF1,2 mutant is overcome by growth with S.

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aureus. Moreover, these results suggest the presence of a PrrF-independent iron

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regulatory pathway that controls AQ production in P. aeruginosa.

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Expression of anthranilate degradation genes is not altered by co-culture with

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S. aureus. While the PrrF sRNAs positively affect AQ production through modulation of

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antA expression in mono-culture (25), our results above show that PrrF is not required

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for production of these metabolites in co-culture (Figure 1C). We therefore

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hypothesized that co-culture with S. aureus enhanced iron-regulated expression of antA

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in the ∆prrF1,2 mutant, allowing for iron-regulated AQ production. In agreement with

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previous work (25), real time PCR (RT-PCR) analysis demonstrated that iron induces

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expression of antA in both a PrrF-dependent and PrrF-independent manner (Figure

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4A). However, iron induction of antA was not enhanced by co-culture with S. aureus,

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suggesting that modulation of this specific regulatory pathway is not the source of

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restored PQS production in the ∆prrF1,2 mutant. Iron was also previously shown to

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induce expression of antR, encoding a positive LysR-type activator of the antABC

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operon (25). In contrast to antA, iron induction of antR was almost completely

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dependent upon an intact prrF locus (25). Our current work corroborates this finding, as

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we observed significant iron induction of antR in the wild type, but no induction of antR

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in the ∆prrF1,2 mutant (Figure 4B). Notably, co-culture of the ∆prrF1,2 mutant with S.

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aureus did not restore iron-regulated expression of antR (Figure 4B). Thus, our data

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indicate that increased AQ production by the ∆prrF1,2 mutant upon co-culture with S.

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aureus is not due to changes in antA or antR expression.

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To determine if increased AQ production in iron-depleted conditions was due to

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increased expression of genes for AQ synthesis, we next analyzed pqsA and pqsH

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expression. As previously observed (28), pqsA mRNA levels were significantly

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increased by iron depletion in both the wild type and the ∆prrF1,2 mutant (Figure 4C).

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Iron-regulated expression of pqsH was also observed in both strains (Figure 4D).

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Unexpectedly, co-culture with S. aureus eliminated iron-regulated expression pqsA and

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pqsH, both in the wild type and the ∆prrF1,2 mutant (Figure 4C-D). While the rationale

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for these regulatory data remain unclear at this time, they demonstrate the capacity of

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S. aureus to modulate iron regulatory pathways in P. aeruginosa. Overall, our data

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indicate that additional iron regulatory pathways control AQ production in P. aeruginosa,

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and present the potential for S. aureus to enhance regulation via these pathways during

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co-culture.

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Iron-regulated antimicrobial activity is retained in CF isolates. We previously

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analyzed a series of clonal, longitudinally-collected CF isolates and demonstrated that

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their iron acquisition and regulatory pathways were substantially altered during CF lung

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infection (28). We additionally showed that one of the later CF isolates, termed JSRI-2,

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produced substantially less AQ than its “parent” isolate, termed JSRI-1. Moreover, AQ

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production by the JSRI-2 strain was not responsive to iron (28). We therefore

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determined if these strains retained the ability to suppress S. aureus growth. Our data

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show that, as expected, JSRI-2 was less efficient at suppressing S. aureus growth than

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either JSRI-1 or the PAO1 laboratory strain, regardless of growth medium (Figure 5A).

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However, iron-regulated growth stasis of S. aureus was still observed in the JSRI-2

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isolate (Figure 5A), indicating AQ production could be stimulated by growth in iron-

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depleted medium. When we quantified this activity using our transwell co-culture assay,

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we observed significant growth suppression of S. aureus in low iron by both the JSRI-1

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and JSRI-2 strains (Figure 5B). Moreover, this assay showed a significantly reduced

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ability of JSRI-2 to suppress S. aureus growth as compared to JSRI-1 or PAO1 (Figure

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5B). Thus, this later-stage CF isolate, while reduced for this activity, retained the ability

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to mediate iron-regulated antimicrobial activity against S. aureus.

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Since AQ production by the JSRI-2 isolate was previously reported to be diminished

328

and unresponsive to iron, we determined if this strain’s defect in AQ production could be

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overcome by S. aureus co-culture, as was observed in the ∆prrF1,2 mutant (Figure

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1C). TLC analysis of culture supernatants from these strains demonstrated that co-

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culture with S. aureus greatly enhanced production of both PQS and the distinct pqsA-

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dependent metabolite by JSRI-2 (Figure 5C). These data demonstrate that, despite our

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earlier findings that AQ production was substantially reduced in this isolate when grown

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in mono-culture (28), its capacity for iron-regulated antimicrobial activity was conserved.

335 336

PAO1 produces an iron-regulated C9-PQS metabolite. Previously and in our

337

current report, we have observed an iron-regulated metabolite whose production is

338

dependent upon pqsA but migrates faster than PQS (28). TLC analysis of several AQ

339

standards, which were verified by high resolution tandem mass spectrometry (Figure

340

S3), demonstrated that this metabolite was not HHQ, HQNO, or anthranilate (ANT)

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(Figure 6A). Since the levels of this metabolite correlated with antimicrobial activity in

342

the above described studies, we analyzed the contribution of known PQS biosynthetic

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genes toward its production. As expected, TLC analysis of culture supernatants

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demonstrated that this metabolite was not produced by P. aeruginosa mutants deleted

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for either pqsA or pqsD (Figure 6A), which catalyze the first and second steps in HHQ

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and PQS biosynthesis (29, 31). Also as expected, deletion of pqsE, which is required for

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quorum signaling by HHQ and PQS, but not their synthesis, had no impact on the

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production of this metabolite (Figure 6A). Moreover, pqsL, which allows for production

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of the Gram-positive antimicrobial HQNO, was not required for the production of this

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metabolite (Figure 6A). Combined, these data suggest that this metabolite is a potential

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analog of either HHQ or PQS, which both require PqsA and PqsD for their synthesis.

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We next analyzed a deletion mutant for pqsH generated in the PA14 P. aeruginosa

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laboratory strain background, a generous gift from the laboratory of Deborah Hogan.

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PqsH catalyzes the final step in PQS production (36, 51), adding a 3-hydroxyl to the

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quinolone moiety. Our analysis of the ∆pqsH mutant demonstrates this gene is

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absolutely required for production of this metabolite (Figure 6A), indicating it is in fact

357

an analog of PQS. We therefore hypothesized that this metabolite is a PQS analog with

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an alkyl chain of different length, such as 3-hydroxy-2-nonyl-4(1H)-quinolone (C9-PQS)

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that was previously reported to be produced by P. aeruginosa (36).

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To further investigate the production of C9-PQS in our culture conditions, we

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analyzed PAO1 supernatants by ultra performance liquid chromatography (UPLC)

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coupled to high resolution mass tandem mass spectrometry (MS/MS). This analysis

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identified an ion with a mass to charge (m/z) value of 288.2 corresponding to C9-PQS

364

(Figure S4A). Analysis of the tandem mass spectrum of this precursor ion revealed the

365

formation of product ions at m/z values 175.1 and 188.1, which were also observed in

366

the PQS standard (Figure S3C), confirming the presence of C9-PQS in our samples

367

(Figure S4B). Comparative analysis further showed that C9-PQS production was

368

regulated by iron at levels similar to the unknown AQ detected by TLC (Table 2). Two

369

other related AQ structures, one with an identical m/z of 288.2 and another with a m/z

Page 17

370

value of 286.2, were also detected via unique chromatographic retention times and

371

diagnostic tandem mass spectra (Figure S4). These precursor ions fragmented into

372

distinct product ions that were characteristic of the HQNO core quinolone structure

373

(Figure S3B) and were assigned as C9-QNO (m/z 288.2) and monounsaturated C9:1-

374

QNO (m/z 286.2) (Figure S4C and S4E). The unique chromatographic retention times,

375

accurate mass measurements, and diagnostic fragmentation data obtained from the

376

UPLC-MS/MS platform provided structural data that could distinguish C9-PQS from

377

closely-related AQ structures (Figure S4). Combined with our genetic analysis above,

378

these data indicated that the iron-regulated fluorescent metabolite observed by TLC in

379

this and previous reports was C9-PQS.

380 381

Multiple pqs genes contribute to iron-regulated antimicrobial activity against

382

S. aureus. Since multiple AQs are produced by P. aeruginosa with a suite of different

383

activities, we next determined antimicrobial activity by each of the pqs mutants against

384

S. aureus. While antimicrobial activity was not observed for the ∆pqsA mutant on agar

385

plates (Figure 6B), we did observe some level of iron-regulated growth suppression by

386

this mutant using our transwell assay in this study set (Figure 6C). However, the ability

387

of the ∆pqsA mutant to suppress S. aureus grow was significantly reduced as compared

388

to PAO1 (Figure 6C, p < 0.000005). The ∆pqsD, ∆pqsE, and ∆pqsL mutants also

389

showed significant defects in suppressing S. aureus growth when compared to the wild

390

type (Figure 6B-C), indicating this phenotype is dependent upon the activity of multiple

391

AQs. Surprisingly, the ∆pqsH, which did not produce any PQS or C9-PQS (Figure 6A),

392

demonstrated iron-regulated growth suppression of S. aureus at levels similar to its

Page 18

393

PA14 parent strain (Figure 6B-C), indicating PQS is an indirect correlate of anti-

394

staphylococcal activity. Thus, our data demonstrate that iron-regulated antimicrobial

395

activity of P. aeruginosa is multifactorial, yet is independent of the PQS metabolite

396

produced by this pathway.

397 398

DISCUSSION

399

Iron is an essential nutrient and is limiting in most environments, especially in the

400

context of infections. Therefore, it is not surprising that P. aeruginosa has adapted

401

several mechanisms to acquire this nutrient in the presence of competing

402

microorganisms, as well as from the human host. AQs play many important functions in

403

P. aeruginosa, including signaling to induce expression of virulence determinants, as

404

well as growth suppressing activities (52). Here, we show that iron-regulated production

405

of AQs contributes to P. aeruginosa’s ability to suppress growth of another microbial

406

pathogen, S. aureus. It was previously reported that S. aureus could serve as an iron

407

source for P. aeruginosa, purportedly due to lysis and the subsequent releases of iron

408

stores during co-culture (53). Our finding that antimicrobial activity against S. aureus is

409

enhanced by iron depletion further supports the idea that AQs contribute to iron

410

acquisition, particularly in polymicrobial environments. In the context of CF lung

411

infection, this iron competition strategy may be critical for P. aeruginosa’s ability to

412

supplant other microflora, allowing for the observed shift in microbial population.

413

Perhaps more strikingly, our data demonstrate that co-culture with S. aureus alters

414

the iron-regulatory pathways that control AQ production in P. aeruginosa. Specifically,

415

co-culture of the ∆prrF1,2 mutant with S. aureus restores iron-regulated AQ production,

Page 19

416

demonstrating a PrrF-independent pathway mediates iron-regulated antimicrobial

417

production. Our initial analysis of iron-regulated expression of both antA and antR

418

demonstrates that S. aureus does not have a significant impact on these pathways

419

(Figure 4). Further confounding this analysis was the finding that co-culture with S.

420

aureus reduces iron-regulated expression of both pqsA and pqsH (Figure 4). Thus, it is

421

likely that co-culture with S. aureus directs the expression of other genes involved in

422

either AQ biosynthesis or catabolism. Alternatively, this phenomenon may be due to

423

iron-dependent changes that occur post-transcriptionally. Regardless, our current

424

understanding of iron regulation in P. aeruginosa is largely based on studies with pure

425

cultures. Combined with the findings of earlier studies (6, 30), this work demonstrates

426

the need to reevaluate well-established paradigms in iron regulation to determine how

427

polymicrobial environments alter these pathways.

428

Our study has also provided evidence that the iron-regulated AQ observed in

429

previous (28) and current studies is a C9 analog of PQS. Like PQS, this species is

430

eliminated by deletion of pqsA, pqsD, or pqsH, yet is still present in the ∆pqsE and

431

∆pqsL mutants (Figure 6A). Moreover, UPLC-MS/MS analysis identified C9-PQS in

432

PAO1 supernatants, and demonstrated that this species is induced by iron-depletion

433

(Table 2). However, it is not yet clear why this species is so much more predominant

434

than the C7-PQS that has been observed in previous analyses (25, 27). This particular

435

iron-regulated AQ has been detected in supernatants of every strain we have analyzed

436

to date, indicating its presence is the result of our experimental conditions, rather than

437

strain differences. The potential for this distinct AQ to have a biological impact is

Page 20

438

especially intriguing, and we are currently working toward further defining the regulation

439

of this and other AQs by UPLC-MS/MS.

440

The iron-regulated C9-PQS analog observed in these studies correlated very

441

strongly with antimicrobial activity against S. aureus (Figures 1 and 5). Thus, it was

442

surprising to find that the ∆pqsH mutant, which does not make this metabolite, is not

443

defective for S. aureus growth suppression (Figure 6). Instead, it appears that this

444

activity is likely due to a combination of the signaling (∆pqsE) and direct growth stasis

445

(∆pqsL) capabilities of these diverse AQ molecules. With this in mind, determining how

446

iron affects the production of each of these AQ molecules is likely to shed further light

447

on the mechanism by which P. aeruginosa regulates AQ-dependent antimicrobial

448

activity.

449

The implications of this study for CF disease and the interplay between residents of

450

the CF lung are important to consider. JSRI-2, which represents a later-stage CF

451

isolate, was previously shown to have diminished AQ production in monoculture (54).

452

This finding was notable in consideration of a previous report, in which PQS was

453

consistently detected in sputum from several late stage CF patients (45). Here, we show

454

that co-culture with S. aureus can restore AQ production to strains previously thought

455

unable to produce this metabolite (Figure 5). Reduction in AQ production by the JSRI-2

456

isolate grown in mono-culture may suggest that this activity becomes more dependent

457

upon the presence of S. aureus in later stages of disease. In light of these data,

458

reported loss of other virulence traits by late stage CF isolates should be reconsidered,

459

as polymicrobial cultures could have similar effects on the expression of additional

460

virulence determinants.

Page 21

461

Increased production of AQs by the JSRI-2 isolate in the presence of S. aureus has

462

compelling implications for the mechanism underlying exacerbations in CF lung

463

infections. Previous work has noted that co-infection with both of these pathogens

464

correlated strongly with rapidly decreased lung function (8). Our studies suggest that

465

iron availability in the CF lung is also likely to be a determinant for these exacerbations.

466

As such, it will be interesting to consider how disruption of iron acquisition affects this

467

activity in future studies. Our initial analysis of siderophore biosynthesis mutants

468

indicates that eliminating this mode of iron uptake does not enhance antimicrobial

469

activity (Nguyen and Oglesby-Sherrouse, unpublished). Perhaps there are differences

470

in limiting environmental iron, such as through the activity of iron chelators, versus

471

limiting P. aeruginosa’s ability to acquire this element in iron-limiting environments.

472

A notable finding from our study is that co-culture is required for iron-regulated

473

antimicrobial activity (Figure S1), indicating a complex interplay between P. aeruginosa

474

and S. aureus allows for this activity. Several reports provide additional evidence for an

475

exchange between these two bacterial species. Korgaonkar, et. al demonstrated that

476

PQS production by P. aeruginosa was enhanced by co-culture with S. aureus (6). This

477

report additionally found that agtR, a response regulator involved in metabolite sensing

478

and uptake, was required for this activity, suggesting metabolite sensing as a possible

479

means for bacterial cross-talk. An additional report indicated that S. aureus secretes

480

one or more proteins to manipulate the growth characteristics of P. aeruginosa (55). It is

481

therefore likely that iron-regulated AQ production is not the sole driver of interactions

482

between these two bacterial species. Future studies into how S. aureus alters the

483

phenotypes of different P. aeruginosa strains are likely to increase our understanding of

Page 22

484

how these two microbial pathogens establish equilibrium and cause disease during

485

polymicrobial infections.

486 487

ACKNOWLEDGEMENTS

488

We would like to thank Dr. Eb Pesci for providing isogenic pqs mutants and deletion

489

constructs; to Dr. Deborah Hogan for providing PA14 and ∆pqsH strains for our studies;

490

and to Dr. Mark Shirtliff for sharing the M2-MRSA, S. mutans, and H. influenzae strains.

491

We would also like to thank Geoffrey Heinzl and Luke Brewer for thoughtful discussion

492

during preparation of this manuscript. This work was supported by NIH-NIAID K22 grant

493

AI089776 (to AOS), NIH-NIAID Contract HHSN272201000046C (to MAK), the

494

University of Maryland School of Pharmacy Mass Spectrometry Center (SOP1841-

495

IQB2014 – to MAK), and start-up funds from the University of Maryland School of

496

Pharmacy (to AOS and MAK).

497 498

Page 23

499 500

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TABLES Table 1. Strains used in this study Strain

Description

PAO1

Wild type P. aeruginosa strain used for mutational analysis in this and previous studies.

∆pqsA

Deletion of pqsA gene generated in PAO1.

(54)

∆pqsD

Deletion of pqsD gene generated in PAO1.

E. Pesci

∆pqsE

Deletion of pqsE gene generated in PAO1.

(42)

∆pqsL

(43)

∆pqsH

Deletion of pqsL gene generated in PAO1. Burn wound isolate from 1995 at the Massachusetts General Hospital, Boston. Deletion of pqsH gene generated in PA14.

PAK

Wild type P. aeruginosa strain.

JSRI-1

CF P. aeruginosa lung isolate from 8-year-old patient

(54)

JSRI-2

CF P. aeruginosa lung isolate from 17year-old patient

(54)

MRSA-M2

Methicillin-resistant isolate of S. aureus isolated from an osteomyelitis patient in Galveston, Texas.

(56)

UA159

Streptococcus mutans serotype c strain

(49)

1128

Haemophilus influenzae strain originally isolated from a child with otitis media

(48)

PA14

Source

698

Page 33

(58)

(57) D. Hogan M. Vasil

699

Table 2. Quantification of PQS and C9-PQS

Metabolite

PQS1

C9-PQS1

2

C9-PQS

Growth Mono-culture Condition PAO1

∆pqsA

∆prrF1,2

PAO1

∆pqsA

∆prrF1,2

Low Iron

2551 ± 321**

932 ± 104

2405 ± 491*

3075 ± 428**

743 ± 79

2411 ± 503*

High Iron

935 ± 13

620 ± 59

912 ± 159

1167 ± 57

595 ± 66

1171 ± 82

Low Iron

11481 ± 1631**

937 ± 107

5722 ± 761**

14204 ± 1342*** 720 ± 71

9626 ± 1082**^

High Iron

1827 ± 116

616 ± 64

1896 ± 429

2298 ± 380

610 ± 68

2662 ± 749

Low Iron

1251 ± 139***

0.5 ± 1

83 ± 48

N.D.

N.D.

N.D.

High Iron

315 ± 119

0±0

115 ± 67

N.D.

N.D.

N.D.

Co-culture

1

As determine by densitometry of thin layer chromatography. C9-PQS refers to the unknown metabolite observed in Figure 1C. Standard deviation are from 3 biological replicates. 2 As determined by UPLC-MS/MS (described in the materials and methods). Standard deviations are from 5 biological replicates. Asterisks (*) indicate the following p values when comparing high and low iron conditions: p < 0.05*; p < 0.005**; p < 0.0005*** indicates significance when comparing high and low iron conditions The carrot (^) indicate significance between mono-culture and co-culture with S. aureus p