- Research article
- Open Access
Molybdate transporter ModABC is important for Pseudomonas aeruginosa chronic lung infection
© Périnet et al. 2016
Received: 23 October 2015
Accepted: 6 January 2016
Published: 12 January 2016
Mechanisms underlying the success of Pseudomonas aeruginosa in chronic lung infection among cystic fibrosis (CF) patients are poorly defined. The modA gene was previously linked to in vivo competitiveness of P. aeruginosa by a genetic screening in the rat lung. This gene encodes a subunit of transporter ModABC, which is responsible for extracellular uptake of molybdate. This compound is essential for molybdoenzymes, including nitrate reductases. Since anaerobic growth conditions are known to occur during CF chronic lung infection, inactivation of a molybdate transporter could inhibit proliferation through the inactivation of denitrification enzymes. Hence, we performed phenotypic characterization of a modA mutant strain obtained by signature-tagged mutagenesis (STM_modA) and assessed its virulence in vivo with two host models.
The STM_modA mutant was in fact defective for anaerobic growth and unable to use nitrates in the growth medium for anaerobic respiration. Bacterial growth and nitrate usage were restored when the medium was supplemented with molybdate. Most significantly, the mutant strain showed reduced virulence compared to wild-type strain PAO1 according to a competitive index in the rat model of chronic lung infection and a predation assay with Dictyostelium discoideum amoebae. As the latter took place in aerobic conditions, the in vivo impact of the mutation in modA appears to extend beyond its effect on anaerobic growth.
These results support the modABC-encoded transporter as important for the pathogenesis of P. aeruginosa, and suggest that enzymatic machinery implicated in anaerobic growth during chronic lung infection in CF merits further investigation as a potential target for therapeutic intervention.
Pseudomonas aeruginosa is an environmental bacterium and the most common cause of chronic lung infection among cystic fibrosis (CF) patients . Its success in causing opportunistic infection and its persistence capacities are presumably attributable to a large 6.3–6.9 Mbp genome regulated by more than 550 transcriptional regulators, which allow adaptation to diverse environments and growth conditions [2, 3]. P. aeruginosa also produces a wide repertoire of molecules and sensors for nutrient uptake, adhesion, mobility, biofilm formation and antibiotic resistance that are all key components of in vivo proliferation .
It was shown that the CF mucus is oxygen-depleted  and may carry strict anaerobes . Hence, during chronic lung infection in CF, P. aeruginosa is exposed to microaerophilic  or anaerobic conditions , which are well suited for biofilm formation . Denitrification, the main source of energy production under anaerobic conditions, is based upon the reduction of oxidized forms of nitrogen (preferentially nitrates, NO3) by metalloenzymes such as nitrate reductase [10, 11]. Molybdate (MoO4 2−) is the usable form of trace-element molybdenum (Mo)  and resembles sulfate, phosphate, tungstate and vanadate in molecular size, shape, charge and hydrogen-binding properties . Mo is incorporated into the molybdenum cofactor (MoCo) and was found to be essential for the activity of molybdoenzymes. These enzymes catalyze various oxidation/reduction reactions and are implicated in the metabolism of nitrogen, carbon and sulfur. All nitrate reductases required for P. aeruginosa anaerobic growth require a MoCo cofactor , which can contain either Mo or tungsten (W) .
A previous PCR-based signature-tagged mutagenesis (PCR-STM) mutant screen allowed the identification of 148 genes presumably essential for in vivo survival of P. aeruginosa PAO1 in a rat model of chronic lung infection . One of these genes was modA. In E. coli, molybdate and tungstate are internalized using an ATP-binding cassette transporter, ModABC , where ModA is the periplasmic binding protein with a high affinity for Mo/W, ModB is the integral membrane channel protein and ModC is the energizer protein . In P. aeruginosa, modA was recently demonstrated to be essential for molybdate acquisition and anaerobic growth using a deletion mutant . Here, with the goal of gaining further information on the role of ModABC in the virulence of P. aeruginosa, we present a characterization of the PAO1 transposon mutant STM_modA. We then assess the relevance of modA as a therapeutic target by testing virulence attenuation of STM_modA in two host models: the rat model of chronic lung infection, which best represents the context of CF lung infections, and the amoeba predation assay, which takes place in aerobic conditions. Results suggest that modA is important for the pathogenesis of P. aeruginosa in both host models.
Bacterial strains, plasmids, primers and culture conditions
Strains and plasmids
Strain or plasmid
Source or reference
E. coli strain
Chemically competent cells
New England Biolabs
P. aeruginosa strains
PAO1293, CmS, wild-type, derivative of PAO2 which originates from PAO1
PAO1 STM_modA::miniTn5-Km2 mutant inactivating modA (PA1863), KmR
PAO1 STM_modA with pUCP19::modABC plasmid (PA1863, PA1862, PA1861)
CbR, cloning vector and used for CI
CbR, KmR, modABC genes on pUCP19 vector
For routine cultures, P. aeruginosa and E. coli were grown aerobically at 37 °C in tryptic soy broth (TSB) or Luria–Bertani (LB) broth (EMD Serono). When needed, culture medium was supplemented with 1.5 (w/v) % bacto agar, kanamycin (Km 150 μg ml−1 for STM_modA; Calbiochem), ampicillin (Ap 100 μg ml−1 for DH10B transformants; Sigma-Aldrich) or carbenicillin (Cb 200 μg ml−1 for P. aeruginosa transformants; Thermo-Fisher).
For growth experiments, P. aeruginosa was grown aerobically under vigorous shaking (250 rpm) in 100 ml LB using a 1:10 pre-culture to Erlenmeyer volume ratio. For anaerobic growth, nephelo flasks sealed with silicone stoppers containing 100 ml LB were flushed with argon for 45 min and needle-inoculated with 1 ml of pre-culture before incubation with weak agitation (120 rpm) to prevent precipitation. Media for aerobic and anaerobic growth were supplemented with potassium nitrate (KNO3; 15 mM; Merck) and, when needed, sodium molybdate (100 μM; Sigma-Aldrich). Growth was monitored by spectrophotometric measurements of optical density at 600 nm. Growth experiments were repeated three times.
Restriction enzymes, Q5 polymerase and Gibson assembly cloning kit were purchased from New England Biolabs. The QIAprep Spin Miniprep Kit (Qiagen) was used for plasmid isolation and the DNeasy Blood and Tissue kit (Qiagen) was used for genomic DNA isolation. PCR reactions were performed in an iCycler (Bio-Rad); primers used in this study are listed in Additional file 1.
Spectrophotometric determination of nitrate in culture medium after anaerobic growth
To determine if the STM_modA mutant was able to use nitrates during anaerobic growth, the concentration of residual nitrates in the medium was quantified after overnight growth based on the principle of chemical reduction of nitrate and its spectrophotometric detection using the Griess reaction . Vanadium (III) chloride (Sigma-Aldrich) was used for the reduction of nitrates to nitrites. Sulfanilamide and N-(1-naphtyl)-ethylenediamine (Sigma-Aldrich) were used in the composition of the Griess reagent. Nitrite concentration was subtracted from the total nitrate and nitrite concentration to obtain the nitrate concentration alone.
Complementation of the STM_modA mutation
Cloning of the modABC operon into the pUCP19 vector was done using the Gibson Assembly Master Mix (New England Biolabs) following manufacturer’s instructions. Two overlapping PCR fragments (Additional file 1) containing the entire modABC operon plus a 448 nt upstream region (2.1 kb final insert size) and an overlapping section of the KpnI and PstI restriction sites were ligated with the digested vector. The recombinant plasmid was introduced by heat shock into E. coli NEB 5α chemically competent cells (Table 1). The plasmid transfer was first confirmed by EcoRI and PstI digestion followed by DNA sequencing. The plasmid was then recovered and electroporated into the P. aeruginosa STM_modA mutant. Plasmid insertion in the mutant strain was confirmed using digestions with the same restriction enzymes.
Biofilm formation assay
Multiple phenotypic tests were performed for this study (see reference  for more information on the protocols used), but only biofilm formation showed variation among strains. To measure the quantity of biofilm produced by wild-type strain PAO1, STM_modA, and the complemented STM_modA, a 96-plate rapid biofilm formation assay was performed as previously described . Briefly, strains were grown overnight in LB supplemented when needed with Km or Cb. The M63 culture medium for biofilm formation  was supplemented when needed with 100 μM sodium molybdate and 15 mM KNO3. Biofilms were stained with crystal violet after a 6-hour static incubation time. Biofilm formation was quantified after stain dissolution in 2 × 200 μl 95 % v/v ethanol . The experiment was done three times, with eight repetitions for each strain and condition. Statistical significance was assessed using an ANOVA in GraphPad Prism 6.0.
The rat model in this study was used in a protocol approved by the “Comité de protection des animaux de l’Université Laval” (certificate 2011194-3, IACUC is a Canadian Council on Animal Care certificate holder).
Preparation of agarose bead-embedded bacteria and in vivo competitive index
The previously described rat model of chronic lung infection  using agarose bead-embedded bacteria was optimized as described elsewhere . Sprague–Dawley rats of 450–500 g in weight were sedated (ketamine/xylazine IP injection, 10 mg/100 g) and a local anesthetic (lidocaine) was applied to the vocal cords. Animals were then intubated and inoculated with 120 μl of bead preparation containing PAO1 + pUCP19 (CbR) strain and the STM_modA mutant in equal parts (1.6 × 106 CFU ml−1 per strain) for competitive index (CI) determination. At day 7 post-infection, rats were euthanized by barbiturate overdose (Euthanyl IP injection, 120 mg/kg) and homogenized lungs were diluted and plated in triplicate on TSA to quantify the total number of viable P. aeruginosa cells (TSA supplemented with 200 μg Cb ml−1 for the wild-type selection or TSA supplemented with 150 μg Km ml−1 for the mutant selection). In vivo CI was calculated as the ratio of mutant to wild-type bacteria recovered in vivo and adjusted according to the input ratio. The final CI data was represented as the geometric mean for each group of six animals and statistical significance was assessed with a two-tailed Mann–Whitney test on GraphPad Prism 6.0 software.
Amoeba predation assay
A predation assay was used to determine the bacterial capacity to resist to amoeba grazing . The D. discoideum amoeba (DH1-10) was grown in HL5 medium (14.3 g l−1 of bactopeptone, 7.15 g l−1 of yeast extract, 18 g l−1 of d-(+)-monohydrate maltose, 0.641 g l−1 of Na2HPO4•2H2O, and 0.490 g l−1 of KH2PO4)  supplemented with 15 µg ml−1 tetracycline. The confluence of amoebae was about 60 % the day of the experiment. A volume of 300 µL of P. aeruginosa liquid pre-culture (OD600 = 0.9) was spread in a uniform lawn on a Petri dish containing SM 1/10 agar (1 g l−1 of bactopeptone, 0.1 g l−1 of yeast extract, 0.22 g l−1 of KH2PO4, 0.1 g l−1 of K2HPO4, 0.1 g l−1 of MgSO4∙7H2O, 2 g l−1 of Bacto agar, and 1 g l−1 of glucose). Serial 1/10 dilutions of 50,000 amoeba cells/5 µL down to 0 cell/5 µl were deposited on the bacterial lawn. Petri dishes were incubated at 21 °C for 7 days and phagocytic plaques due to amoeba grazing were monitored. The experiment was repeated three times.
Results and discussion
Genetic characterization of the P.aeruginosa STM_modA mutant
modA is required for anaerobic growth and nitrate utilization
It was previously demonstrated that mod mutations in E. coli caused pleiotropic effects on molybdo enzyme activity, including nitrate reductase activity . These effects were reversible in the presence of high concentrations of molybdate, which can be internalized by the sulfate transport system under appropriate conditions [16, 34]. However, sulfur compounds have been shown to inhibit the sulfate transport system, thus in protein-rich medium (such as LB), molybdate is likely internalized by another, less specific transporter, presumably the selenite transport system . A novel permease (PerO) internalizing molybdate at micromolar concentrations was identified in Rhodobacter capsulatus . PerO also imports sulfate, tungstate and vanadate, suggesting a general oxyanion transporter function. PerO has 30 % AA sequence identity with P. aeruginosa PAO1 PA3839, which may be responsible for the nonspecific uptake of molybdate when added at micromolar concentrations, as was done here for the modA mutant.
modA and biofilm formation
modA is important for virulence during chronic lung infection
modA is important for virulence in the amoeba model
In order to further investigate the idea that the role of modA in virulence may be affected by interaction with the host, we decided to use a markedly different host model. D. discoideum is an alternative model for the study of bacterial virulence where amoebae feed by bacterial uptake using phagocytosis [28, 39]. P. aeruginosa has universal virulence factors used to infect phylogenetically diverse hosts, which makes the amoeba model particularly well suited to study its virulence [28, 39]. Previously identified virulence factors using this model, related to quorum sensing and the type III secretion system, were also essential for infection in mammalian and insect models . Many bacterial virulence factors are active against predation by D. discoideum, thus virulence is assumed to be inversely proportional to amoeba grazing [29, 39]. In addition, the predation assay is performed in aerobic conditions where no growth difference has been noticed between PAO1 and the STM_modA mutant.
Metabolic and molecular basis of STM_modA multi-host virulence
Results presented here independently confirmed that transport of the trace anion molybdate is essential for the activity of molybdoenzymes such as nitrate reductase in P. aeruginosa . In anaerobic conditions, molybdate uptake was restored in STM_modA after the addition of molybdate to the culture medium, which suggests less specific internalization of the molecule by a system other than ModABC. The lack of anaerobic growth in STM_modA was hypothesized to be directly linked to the maintenance defect observed in the rat model of chronic lung infection (CI = 0.004) because this model mimics CF lung infections, which present evidence of oxygen-depletion [7–9, 41, 42]. This hypothesis, however, does not hold in the case of the amoeba predation assay, where a strong virulence defect was observed in at least partly aerobic conditions; oxygen availability may be limited in a fully grown bacterial lawn. For the biofilm formation assay, our results showed that STM_modA is unable to produce as much biofilm as PAO1 in vitro, and that this effect is molybdate-independent. As biofilm is a crucial component of P.aeruginosa chronic lung infections , reduced biofilm production in STM_modA may be related to the virulence defect observed in the rat model of chronic lung infection, in conjunction with anaerobic growth defectiveness. The virulence defect observed in the amoeba model, on the other hand, could be linked to this biofilm defect alone, as for other biofilm-defective mutants shown to have attenuated virulence in the D. dictostelium model . Even without consideration for the biofilm results, which have been discussed at length earlier, the amoeba predation assay suggests that the impact of the mutation in modA may go beyond its effect on anaerobic growth, perhaps due to pleiotropic effects of this gene.
This study is not the first report of an impact on virulence for a gene implicated in molybdate homeostasis in P. aeruginosa. PA1006 encodes a protein of unknown function implicated in molybdate homeostasis that is critical for biofilm maturation and virulence in a lettuce model and in a burned mouse acute virulence model . Other genes implicated in molybdenum utilization and transport (several moe, moa and mod genes) have been linked to a pathogenesis defect in Mycobacterium tuberculosis . These genes were required for intracellular growth in macrophages or maintenance in the organs. Interestingly, modA has also been identified in a STM experiment with M. tuberculosis using a mouse model of acute lung infection with an intravenous administration route .
We have shown that the inactivation of the modA gene in a transposon mutant caused a significant defect in P. aeruginosa PAO1 for the establishment of chronic lung infection and for resistance to D. dictostelium predation. This study complements previous work  in providing evidence that molybdate uptake is important for anaerobic growth and multi-host virulence in P. aeruginosa. Since the capacity to thrive in anaerobic conditions is relevant to CF lung infections, the modABC-encoded transporter may represent a potential target for therapeutic intervention and deserves further investigation.
SP carried out all the experiments except for host model manipulation, performed statistical analyses and drafted the manuscript. JJ performed statistical analyses and drafted the manuscript. IKI performed the experiments with the rat model. MMO performed the amoeba predation assay. All authors read and approved final manuscript.
We are grateful to Anne Sebilo and Manon Couture for support and material used for anaerobic growth condition. We also thank Halim Maaroufi and Brian Boyle for collaboration on this project. The research in R.C.L.’s laboratory is funded by the Canadian Institute for Health Research as part of the CIHR Genomics Program operating grant (MOP-86644) and by Cystic Fibrosis Canada. RCL is a Fonds de la Recherche du Québec - Santé (FRQS) Research Scholar of Exceptional Merit. S.P. is a Master’s scholar from CIHR. J.J. is a Cystic Fibrosis Canada postdoctoral fellow. S.J.C. is a research scholar of the FRQS.
The authors declare that they have no competing interests.
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