Phylogenetic analysis of MgtC-like proteins fromPseudomonas species

The genome ofP.aeruginosa strain PAO1 encodes two MgtC-like proteins, PA4635 and PA2558, which both belong to the same phylogenetic subgroup asSalmonella MgtC [16]. InFig 1A, we provided a phylogenetic analysis of this subgroup that focused on proteins from bacterial pathogens able to replicate in macrophages for which MgtC role in virulence has been studied, as well as proteins from opportunistic bacteria infecting CF patients.

AsmgtC sequences may have been acquired by horizontal gene transfer [16], we investigated the distribution ofmgtC coding sequence inPseudomonas genomes and performed phylogenetic analysis. MgtC appeared to be conserved in allP.aeruginosa strains present in thePseudomonas genome database [23,24], including the atypical PA7 strain [24]. In addition, MgtC was found in the insect pathogenPseudomonas entomophila as well as the closely relatedPseudomonas putida (> 85% identity with PA4635,Fig 1B). In contrast, otherPseudomonas species harbor MgtC-like proteins that largely differ from PA4635 (below 40% identity,Fig 1B) or do not harbor any MgtC-like protein (Pseudomonas syringae). This analysis indicates a strong association of MgtC withPseudomonas species that are pathogenic for humans and insects, suggesting a putative role during animal/human host infection.

To address the role of MgtC inP.aeruginosa, we constructed an in-frame deletion inmgtC (PA4635) to avoid any polar effect on the expression of the downstreamPA4636 gene (S1 Fig). The resulting ΔmgtC mutation was complemented by a single copy of themgtC gene with its own promoter inserted into the uniqueattB site in strain PAO1 (S1 text).

MgtC is important forP.aeruginosa virulence in theDanio rerio infection model in a macrophage-dependent manner

To evaluate the role of MgtC inP.aeruginosa virulence, we used the zebrafish (Danio rerio) embryo model. This model, which has been used for various intracellular and extracellular bacterial pathogens, is a model of choice to investigate the contribution of cells from the innate immune system during infection [25]. TheDanio has been successfully used to monitor the role ofP.aeruginosa determinants in virulence based on survival curves of larvae infected with mutants deficient in type III secretion system (T3SS) or Quorum Sensing [20,26]. Bacteria producing a fluorescent protein were injected intravenously in the caudal vein of embryos (Fig 2A) at 30 hours post-fertilization, a time where macrophages, but not neutrophils, are fully functional and capable of engulfing invading bacteria [27]. The survival curves of infected embryos indicated that MgtC is a critical virulence determinant in this model since themgtC mutant is significantly attenuated as compared to the wild-type PAO1 strain (Fig 2B). In agreement, fluorescence microscopy of 18 hpi infected embryos showed a lower bacterial burden with PAO1 strain thanmgtC mutant (Fig 2C).

Confocal microscopy at early time after injection allowed us to visualize bacteria phagocytosed by macrophages close to the site of injection (Fig 2D). Macrophages can be depleted from zebrafish embryos with a validated method that uses antisens oligonucleotides (morpholinos) against the myeloid transcription factor genepu.1 [28,29]. Macrophage-depleted zebrafish embryos have been shown to be hypersusceptible toP.aeruginosa infection [20]. In addition, it has been shown that macrophage depletion restored the virulence of the attenuated T3SS mutant in zebrafish embryos [20]. We carried out experiments with Tg(mpeg1::mCherry) embryos in which macrophages are visualized as red cells [29]. Therefore, macrophage depletion can be checked uponpu.1 morpholinos injection (S2 Fig). Interestingly, the survival curve of macrophage-depleted embryos was similar upon infection with wild-type and mutant strains (Fig 2E andS3 Fig), indicating that macrophage depletion suppresses the difference between wild-type and mutant strain. Taken together, these results suggest that MgtC acts by protectingP.aeruginosa against macrophages.

MgtC contributes toP.aeruginosa resistance to macrophage killing in J774 macrophages

The virulence ofP.aeruginosa is associated with its ability to resist the innate immune system notably because of a cytotoxic action towards macrophages and the T3SS plays a major role inPseudomonas cytotoxicity [30]. We thus tested the cytotoxicity of themgtC mutant on J774 macrophages and showed that the mutant is slightly but significantly less cytotoxic than the wild-type strain or the complemented strain (Fig 3A). However, the reduction of cytotoxicity was much lower than the one observed with a T3SS mutant (Fig 3A). Given our results in zebrafish embryos, we hypothesized that MgtC may play a role in the ability ofP.aeruginosa strains to resist killing by phagocytic cells. We therefore tested the sensitivity ofP.aeruginosa strains to the bactericidic activity of J774 macrophages. Whereas the T3SS mutant behaves similarly than the wild-type strain towards the killing by macrophages after phagocytosis, our results indicated that themgtC mutant is more sensitive to macrophage killing than the wild-type or complemented strain, with a striking difference at time 1.5 hr (Fig 3B). The survival decrease of the wild-type strain at the second time point may account for a slower killing, but may also be due in part to the damage of infected macrophages.

Salmonella MgtC can inhibit the bacterial F-ATP synthase and modulate physiological ATP levels and cytosolic pH, which has been proposed to play a role in the ability ofSalmonella strain to replicate in macrophages [5]. Moreover, acidification of the phagosome has been proposed to promoteSalmonella MgtC production by increasing bacterial ATP level [7]. The acidification of the phagosome is dependent on the activity of the host vacuolar ATPase, which can be specifically inhibited by the inhibitor bafilomycin A1 [31]. To decipher the contribution of phagosome acidification in the intramacrophage role ofPseudomonas MgtC protein, macrophage infection was performed in presence of bafilomycin A1 (Fig 3C). The addition of bafilomycin A1 did not affect significantly the mutant strain whereas the survival rate of the PAO1 strain or the complemented strain was significantly reduced. As a consequence, the survival rate of wild-type and mutant strains did not differ when cells are treated with vacuolar ATPase inhibitor.

To visualize intracellular bacteria upon infection of J774 cells, we imaged slides of fixed macrophages infected with fluorescent bacteria (Fig 4A). Moreover, bacterial counts on images were made at time 0 and 2 hrs after phagocytosis (Fig 4B). Our results indicated that the number of bacteria per cell after phagocytosis is slightly higher for the mutant strain than for the wild-type strain. We further quantified the phagocytosis rate with CFUs counts and showed that themgtC mutant is phagocytosed at a slightly but significantly higher rate than the wild-type strain (S4 Fig). The evolution of the patterns between time 0 and time 2 hrs (Fig 4B) indicated that the bacterial number per cell rather increased with the wild-type but not with the mutant. These experiments, which do not discriminate between live and dead/dying bacteria, suggested a survival of wild-type bacteria with limited bacterial multiplication.

We also investigated the behavior of themgtC mutant towards non-phagocytic cells that can be targeted by the pathogen during the infection [22]. Gentamycin protection assays performed to measure internalisation in HeLa cells did not show significant difference between wild-type andmgtC mutant strains (S4 Fig).

Cumulatively,ex vivo experiments indicate that, similarly to intracellular pathogens, the MgtC virulence determinant ofP.aeruginosa plays a role towards macrophages. Wild-type bacteria resisted better to phagocytosis and to bacterial killing by the phagosome than themgtC mutant. Moreover, our results indicate an interplay betweenPseudomonas MgtC role in macrophages and phagosome acidification.

Expression ofP.aeruginosa mgtC is induced within macrophages

We first investigated the regulation ofmgtC expression in liquid media depending on Mg²⁺ concentration and pH. Quantitative RT-PCR experiment showed thatmgtC gene (PA4635) is strongly induced by low Mg²⁺ environment (Fig 5A). Moreover, in low Mg²⁺ condition, the level ofmgtC gene expression can be further increased by lowering the pH (Fig 5A). On the other hand, the othermgtC-like gene (PA2558) was not regulated by Mg²⁺ nor pH (Fig 5B). We then quantified themgtC mRNA level from infected macrophages. Importantly, we showed thatmgtC is highly induced in macrophages, which agrees with a specific role in this step. On the other handPA2558, as well as the T3SS genepcrV or the house-keeping genegyrA (S5 Fig), were similarly expressed in liquid medium and macrophages, and are therefore not induced in macrophages. Bafilomycin A1 treatment of macrophages, which decreases phagosome acidification, loweredmgtC expression intracellularly, which is consistent with the pH regulation observedin vitro. Taken together, these results indicate thatmgtC is specifically expressed in macrophages and that phagosome acidification contributes to an optimal expression of the gene.

Role of MgtC forP.aeruginosa growth in low magnesium medium and biofilm formation on abiotic surface

Bacterial growth ofP.aeruginosa strains was measured in minimal medium deprived for magnesium (without added Mg²⁺ or with 10 μM Mg²⁺) or containing 1 mM Mg²⁺ (corresponding to physiological concentration). Growth of themgtC mutant was significantly impaired in low Mg²⁺ media (Fig 6A andFig 6B), while it did grow similarly to the PAO1 wild-type strain in high Mg²⁺ medium (Fig 6C). The complemented strain displayed a wild-type growth in these conditions. Hence, MgtC contributes to the adaptation ofP.aeruginosa to low Mg²⁺ environment, similarly to MgtC homologues from intracellular pathogens [1]. This result is consistent with the induction ofmgtC gene expression in low Mg²⁺ environment (Fig 5A). To test whether the phenotype observed with themgtC mutant in macrophages may be related to the growth defect upon Mg²⁺ limitation, the killing assay by J774 macrophages was repeated upon addition of 25 mM Mg²⁺ in the DMEM medium. However, addition of Mg²⁺ did not rescue the enhanced sensitivity of themgtC mutant to macrophages (S6 Fig), suggesting that the increased killing of the mutant is not related to intracellular Mg²⁺ limiting environment.

P.aeruginosa ability to form biofilm is a crucial virulence determinant, more particularly in chronic infection. Interestingly, biofilm formation has been shown to be sensitive to EDTA through chelation of divalent cations including Mg²⁺ [18]. To check the role of both MgtC and Mg²⁺ ions during biofilm formation, wild-type, mutant and complemented strains were grown in different Mg²⁺ concentrations in glass tubes for 24 h and bacterial adherence to the glass was visualized (Fig 7A) and quantified using crystal violet staining (Fig 7B) to infer the ability of strains to form biofilm. Profiles of the three strains were the same in both assays if bacteria are grown in high Mg²⁺, indicating similar biofilm formation at physiological Mg²⁺ concentration. However, in the absence of Mg²⁺, wild-type and complemented strains poorly attached to the glass, indicating that Mg²⁺ is required for biofilm initiation byP.aeruginosa. In contrast, the ΔmgtC mutant was still able to form biofilm. The increased biofilm formation for the ΔmgtC mutant comparatively to the wild-type strain was also observed in the presence of 10 μM Mg²⁺. Cumulatively, these results indicate that when expressed, i.e. under Mg²⁺ limitation, MgtC limits biofilm formation. Since exopolysaccharides (EPS) are essential biofilm matrix components, important for initial adherence and biofilm formation [32,33], EPS production was measured by Congo red assay [34] in low and high Mg²⁺ (S7 Fig). Whereas, all strains produced similar EPS level at 1 mM Mg²⁺, the mutant strain produced significantly more EPS than wild-type and complemented strain in low Mg²⁺ medium.

We also investigated whether the increased biofilm formation of themgtC mutant strain under Mg²⁺ limitation may be related to altered properties of the flagellum or the type IV pili, which are major adhesins required for initial attachment [35]. Our results indicated that the twitching and swimming motilities, which depend on type IV pili and flagellum, respectively, were similar in wild-type, mutant and complemented strain (S8 Fig). On the other hand, we observed thatP.aeruginosa ΔmgtC bacteria grown under Mg²⁺starvation were more elongated than wild-type bacteria (S9 Fig). Hence the increased biofilm formation of themgtC mutant strain under Mg²⁺ limitation may be due to increased EPS production and/or bacterial cell elongation.

Heterologous production of theSalmonella MgtR peptide in PAO1 mimics themgtC mutant virulence phenotypes

MgtR is a membrane peptide (30 amino-acid long) encoded downstream ofmgtC inS. Typhimurium that regulates negatively MgtC expression [8]. MgtR interacts directly withSalmonella MgtC in a bacterial two-hybrid system and over-expression ofmgtR in a wild-typeSalmonella strain reduced significantly the ability of the strain to grow within macrophages, thus displaying an anti-infective effect [8]. Site-directed mutagenesis and structural analysis have suggested that MgtR interacts with the 4th transmembrane domain of MgtC [8,36]. Examination of the DNA region adjacent toPA4635 as well as Blast analyses did not reveal any peptide similar to MgtR inP.aeruginosa. Because the 4th transmembrane domain of MgtC is well conserved betweenS. Typhimurium andP.aeruginosa (S10 Fig), we have tested whether theSalmonella MgtR peptide could interact withP.aeruginosa MgtC (PaMgtC) using the bacterial two-hybrid system BACTH [37]. The interaction between T18-PaMgtC and T25-MgtR fusion proteins was evaluated by measuring β-galactosidase activity (Fig 8A). Negative controls consisted in T18-PaMgtC with empty pKT25 (Fig 8A) and empty pUT18 with T25-MgtR (S11 Fig). The high level of β-galactosidase activity, which was 60 fold the value of the negative control, was indicative of an interaction between the MgtR peptide andPaMgtC in this system. On the other hand,PaMgtC did not interact with another membrane peptide, theSalmonella KdpF peptide (S11 Fig), indicating that the interaction with MgtR is not due to sticky properties ofPaMgtC.

We then transformed the wild-typeP.aeruginosa strain with pMgtR, a plasmid that harbors theSalmonella mgtR gene [8]. Expression ofmgtR in PAO1 was verified by RT-PCR (Fig 8B). The virulence of the strain was evaluated using the zebrafish embryo model. As shown inFig 8C, a wild-type PAO1 strain that expressedmgtR was attenuated comparatively to a control PAO1 strain that harbored an empty vector. On the other hand, no effect of the pMgtR plasmid was detected in the context of the PAO1mgtC mutant strain. Expression ofmgtR also increased significantly the killing of wild-type PAO1 strain, whereas no significant effect was found in the presence of themgtC mutation (Fig 8D). In addition, a mild but significant increase in phagocytosis was observed (S12 Fig). Taken together, these results suggest that heterologous production of theSalmonella MgtR peptide can lower PAO1 virulence and resistance to macrophage killing by targeting theP.aeruginosa MgtC. Regarding other phenotypes related to MgtC defect inP.aeruginosa, production of theSalmonella MgtR peptide did not modulate bacterial growth in magnesium deprived medium (S13 Fig). This correlates with results obtained inSalmonella and is consistent with the finding of a dual role for MgtC [15].

Bacterial strains and growth conditions

Bacterial strains and plasmids are described inS1 Table.P.aeruginosa was grown at 37°C in Luria broth (LB) or at 30°C in MM63 medium supplemented with 0.5% casamino acids, 0.2% glucose, and MgSO₄ (10 μM or 1mM) or without MgSO₄. Plasmids were introduced inP.aeruginosa by conjugation, using anE.coli strain containing pRK2013. Recombinant bacteria were selected onPseudomonas isolation agar (PIA).P.aeruginosa mutants obtained by using the pKNG101 suicide vector were selected on plates containing 6% sucrose. Antibiotic concentrations (μg/ml) were: forE.coli: ampicillin, kanamycin, streptomycin, gentamicin, 50; tetracycline, 40; forP.aeruginosa: gentamicin, 50 or 125 (plates); tetracycline, 200; streptomycin, 2000.

Construction of aP.aeruginosa mgtC mutant and complemented strain

To generate a non-polar deletion of themgtC (PA4635) gene, 526 bp upstream and 580 bp downstream of themgtC gene were amplified, cloned in pCR2.1 and subcloned in pKNG101 suicide vector giving the mutator pSBC44 which was mobilized in the wild-typeP.aeruginosa strain PAO1 to select for double recombination event (S1 Text).

Complementation by a single copy of themgtC⁺ gene under its own promoter was carried out with the miniCTX integration method atattB region [51]. The entiremgtC gene with the 525 bp-upstream region, in which a σ⁷⁰ dependent promoter was predicted (S1 Fig), was amplified, cloned in the pCR2.1 giving pSBC46, which was subcloned in mini-CTX1 plasmid vector giving pSBC47. Chromosomal insertion of pSBC47 carryingmgtC⁺ and excision of plasmid DNA sequences resulted in an unmarked integrant (S1 Text).

Infection ofDanio rerio embryos

Experiments were performed using thegolden zebrafish mutant [52] or the Tg(mpeg1::mCherry) [29] zebrafish line and maintained under standard conditions (supplemental material). Bacterial strains, which were freshly streaked out from glycerol stocks, were grown in LB medium to mid-log phase (DO = 0.6), recovered by centrifugation and washed twice in Phosphate-Buffered Saline (PBS). Suspensions were homogenized through a 26-gauge needle and resuspended in PBS at about 10⁹ bacteria/ml added with 10% phenol red to aid visualization of the injection process. Infections were carried out by microinjection of 1–2 nl of bacterial suspensions into the caudal vein of 30 hpf embryos, which were previously dechorionated and anesthetized with 0.02% tricaine. For survival kinetics after infection, the number of dead embryos was determined visually based on the absence of heartbeat. Depletion of macrophages was carried out upon microinjection ofpu.1 morpholinos into zebrafish eggs and visualized by fluorescence microscopy (S1 Text).

Microscopic analysis of zebrafish embryos

For live imaging, anesthetized infected embryos were mounted in 35 mm dishes and immobilized with 1% low-melting point agarose. Direct visualization is performed using an Olympus MVX10 epifluorescent microscope equipped with a digital color camera (Olympus XC50). Fluorescence and bright-field images are acquired and processed with CellSens (Olympus) and assembled using GIMP 2.6 freeware to adjust levels and brightness and to remove out-of-focus background fluorescence.

For fixed samples observations, anesthetized embryos are fixed overnight at 4°C with 4% paraformaldehyde in PBS, then washed twice in PBS and transferred gradually from PBS to 50% glycerol-PBS. Fixed fishes were positioned flat onto depression transparent slides covered with a cover slip for observation with 40x Leica Apo oil 1.15 NA or 63x Leica Apo oil 1.33 NA objectives and using a Leica DM2500CSQ upright microscope equiped with a Leica TCS SPE confocal scan head and fluorescence and differential interference contrast (DIC) optics. Widefield fluorescence and DIC images were captured and assembled by LAS-AF software (Leica Microsystems).

Ethics statement

All animal experiments described in the present study were conducted at the University Montpellier 2 according to European Union guidelines for handling of laboratory animals (http://ec.europa.eu/environment/chemicals/lab_animals/home_en.htm) and were approved by the Direction Sanitaire et Vétérinaire de l'Hérault and Comité d'Ethique pour l'Expérimentation Animale under reference CEEA-LR-13007. The breeding of adult fish adhered to the international guidelines specified by the EU Animal Protection Directive 2010/63/EU and adult zebrafish were not sacrificed for this study. All experiments were performed before the embryos free-feeding stage and did not fall under animal experimentation law according to the EU Animal Protection Directive 2010/63/EU. For survival curves, cardiac rhythm was used as a clinical criterion to fix the endpoint at which embryos are euthanized using the anaesthetic Tricaine up to a lethal dose (500 mg/ml). Embryos that survive infection were anaesthetized with Tricaine up to a lethal dose before bleach treatment.

Cytotoxicity towards macrophages

The cytotoxicity ofP.aeruginosa strains grown to mid-log phase in LB broth was assayed by using J774 macrophages as described in [53], except that macrophages were infected for 1.5 hr at a multiplicity of infection (MOI) of 20. The percentage of LDH release was calculated relatively to that of the uninfected control, which was set at 0% LDH release, and that of uninfected cells lysed with Triton X-100, which was set at 100% LDH release.

Macrophage-mediated bactericidal assay

The killing ofP.aeruginosa strains by J774 macrophages was carried out essentially as described previously [54]. Mid-log phaseP.aeruginosa grown in LB broth was centrifuged and resuspended in PBS to infect J774 macrophages grown in DMEM medium supplemented with 10% FBS at a MOI of 10. After centrifugation of 24-well culture plates, bacterial phagocytosis was allowed to proceed for 30 min before washing three times with sterile PBS and adding fresh DMEM media supplemented with 400 μg ml^-1 gentamicin. Macrophages were lysed after 1 h 30 min and 2 h 30 by using 1% Triton X-100 and the number of viable bacteria was determined by subsequent plating onto LB agar plates. The percentage of survival represents the ratio between the number of bacteria at time 1.5 hr or 2.5–3 hrs and the number of bacteria internalized after phagocytosis. In case of treatment with bafilomycin A1, the inhibitor was added at a concentration of 100 nM 30 min before phagocytosis and was also added after phagocytosis. Bafilomycin does not exhibit any bacterial toxicity at the working concentration. In experiments with strains expressingmgtR, macrophages were lysed after 20 min or 2 hrs of gentamicin treatment and the ratio between bacteria enumerated at the two time points was calculated.

Analysis of intracellular bacteria by fluorescence microscopy

For analysis of bacteria in fixed cells, infection of J774 macrophages was carried out as described above for the CFU assay, except that macrophages were seeded on glass coverslips and infected at a MOI of 5 to 10 with bacteria that were previously passed four times through a 26G needle. Because fluorescent bacterial strains carry a plasmid-encoded gentamycin resistant gene, amikacin (300 μg ml^-1) was used to kill extracellular bacteria. For time 0, cells have been in contact with amikacin for 20 min. At the end of the incubation period, the cells were fixed with 4% paraformaldehyde in PBS for at least 1 hr. Cells were washed three times with PBS and mounted on glass slides in Vectashield with DAPI (Vector Laboratories, Inc). The slides were examined using an upright fluorescence microscope (Axioimager Z2, Zeiss) equipped with an Apotome 1 for optical sectioning. A 63X Apochromat Objective (NA 1.4) was used, transmitted light was acquired using differential interference contrast (DIC), and m-Cherry fluorescence was acquired using a texas red filter set. Z-stacks were systematically acquired using a Z-step of 240 nm. Counting was performed taking into account a maximum projection of the Z-stack. Images were processed using ZEN blue software (Zeiss).

RNA extraction and quantitative RT-PCR (qRT-PCR)

For the study of expression ofPA4635 andPA2558 genes, RNA was prepared from mid-logarithmic bacterial cultures grown for 1 hour in NCE-minimal medium supplemented with 0.1% casamino acids, 38 mM glycerol and 10 μM MgSO₄ or 1 mM MgSO₄ [8] at pH 7 or pH 5.7. FormgtR expression study, RNA was prepared from mid-logarithmic bacterial cultures grown in LB. For bacterial RNA extraction from infected J774, 4.10⁶ macrophages were seeded into a 100 cm² tissue culture dish and infected at an MOI of 10 as described above. One hour after phagocytosis, cells were washed three times with PBS, lysed with 0.1% Triton X100 and pelleted by centrifugation at 13 000 rpm for 10 min at 15°C. Bacteria were resuspended by adding 500 μl PBS and transferred to a new tube for RNA preparation (thus discarding non resuspended cellular debris).

RNA was prepared and reverse transcribed as previously described [55] except that bacteria were incubated only 5 min with RNA protect reagent and were disrupted with lyzozyme. Controls without reverse transcriptase were done on each RNA sample to rule out possible DNA contamination. Quantitative real-time PCR was performed using a Light Cycler 480 SYBR Green I Master mix and a 480 Light Cycler instrument (Roche). PCR conditions were as follows: 3 min denaturation at 98°C, 45 cycles of 98°C for 5 sec, 60°C for 10 sec and 72°C for 10 sec. PCR conditions formgtR and 16S rRNA control gene were as follows: 25 cycles of 95°C for 30 sec, 46°C (59°C for 16S rRNA) for 20 sec and 72°C for 30 sec. The sequences of primers used for RT-PCR are listed inS1 Table.

Internalization in non-phagocytic cells

Invasion assay of HeLa cells were done as described [22] with mid-log phaseP.aeruginosa grown in LB broth.

Adherence assay on inert surfaces and analysis of exopolysaccharide (EPS) production

The adherence assay was performed as previously described [56] except that the biofilm visualisation was done in glass tubes. O/N cultures were done in 2 ml of MM63 medium supplemented with glucose, Casamino acids and 1 mM MgSO₄, cells were washed with PBS before resuspension in the same medium with various concentrations of MgSO₄. The absorbance of the culture was measured at 600 nm (A₆₀₀) before attached bacteria were stained with 0.1% crystal violet for a period of 15 min and washed twice with water. The stain was then dissolved in ethanol and absorbance was measured at 600 nm.

For liquid Congo red (CR) assays, bacteria were grown as previously in a minimal medium containing 40 μg/ml CR, under vigorous agitation. At 24 h, the A₆₀₀ (absorbance at 600 nm) was determined and the bacterial cells were pelleted by centrifugation at 15493 g. For quantification of CR binding, the A₄₉₀ of the supernatant of each sample was determined. The ratio of A₄₉₀/A₆₀₀ indicates the EPS production.

Bacterial two-hybrid analysis

TheP.aeruginosa mgtC gene, amplified using PAO1 chromosomal DNA and primers PA4635-pUT18-F/PA4635-pUT18-R, was cloned at theHindIII andEcoRI sites of the pUT18 vector, to produce a fusion proteinPaMgtC-T18. This recombinant plasmid was cotransformed with a plasmid producing a fusion protein T25-MgtR [8] into BTH101 bacteria. Quantification of interaction between fusion proteins was carried out at 30°C as described previously [8]. Values are average from at least six independent cultures. A level of β-galactosidase activity at least fivefold higher than that measured for vectors indicates an interaction.

Statistical analysis

Statistical analyses of comparisons between survival curves were performed using the log rank test with Prism 4.0 (Graphpad, Inc). Statistical significance was assumed atP values <0.05. Forex vivo experiments (J774 or HeLa cells), paired Student’s t test was performed using Excel software (Microsoft). In the figures, * meansP values <0.05, ** <0.01, and *** <0.001.

• 1.Alix E, Blanc-Potard ABMgtC: a key player in intramacrophage survival. Trends Microbiol. 2007; 15: 252–256. [DOI] [PubMed] [Google Scholar]
• 2.Blanc-Potard AB, Groisman EATheSalmonella selC locus contains a pathogenicity island mediating intramacrophage survival. EMBO J. 1997; 16: 5376–5385. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 3.Lawley TD, Chan K, Thompson LJ, Kim CC, Govoni GR, et al. Genome-wide screen forSalmonella genes required for long-term systemic infection of the mouse. PLoS Pathog. 2006; 2: e11 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 4.Thompson JA, Liu M, Helaine S, Holden DWContribution of the PhoP/Q regulon to survival and replication ofSalmonella enterica serovar Typhimurium in macrophages. Microbiology. 2011; 157: 2084–2093. 10.1099/mic.0.048926-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 5.Lee EJ, Pontes MH, Groisman EAA bacterial virulence protein promotes pathogenicity by inhibiting the bacterium's own F1Fo ATP synthase. Cell. 2013; 154: 146–156. 10.1016/j.cell.2013.06.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 6.Garcia Vescovi E, Soncini FC, Groisman EAMg2+ as an extracellular signal: environmental regulation ofSalmonella virulence. Cell. 1996; 84: 165–174. [DOI] [PubMed] [Google Scholar]
• 7.Lee EJ, Groisman EAControl of aSalmonella virulence locus by an ATP-sensing leader messenger RNA. Nature. 2012; 486: 271–275. 10.1038/nature11090 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 8.Alix E, Blanc-Potard ABPeptide-assisted degradation of theSalmonella MgtC virulence factor. EMBO J. 2008; 27: 546–557. 10.1038/sj.emboj.7601983 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 9.Buchmeier N, Blanc-Potard A, Ehrt S, Piddington D, Riley L, et al. A parallel intraphagosomal survival strategy shared by mycobacterium tuberculosis andSalmonella enterica. Mol Microbiol. 2000; 35: 1375–1382. [DOI] [PubMed] [Google Scholar]
• 10.Lavigne JP, O'Callaghan D, Blanc-Potard ABRequirement of MgtC forBrucella suis intramacrophage growth: a potential mechanism shared bySalmonella enterica andMycobacterium tuberculosis for adaptation to a low-Mg2+ environment. Infect Immun. 2005; 73: 3160–3163. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 11.Maloney KE, Valvano MAThemgtC gene ofBurkholderia cenocepacia is required for growth under magnesium limitation conditions and intracellular survival in macrophages. Infect Immun. 2006; 74: 5477–5486. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 12.Grabenstein JP, Fukuto HS, Palmer LE, Bliska JBCharacterization of phagosome trafficking and identification of PhoP-regulated genes important for survival ofYersinia pestis in macrophages. Infect Immun. 2006; 74: 3727–3741. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 13.Retamal P, Castillo-Ruiz M, Mora GCCharacterization of MgtC, a virulence factor ofSalmonella enterica Serovar Typhi. PLoS One. 2009; 4: e555110.1371/journal.pone.0005551 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 14.Belon C, Gannoun-Zaki L, Lutfalla G, Kremer L, Blanc-Potard ABMycobacterium marinum MgtC plays a role in phagocytosis but is dispensable for intracellular multiplication. PLoS One. 2014; 9: e11605210.1371/journal.pone.0116052 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 15.Rang C, Alix E, Felix C, Heitz A, Tasse L, et al. Dual role of the MgtC virulence factor in host and non-host environments. Mol Microbiol. 2007; 63: 605–622. [DOI] [PubMed] [Google Scholar]
• 16.Blanc-Potard AB, Lafay BMgtC as a horizontally-acquired virulence factor of intracellular bacterial pathogens: evidence from molecular phylogeny and comparative genomics. J Mol Evol. 2003; 57: 479–486. [DOI] [PubMed] [Google Scholar]
• 17.Ripoll F, Pasek S, Schenowitz C, Dossat C, Barbe V, et al. Non mycobacterial virulence genes in the genome of the emerging pathogenMycobacterium abscessus. PLoS One. 2009; 4: e566010.1371/journal.pone.0005660 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 18.Banin E, Brady KM, Greenberg EPChelator-induced dispersal and killing ofPseudomonas aeruginosa cells in a biofilm. Appl Environ Microbiol. 2006; 72: 2064–2069. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 19.Mulcahy H, Lewenza SMagnesium limitation is an environmental trigger of thePseudomonas aeruginosa biofilm lifestyle. PLoS One. 2011; 6: e2330710.1371/journal.pone.0023307 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 20.Brannon MK, Davis JM, Mathias JR, Hall CJ, Emerson JC, et al.Pseudomonas aeruginosa Type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos. Cell Microbiol. 2009; 11: 755–768. 10.1111/j.1462-5822.2009.01288.x [DOI] [PMC free article] [PubMed] [Google Scholar]
• 21.Garcia-Medina R, Dunne WM, Singh PK, Brody SLPseudomonas aeruginosa acquires biofilm-like properties within airway epithelial cells. Infect Immun. 2005; 73: 8298–8305. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 22.Sana TG, Hachani A, Bucior I, Soscia C, Garvis S, et al. The second type VI secretion system ofPseudomonas aeruginosa strain PAO1 is regulated by quorum sensing and Fur and modulates internalization in epithelial cells. J Biol Chem. 2012; 287: 27095–27105. 10.1074/jbc.M112.376368 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 23.Winsor GL, Lam DK, Fleming L, Lo R, Whiteside MD, et al.Pseudomonas Genome Database: improved comparative analysis and population genomics capability for Pseudomonas genomes. Nucleic Acids Res. 2011; 39: D596–600. 10.1093/nar/gkq869 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 24.Roy PH, Tetu SG, Larouche A, Elbourne L, Tremblay S, et al. Complete genome sequence of the multiresistant taxonomic outlierPseudomonas aeruginosa PA7. PLoS One. 2010; 5: e884210.1371/journal.pone.0008842 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 25.Torraca V, Masud S, Spaink HP, Meijer AHMacrophage-pathogen interactions in infectious diseases: new therapeutic insights from the zebrafish host model. Dis Model Mech. 2014; 7: 785–797. 10.1242/dmm.015594 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 26.Clatworthy AE, Lee JS, Leibman M, Kostun Z, Davidson AJ, et al.Pseudomonas aeruginosa infection of zebrafish involves both host and pathogen determinants. Infect Immun. 2009; 77: 1293–1303. 10.1128/IAI.01181-08 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 27.Lieschke GJ, Oates AC, Crowhurst MO, Ward AC, Layton JEMorphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrafish. Blood. 2001; 98: 3087–3096. [DOI] [PubMed] [Google Scholar]
• 28.Clay H, Davis JM, Beery D, Huttenlocher A, Lyons SE, et al. Dichotomous role of the macrophage in earlyMycobacterium marinum infection of the zebrafish. Cell Host Microbe. 2007; 2: 29–39. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 29.Bernut A, Herrmann JL, Kissa K, Dubremetz JF, Gaillard JL, et al.Mycobacterium abscessus cording prevents phagocytosis and promotes abscess formation. Proc Natl Acad Sci U S A. 2014; 111: E943–952. 10.1073/pnas.1321390111 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 30.Engel J, Balachandran PRole ofPseudomonas aeruginosa type III effectors in disease. Curr Opin Microbiol. 2009; 12: 61–66. 10.1016/j.mib.2008.12.007 [DOI] [PubMed] [Google Scholar]
• 31.Bowman EJ, Siebers A, Altendorf KBafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci U S A. 1988; 85: 7972–7976. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 32.Friedman L, Kolter RGenes involved in matrix formation inPseudomonas aeruginosa PA14 biofilms. Mol Microbiol. 2004; 51: 675–690. [DOI] [PubMed] [Google Scholar]
• 33.Vasseur P, Vallet-Gely I, Soscia C, Genin S, Filloux AThepel genes of thePseudomonas aeruginosa PAK strain are involved at early and late stages of biofilm formation. Microbiology. 2005; 151: 985–997. [DOI] [PubMed] [Google Scholar]
• 34.Ma L, Jackson KD, Landry RM, Parsek MR, Wozniak DJAnalysis ofPseudomonas aeruginosa conditionalpsl variants reveals roles for thepsl polysaccharide in adhesion and maintaining biofilm structure postattachment. J Bacteriol. 2006; 188: 8213–8221. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 35.O'Toole GA, Kolter RFlagellar and twitching motility are necessary forPseudomonas aeruginosa biofilm development. Mol Microbiol. 1998; 30: 295–304. [DOI] [PubMed] [Google Scholar]
• 36.Jean-Francois FL, Dai J, Yu L, Myrick A, Rubin E, et al. Binding of MgtR, aSalmonella transmembrane regulatory peptide, to MgtC, aMycobacterium tuberculosis virulence factor: a structural study. J Mol Biol. 2014; 426: 436–446. 10.1016/j.jmb.2013.10.014 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 37.Karimova G, Pidoux J, Ullmann A, Ladant DA bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A. 1998; 95: 5752–5756. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 38.Geiger T, Francois P, Liebeke M, Fraunholz M, Goerke C, et al. The stringent response ofStaphylococcus aureus and its impact on survival after phagocytosis through the induction of intracellular PSMs expression. PLoS Pathog. 2012; 8: e100301610.1371/journal.ppat.1003016 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 39.Prajsnar TK, Hamilton R, Garcia-Lara J, McVicker G, Williams A, et al. A privileged intraphagocyte niche is responsible for disseminated infection ofStaphylococcus aureus in a zebrafish model. Cell Microbiol. 2012; 14: 1600–1619. 10.1111/j.1462-5822.2012.01826.x [DOI] [PMC free article] [PubMed] [Google Scholar]
• 40.Eriksson S, Lucchini S, Thompson A, Rhen M, Hinton JCUnravelling the biology of macrophage infection by gene expression profiling of intracellularSalmonella enterica. Mol Microbiol. 2003; 47: 103–118. [DOI] [PubMed] [Google Scholar]
• 41.Rathman M, Sjaastad MD, Falkow SAcidification of phagosomes containingSalmonella typhimurium in murine macrophages. Infect Immun. 1996; 64: 2765–2773. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 42.Porte F, Liautard JP, Kohler SEarly acidification of phagosomes containingBrucella suis is essential for intracellular survival in murine macrophages. Infect Immun. 1999; 67: 4041–4047. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 43.Skurnik D, Roux D, Aschard H, Cattoir V, Yoder-Himes D, et al. A comprehensive analysis of in vitro and in vivo genetic fitness ofPseudomonas aeruginosa using high-throughput sequencing of transposon libraries. PLoS Pathog. 2013; 9: e100358210.1371/journal.ppat.1003582 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 44.Guina T, Wu M, Miller SI, Purvine SO, Yi EC, et al. Proteomic analysis ofPseudomonas aeruginosa grown under magnesium limitation. J Am Soc Mass Spectrom. 2003; 14: 742–751. [DOI] [PubMed] [Google Scholar]
• 45.Martin-Orozco N, Touret N, Zaharik ML, Park E, Kopelman R, et al. Visualization of vacuolar acidification-induced transcription of genes of pathogens inside macrophages. Mol Biol Cell. 2006; 17: 498–510. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 46.Song B, Leff LGInfluence of magnesium ions on biofilm formation byPseudomonas fluorescens. Microbiol Res. 2006; 161: 355–361. [DOI] [PubMed] [Google Scholar]
• 47.Robertson EJ, Wolf JM, Casadevall AEDTA inhibits biofilm formation, extracellular vesicular secretion, and shedding of the capsular polysaccharide glucuronoxylomannan byCryptococcus neoformans. Appl Environ Microbiol. 2012; 78: 7977–7984. 10.1128/AEM.01953-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 48.Yoon MY, Lee KM, Park Y, Yoon SSContribution of cell elongation to the biofilm formation ofPseudomonas aeruginosa during anaerobic respiration. PLoS One. 2011; 6: e1610510.1371/journal.pone.0016105 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 49.Pontes MH, Lee EJ, Choi J, Groisman EASalmonella promotes virulence by repressing cellulose production. Proc Natl Acad Sci U S A. 2015; 112: 5183–5188. 10.1073/pnas.1500989112 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 50.Yang Y, Labesse G, Carrere-Kremer S, Esteves K, Kremer L, et al. (2012) The C-terminal domain of the virulence factor MgtC is a divergent ACT domain. J Bacteriol194: 6255–6263. 10.1128/JB.01424-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 51.Hoang TT, Kutchma AJ, Becher A, Schweizer HPIntegration-proficient plasmids forPseudomonas aeruginosa: site-specific integration and use for engineering of reporter and expression strains. Plasmid. 2000; 43: 59–72. [DOI] [PubMed] [Google Scholar]
• 52.Lamason RL, Mohideen MA, Mest JR, Wong AC, Norton HL, et al. SLC24A5, a putative cation exchanger, affects pigmentation in zebrafish and humans. Science. 2005; 310: 1782–1786. [DOI] [PubMed] [Google Scholar]
• 53.Soscia C, Hachani A, Bernadac A, Filloux A, Bleves SCross talk between type III secretion and flagellar assembly systems inPseudomonas aeruginosa. J Bacteriol. 2007; 189: 3124–3132. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 54.Del Porto P, Cifani N, Guarnieri S, Di Domenico EG, Mariggio MA, et al. Dysfunctional CFTR alters the bactericidal activity of human macrophages againstPseudomonas aeruginosa. PLoS One. 2011; 6: e1997010.1371/journal.pone.0019970 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 55.Gannoun-Zaki L, Belon C, Dupont C, Hilbert F, Kremer L, et al. Overexpression of theSalmonella KdpF membrane peptide modulates expression ofkdp genes and intramacrophage growth. FEMS Microbiol Lett. 2014; 359: 34–41. 10.1111/1574-6968.12559 [DOI] [PubMed] [Google Scholar]
• 56.Vallet I, Olson JW, Lory S, Lazdunski A, Filloux AThe chaperone/usher pathways ofPseudomonas aeruginosa: identification of fimbrial gene clusters (cup) and their involvement in biofilm formation. Proc Natl Acad Sci U S A. 2001; 98: 6911–6916. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 57.Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, et al. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 2008; 36: W465–469. 10.1093/nar/gkn180 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 58.Gastebois A, Blanc Potard AB, Gribaldo S, Beau R, Latge JP, et al. Phylogenetic and functional analysis ofAspergillus fumigatus MGTC, a fungal protein homologous to a bacterial virulence factor. Appl Environ Microbiol. 2011; 77: 4700–4703. 10.1128/AEM.00243-11 [DOI] [PMC free article] [PubMed] [Google Scholar]

Supplementary Materials

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.