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Infection and Immunity, October 2006, p. 5477-5486, Vol. 74, No. 10
0019-9567/06/$08.00+0     doi:10.1128/IAI.00798-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The mgtC Gene of Burkholderia cenocepacia Is Required for Growth under Magnesium Limitation Conditions and Intracellular Survival in Macrophages

Kendra E. Maloney1 and Miguel A. Valvano1,2*

Infectious Diseases Research Group, Siebens-Drake Research Institute, Departments of Microbiology and Immunology,1 Medicine, University of Western Ontario, London, Ontario, N6A 5C1, Canada2

Received 17 May 2006/ Returned for modification 27 June 2006/ Accepted 7 July 2006


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ABSTRACT
 
Burkholderia cenocepacia, a bacterium commonly found in the environment, is an important opportunistic pathogen in patients with cystic fibrosis (CF). Very little is known about the mechanisms by which B. cenocepacia causes disease, but chronic infection of the airways in CF patients may be associated, at least in part, with the ability of this bacterium to survive within epithelial cells and macrophages. Survival in macrophages occurs in a membrane-bound compartment that is distinct from the lysosome, suggesting that B. cenocepacia prevents phagolysosomal fusion. In a previous study, we employed signature-tagged mutagenesis and an agar bead model of chronic pulmonary infection in rats to identify B. cenocepacia genes that are required for bacterial survival in vivo. One of the most significantly attenuated mutants had an insertion in the mgtC gene. Here, we show that mgtC is also needed for growth of B. cenocepacia in magnesium-depleted medium and for bacterial survival within murine macrophages. Using fluorescence microscopy, we demonstrated that B. cenocepacia mgtC mutants, unlike the parental isolate, colocalize with the fluorescent acidotropic probe LysoTracker Red. At 4 h postinfection, mgtC mutants expressing monomeric red fluorescent protein cannot retain this protein within the bacterial cytoplasm. Together, these results demonstrate that, unlike the parental strain, an mgtC mutant does not induce a delay in phagolysosomal fusion and the bacterium-containing vacuoles are rapidly targeted to the lysosome, where bacteria are destroyed.


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INTRODUCTION
 
The Burkholderia cepacia complex (Bcc) comprises a group of closely related species that are commonly associated with nosocomial infections and opportunistic infections in patients with chronic granulomatous disease and cystic fibrosis (CF) (10, 36). Lung infections by Bcc species in CF patients result in increased morbidity and mortality (13, 15, 25, 37, 48). A proportion of patients with CF who become infected by this pathogen experience a severe and often lethal necrotizing pneumonia termed "cepacia syndrome" (24). These infections are very difficult to treat due to the inherent resistance of Bcc species to host antimicrobial factors and most clinically relevant antibiotics. Bcc infections are also a cause for concern since there is evidence that there has been patient-to-patient transmission leading to epidemic outbreaks in cystic fibrosis clinics throughout North America and Europe (7, 21, 29, 32). Very little is known about the mechanisms by which Bcc species cause disease. Intracellular survival may contribute to the ability of Bcc strains to persist in the airways of patients with cystic fibrosis. Our laboratory and other laboratories have demonstrated that Bcc strains can survive intracellularly within membrane-bound vacuoles in amoebae and macrophages (38, 39, 44). Bcc strains can also survive within airway epithelial cells (6, 8), and they can be observed within alveolar macrophages in a murine lung infection model (9). Intracellular survival may also be important for transmission since we have shown that Bcc-infected amoebae release membrane-bound vesicles containing viable bacteria that are potentially respirable and could be transported to the lower airways of patients by airflow (38).

Our laboratory has recently described the mgtC gene as one of 109 genes identified by signature-tagged mutagenesis that are required for survival of B. cenocepacia in a rat agar bead model of lung infection (23). The mgtC insertional mutant 6E3 showed a 1,000-fold reduction in recovery from this animal compared to the wild type after a 14-day infection (23). The mgtC gene was originally identified in Salmonella enterica serovar Typhimurium as a component of Salmonella pathogenicity island 3, which is necessary for the intracellular survival of this pathogen (2). This gene has a limited distribution in eubacterial genomes, and phylogenetic analysis suggests that it may be acquired horizontally by intracellular bacterial pathogens (3). However, the physiological function of the MgtC protein has not been elucidated (41, 42, 46). The importance of MgtC for intracellular survival in other organisms (2, 5, 19, 28) and its involvement in B. cenocepacia pathogenesis prompted us to investigate in more detail the physiological role of this protein. In this study, we found that B. cenocepacia MgtC is required for bacterial growth in magnesium-depleted medium and is essential for survival of bacteria within macrophages. Using fluorescence microscopy, we demonstrated that B. cenocepacia cells with an insertionally inactivated mgtC gene colocalize with the fluorescent acidotropic probe LysoTracker Red. In contrast to the parental strain, mgtC mutant cells expressing monomeric red fluorescent protein 1 (mRFP1) also do not retain this protein in their cytoplasm 4 h postinfection. Together, these results demonstrate that unlike wild-type bacteria, mgtC mutants rapidly traffic to the lysosomes, suggesting that mgtC is a critical factor for the intracellular survival of B. cenocepacia.


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MATERIALS AND METHODS
 
Reagents, bacterial strains, and culture conditions. Chemicals and reagents were purchased from Sigma-Aldrich, St. Louis, Mo., unless indicated otherwise. Bacterial strains and plasmids used in this study are described in Table 1. B. cenocepacia strain K56-2 was previously classified as a B. cepacia complex genomovar III strain (22) and was originally isolated from a patient with cystic fibrosis. Escherichia coli and B. cenocepacia strains were cultured at 37°C in Luria-Bertani (LB) broth. B. cenocepacia and E. coli strains carrying plasmid pKMBAD or pKM2 were grown in the presence of 100 µg ml–1 trimethoprim and 100 µg ml–1 chloramphenicol (final concentrations) and in the presence of 50 µg ml–1 trimethoprim and 50 µg ml–1 chloramphenicol (final concentrations), respectively. For growth in Mg2+-depleted medium, strains were grown in modified M56 minimal salts medium consisting of 0.037 M KH2PO4, 0.06 M Na2HPO4, 50 µM FeSO4, and 3 mM (NH4)2SO4 supplemented with 0.2% (final concentration) glycerol, 0.2% Casamino Acids, 20 µg ml–1 tryptophan, 2 µg ml–1 vitamin B1, 0.3 mM Ca(NO3)2, and various concentrations of MgSO4, as indicated below. For some experiments, the growth rate was determined in a 100-well microtiter plate using a Bioscreen C automated microbiology growth curve analysis system (MTX Lab Systems, Inc., Vienna, VA). For growth in low-pH medium, strains were grown in buffered minimal medium (pH 5.5) containing 50 mM 4-morpholineethanesulfonic acid (MES) (pH 5.5), 0.3 mM KCl, 0.1 mM MgSO4, 0.6 µM CaCl2, 3 mM (NH4)2SO4, and 0.5 mM KH2PO4 supplemented with 0.2% (final concentration) Casamino Acids, 2 µg ml–1 vitamin B1, 0.65% glycerol, and 20 µg ml–1 tryptophan.


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  		TABLE 1. Strains and plasmids used in this study

RNA isolation and RT-PCR analysis. Total RNA was isolated from B. cenocepacia strain K56-2 with an RNeasy kit (QIAGEN Inc., Mississauga, Ontario, Canada) by following the manufacturer's instructions. The RNA was treated with DNase I for 30 min at 37°C, followed by inactivation at 75°C for 15 min. Reverse transcription (RT) was performed using a Transcriptor reverse transcriptase kit (Roche Diagnostics, Laval, Quebec, Canada) with reverse primers 2253 (5'-CAGGGCGGGCGCCAGGACGG-3'), 2255 (5'-TTTCATGCACGGCGAGCTGC-3'), 2257 (5'-TGACGAGCAACAGCATCGCG-3'), and 2261 (5'-GATCGGCGACGCCGCAGGCGA-3'). The resulting cDNA was subjected to PCR using Taq DNA polymerase (Roche Diagnostics) and the following primers: 2253 and forward primer 2254 (5'-GCGCGCGGTCAGCCTGACTGAGCGC-3'), 2255 and forward primer 2256 (5'-CGTGTTCGTCGCGACCGGCT-3'), 2257 and forward primer 2258 (5'-ATCGTGCAGCGCTCGGTGAACT-3'), and 2261 and forward primer 2262 (5'-CATTGTCGGCCGCCGCCGCG-3'). The conditions used for amplification were as follows: initial denaturation at 94°C for 3 min, 5 cycles of 45 s at 94°C, 45 s at 68°C, and 1 min at 72°C, and then 30 cycles of 45 s at 94°C, 45 s at 68°C, and 1 min at 72°C and a final extension step of 10 min at 72°C. For each PCR, the appropriate control reaction without reverse transcriptase was included to ensure that the amplification products obtained were a result of cDNA and not of contaminating genomic DNA.

Recombinant DNA methods. The plasmids used in this study are described in Table 1. DNA ligation, restriction endonuclease digestion, and agarose gel electrophoresis were performed by using standard techniques (45). Restriction enzymes and T4 DNA ligase were purchased from Roche Diagnostics. Proofstart and Taq polymerases were purchased from QIAGEN. DNA transformation experiments with E. coli were carried out by the calcium chloride method (12). Plasmids were transferred into B. cenocepacia by triparental mating (14) using the pRK2013 helper plasmid (16).

Construction of an mgtC insertional mutant of B. cenocepacia. A 299-bp internal fragment of mgtC was amplified from B. cenocepacia K56-2 chromosomal DNA with primers 1375 (5'-ATTGTCTAGAGCATGCCTGTTCGTCACGCTC-3') and 1376 (5'-ATTGTCTAGAGGCGTTGGAGACGGGCGTC-3') (XbaI recognition sites in both primers are underlined) using Taq polymerase and the following thermal cycling conditions: 94°C for 4 min, 5 cycles of 94°C for 45 s, 68°C for 45 s, and 72°C for 30 s, and then 25 cycles of 94°C for 45 s, 68°C for 45 s, and 72°C for 30 s plus 5-s increase/cycle and a final extension at 72°C for 10 min. The amplicon was digested with XbaI and ligated into XbaI-digested pGP{Omega}Tp to create pKM3. Plasmid pKM3 was transformed into E. coli SY327 and conjugated into B. cenocepacia K56-2 by triparental mating. Mutants were selected on LB medium plates containing 100 µg ml–1 trimethoprim and 50 µg ml–1 gentamicin. The correct insertion and orientation of the integrated plasmid in the K56-2 genome were verified by Southern blot analysis using a digoxigenin-labeled internal fragment of mgtC as a probe and by PCR amplification using the chromosome-specific primer 1331 (5'-TAGGAATTCCGTCGACGTCGTATGCGACG-3'), the plasmid-specific primer 1300 (5'-TAACGGTTGTGGACAACAAGCCAGGG-3'), and the thermal cycling conditions described above with the extension time extended to 1.5 min.

Cloning of the B. cenocepacia mgtC gene. The mgtC gene was amplified from K56-2 chromosomal DNA using primers 1330 (5'-AGCTGCAGACTCCATCATCGGCTC-3'; PstI recognition site underlined) and 1333 (5'-CGCATATGCGCTTCTTGCACGGCAG-3'; NdeI recognition site underlined). The PCR product was digested with PstI and NdeI and ligated into pKV1, which was also digested with NdeI and PstI. This strategy removed the wecA gene in pKV1, which was replaced by mgtC, and at the same time allowed construction of a 3' in-frame fusion to the flag oligonucleotide. The resulting plasmid, pKM1, encoded an MgtC protein with a C-terminal FLAG epitope (MgtCFLAG). Since pKM1 cannot replicate in B. cenocepacia, the mgtC-flag gene of this plasmid was PCR amplified with primers 1373 (5'-TGCGGAATTCATGCGCTTCTTGCAC; EcoRI recognition site underlined) and 1374 (5'-CGTGTCTAGAGCTTAGCAGCCGGAT-3'; XbaI recognition site underlined), and the product was ligated into the expression vector pKMBAD (Table 1). This vector is a derivative of pMLBAD (30) containing the {Omega}-chloramphenicol resistance cassette from pHP45-{Omega}Cm (1), which was cloned into the unique Asp700 site. The resulting mgtC-flag gene was verified by sequencing at the York University Core Molecular Biology and DNA Sequencing Facility, Toronto, Ontario, Canada. Expression of the MgtC-FLAG fusion in total E. coli membranes was confirmed by Western blotting as described previously (52), with some modifications. Briefly, strains expressing MgtCFLAG were grown overnight at 37°C and subcultured in 250 ml of LB medium with the appropriate antibiotics to obtain a final optical density at 600 nm (OD600) of 0.2. Then the culture was grown with vigorous aeration for 3 h before the expression of MgtCFLAG was induced by adding arabinose to a final concentration of 0.2% (wt/vol) for 1 h. Bacteria were collected by centrifugation and resuspended in 11 ml of 25% sucrose in 25 mM HEPES (pH 7.4) containing the Complete broad-spectrum protease inhibitors (Roche). Cells were then lysed by three passages through a French pressure cell at 10,000 lb/in2. Debris and unbroken cells were removed by centrifugation at 27,200 x g for 15 min, and the clear supernatants were layered on a 60% (wt/wt) sucrose cushion (25 mM HEPES, pH 7.4), followed by centrifugation at 270,000 x g for 2 h. Cell membranes were collected from the interface of the sucrose cushion, and the protein concentration was determined by the Bradford method using the Bio-Rad protein assay (Bio-Rad Laboratories Inc., Hercules, CA). Twenty micrograms of protein was mixed with 3x protein tracking dye and either boiled for 10 min or left at room temperature prior to loading into a sodium dodecyl sulfate (SDS)-14% polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane using standard procedures. Western blot analysis was performed using anti-FLAG M2 monoclonal primary antibody and Alexa Fluor 680 goat anti-mouse immunoglobulin G (Molecular Probes, Eugene, Oreg.) secondary antibody. Images were acquired using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, Nebr.).

Macrophage infections. Murine macrophage-like cell line RAW 264.7 (TIB-71) was obtained from the American Type Culture Collection, Manassas, VA. Macrophage cultures were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cell culture reagents were purchased from Wisent Inc., St. Bruno, Quebec, Canada, unless indicated otherwise. Macrophages were trypsinized and seeded into six-well plates containing glass coverslips. The cells were incubated overnight at 37°C in the presence of 5% CO2 in DMEM supplemented with 10% FBS. Bacteria grown overnight were washed twice with DMEM containing 10% FBS and added to macrophages at a multiplicity of infection of 40. Infected cells were centrifuged at 1,000 rpm for 1 min and incubated for 4 h at 37°C in the presence of 5% CO2. At 4 h postinfection, external bacteria were removed by three washes with RPMI prewarmed to 37°C. The infected monolayers were observed with a microscope. In some experiments, macrophages were treated with 5 µM bafilomycin A1 for 15 min prior to infection or with 10 µM diphenyleneiodonium (DPI) when bacteria were added to the macrophages. For colocalization experiments, infected macrophages were incubated for 1 min with 10 µM (final concentration) LysoTracker Red DND-99 (Molecular Probes, Eugene, Oreg.) in DMEM containing 10% FBS. Fluorescence and phase-contrast images were acquired using a Qimaging (Burnaby, British Columbia, Canada) cooled charged-coupled device camera on an Axioscope 2 microscope (Carl Zeiss, Thornwood, N.Y.) with a x100/1.3 numerical aperture, a Plan-Neofluor objective, and a 50-W mercury arc lamp. Red filter set 15 (Carl Zeiss) was used, which has a 546-nm excitation wavelength and a 590-nm emission wavelength. Images were digitally processed with the Northern Eclipse version 6.0 imaging analysis software (Empix Imaging, Mississauga, Ontario, Canada).

Disk diffusion assays. Logarithmic-phase cells were spread on agar plates with a sterile cotton swab, and sterile paper disks were applied to the surface. Eight-microliter portions of 10% SDS, 250 µg ml–1 polymyxin B, 0.5 M EDTA, 30 mg ml–1 chloramphenicol, 20 mg ml–1 tetracycline, or distilled water were applied to duplicate disks. The plates were incubated at 37°C overnight, and the zones of inhibition were measured.

Sensitivity to hydrogen peroxide, methyl viologen, and SIN-1. Cells were grown to logarithmic phase and diluted to obtain a concentration of 105 cells ml–1. Cells were incubated at room temperature with different concentrations of H2O2, methyl viologen, 3-morpholinosydnonimine (SIN-1), or double-distilled water for 1 h, and serial dilutions were plated on LB agar plates. CFU were counted after overnight incubation at 37°C.


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RESULTS AND DISCUSSION
 
B. cenocepacia K56-2 6E3 harbors a transposon insertion in a gene encoding a member of the MgtC-like protein family. Mutant strain K56-2 6E3, attenuated for virulence in a rat model of chronic lung infection, carries a transposon insertion in an open reading frame homologous to the mgtC gene of S. enterica and Mycobacterium tuberculosis (23). The availability of the genome sequence of strain J2315 (http://www.sanger.ac.uk/Projects/B_cenocepacia/), which is clonally related to K56-2 (35), facilitated the analysis of the mgtC genomic region. In strain J2315, mgtC (BCAM1867) is located on chromosome 2 between bases 2085986 and 2086693, directly upstream of the cepIR quorum-sensing genes (BCAM1868 and BCAM1870) and downstream of a putative multigene operon (Fig. 1). The gene organization of the mgtC region in strains K56-2 and J2315 was the same, as determined by PCR analysis (data not shown). In S. enterica, Yersinia pestis, and Brucella melitensis, mgtC forms an operon with mgtB, a gene encoding a magnesium transporter (3). In contrast, the predicted protein products of the operon upstream of B. cenocepacia mgtC suggest that they form an efflux system (Fig. 1). The first gene of the putative operon, BCAM1862, encodes a LysR-type transcriptional regulator. The second gene (BCAM1863) encodes a conserved putative exported protein. This is followed by a gene encoding a member of the HlyD family of membrane fusion proteins (BCAM1864), which also has regions of homology to the EmrA multidrug resistance efflux pump. BCAM1865 encodes a product that has a fusaric acid resistance protein conserved region, and BCAM1866 encodes a product that contains two outer membrane efflux pump domain repeats consistent with the TolC family of outer membrane proteins (Fig. 1). This gene organization is also found in other Burkholderia species and Ralstonia eutropha. The presence of genes encoding efflux pumps near an mgtC-like gene has also been recognized previously (3). The transcriptional organization of mgtC and its upstream region in strain K56-2 was experimentally determined by RT-PCR amplification, which confirmed that the contiguous genes BCAM1866 and mgtC (BCAM1867) are part of two distinct transcriptional units and that the open reading frames of BCAM1863, BCAM1864, BCAM1865, and BCAM1866 are all cotranscribed (data not shown).


Figure 1
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  		FIG. 1. Genetic and transcriptional organization of the 10-kb region surrounding mgtC in B. cenocepacia strains K56-2 and J2315. Annotation of the putative open reading frames (gray arrows) surrounding the transposon insertion shows that it is located within BCAM1867 (black arrow), directly upstream of the transcriptional activator CepR and acyl homoserine lactone synthase CepI open reading frames. mgtC appears to be a discrete transcriptional unit (B), located downstream of an operon (A) containing five genes encoding hypothetical proteins (BCAM1862, BCAM1863, BCAM 1864, BCAM1865, and BCAM1866). Transcriptional units are represented by thin lines with arrowheads.

Alignment of the amino acid sequences of the MgtC proteins of B. cenocepacia, S. enterica, and M. tuberculosis revealed that the MgtC protein of B. cenocepacia exhibits 38% and 39% amino acid identity with the MgtC proteins of Salmonella and Mycobacterium, respectively, particularly in the N-terminal portion of the protein. This conserved, highly hydrophobic region has been referred to as the "MgtC domain" (3). A phylogenetic analysis of proteins with MgtC domains classified the MgtC protein from Burkholderia fungorum strain LB400, a species closely related to B. cenocepacia and recently reclassified as Burkholderia xenovorans (17), as a member of group 1 or "true MgtC" proteins. Therefore, the Burkholderia MgtC proteins may have a function similar to that of the MgtC proteins of other members of this group, including the MgtC proteins of Salmonella, Mycobacterium, Brucella, and Yersinia species (3).

Disruption of mgtC results in a B. cenocepacia mutant defective for growth in magnesium-depleted medium. In S. enterica, M. tuberculosis, Brucella suis, and Y. pestis, mgtC is essential for growth in low-magnesium environments (2, 5, 19, 28). Therefore, we examined whether the B. cenocepacia mgtC gene was required for survival in Mg2+-depleted medium as well. For this purpose, we first constructed strain KEM1, an isogenic derivative of K56-2 containing an insertionally inactivated mgtC gene. Analysis of growth rates in minimal medium containing various concentrations of Mg2+ demonstrated that in the presence of 5 µM Mg2+, wild-type B. cenocepacia K56-2 reached an OD600 that was 1.5-fold higher than that of mgtC mutant KEM1 (Fig. 2). The growth defect was corrected in a dose-dependent manner with increasing concentrations of Mg2+. At Mg2+ concentrations of 35 µM and above, the mgtC mutant grew to the same OD600 as the parental strain (Fig. 2). Complementation experiments were conducted using strain KEM1 conjugated with pKM2, which carries the cloned B. cenocepacia mgtC gene, and with the vector pKMBAD as a control. Strain KEM1(pKM2) had an intermediate growth phenotype in minimal medium with 10 µM Mg2+ compared to the growth rates of KEM1(pKMBAD) and parental strain K56-2 also containing pKMBAD (Fig. 3A), suggesting that mgtC is involved in either uptake or adaptation to a low-magnesium environment in vitro. The partial complementation of the mgtC defect in KEM1 may be due to the overexpression of mgtC in B. cenocepacia, which appears to have a detrimental effect on growth. We observed that the plasmid expressing mgtC cannot be conjugated into a B. cenocepacia strain containing a functional copy of mgtC. Previously, other workers have reported that expression of the Salmonella mgtC gene in E. coli K-12, which lacks mgtC, allows this bacterium to grow in the presence of very low Mg2+ concentrations (2). However, our results demonstrated that the B. cenocepacia MgtC protein, when expressed as a FLAG-tagged protein fusion, did not restore growth to E. coli K-12 in minimal medium supplemented with 10 µM MgSO4 (data not shown). The lack of complementation of growth in E. coli K-12 was not due to a defect in protein expression, since the MgtCFLAG polypeptide could be detected by Western blot analysis of bacterial membrane fractions using anti-FLAG antibodies (Fig. 4). Therefore, the B. cenocepacia mgtC gene is not functionally identical to the Salmonella mgtC gene.


Figure 2
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  		FIG. 2. mgtC is required for growth in magnesium-depleted medium: growth analysis of wild-type B. cenocepacia strain K56-2 (A) and the mgtC-deficient mutant KEM1 (B) in M56 minimal medium supplemented with different concentrations of magnesium. The growth defect of KEM1 is abrogated upon addition of excess magnesium (B and C). (C) Magnesium-dependent growth of K56-2 and KEM1 after 12 h. The data are averages and standard deviations for three independent experiments done in triplicate.


Figure 3
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  		FIG. 3. Growth defect of the mgtC mutant (KEM1) in magnesium-depleted medium can be complemented by transformation with pKM2 encoding an intact mgtC gene. Bacteria were grown in M56 minimal medium supplemented with 10 µM MgSO4 (A) or 10 mM MgSO4 (B). The data are averages and standard deviations for three replicates.


Figure 4
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  		FIG. 4. FLAG-tagged MgtC can be expressed from a plasmid in E. coli: Western blot analysis of E. coli membrane fractions using anti-FLAG antibodies. The band indicated by an asterisk at approximately 30 kDa is at the predicted position of the MgtC protein with the FLAG epitope. The molecular masses (in kDa) of markers (lanes M) are indicated on the left.

Disruption of mgtC results in a B. cenocepacia mutant defective for survival within murine macrophages. Previous work in our laboratory demonstrated that in contrast to classical intracellular pathogens, Bcc strains survive intracellularly without replication in amoebae (38) and murine macrophages (44). A major drawback of cell infection assays using B. cenocepacia isolates is the difficulty in effectively killing extracellular bacteria with antibiotics due to the extraordinary resistance of these isolates to antimicrobials that are commonly employed to kill extracellular bacteria in classical invasion assays (38, 44, 51). Using microscopic single-cell analyses to assess the viability and distribution of intracellular bacteria in specific compartments that can be labeled with fluorescent probes, we established that B. cenocepacia localizes within membrane-bound vacuoles that do not fuse with lysosomes (26, 27). Therefore, to examine whether the disruption of mgtC affects intracellular survival of B. cenocepacia, RAW 264.7 macrophages pretreated with LysoTracker Red were infected with either parental strain K56-2 or the mgtC mutant KEM1. LysoTracker Red, which consists of a fluorophore linked to a weak base that is only partially protonated at neutral pH, is freely permeant to cell membranes and typically concentrates in acidic organelles. Its mechanism of retention has not been firmly established but is likely to involve protonation in low-pH compartments and retention in the membranes of acidic organelles (34, 53). At 4 h postinternalization, the majority of vacuoles containing KEM1 bacteria colocalized with LysoTracker (Fig. 5A), while most of the vacuoles containing K56-2 did not colocalize with the fluorescent probe (Fig. 5B). Quantitative analysis performed by counting intracellular bacteria in a blinded fashion for an average of 15 macrophage cells per field of view over a total of 21 fields demonstrated that in macrophages infected with KEM1, 76.4% ± 3.6% of the vacuoles containing bacteria colocalized with LysoTracker (Fig. 5C). In contrast, only 34.7% ± 3.9% of vacuoles containing bacteria localized with LysoTracker in the infections with parental strain K56-2. Our data may underrepresent the actual number of bacteria reaching the lysosome since degraded bacteria colocalizing with LysoTracker were not visualized. We recently showed that live Burkholderia cells expressing enhanced green fluorescent protein retained the fluorescence within the bacterial cytoplasm, whereas heat-killed bacteria, which retained the fluorescence if they were kept in buffer, leaked fluorescence to the vacuolar space once they were phagocytized (27). Thus, dispersal of the fluorescent protein throughout the phagosomal lumen can be used as an indication of bacterial cell disruption. We used a similar strategy to assess the viability of mutant strain KEM1 carrying pRed-Cm (Table 1), a plasmid encoding mRFP1. At 4 h postinfection, the lumina of phagosomes containing KEM1(pRed-Cm) bacteria were fluorescently labeled, suggesting that soluble mRFP1 had leaked from the bacterial cytoplasm into the phagosomal lumen (Fig. 6A). In contrast, the majority of the bacterium-containing vacuoles in macrophages infected with K56-2(pRed-Cm) did not fluoresce, as the red fluorescence was retained within the bacterial cytoplasm (Fig. 6B). Also, internalized KEM1 expressing mRFP1 exhibited a variety of abnormal morphologies, such as rounding, filamentation, and a very dense cytoplasm (data not shown), suggesting that the cellular envelope had been compromised. Quantitative analyses demonstrated that after infection with KEM1(pRed-Cm) 95.7% ± 1.5% of the bacterium-containing vacuoles were uniformly fluorescent, in contrast to infections with K56-2(pRed-Cm), where only 23.0% ± 3.6% of the vacuoles showed leakage of bacterially encoded mRFP1 into the vacuolar space (Fig. 6C). To ensure that the leakage of mRFP1 was not due to general compromise of the membrane integrity, bacteria were tested for sensitivity to SDS, polymyxin B, EDTA, tetracycline, and chloramphenicol, as the loss of bacterial cell envelope integrity often results in increased permeability and sensitivity to detergents, cationic peptides, antibiotics, and other toxic compounds (43). No differences between KEM1(pRed-Cm) and K56-2(pRed-Cm) were observed (data not shown), suggesting that the cell envelope of the mgtC mutant remains intact until the bacteria are in the intracellular environment.


Figure 5
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  		FIG. 5. mgtC mutants colocalize with the fluid probe LysoTracker Red. RAW 264.7 macrophages infected with B. cenocepacia KEM1 (A) or K56-2 (B) were observed at 4 h postinfection in macrophages pretreated with LysoTracker Red. Differential interference contrast (DIC), red fluorescence, and merged images are shown in panels A and B. The mgtC mutant bacteria reach an acidic compartment, as shown by colocalization with LysoTracker Red (A). The arrows indicate the locations of bacterium-containing vacuoles. (C) Average percentages of B. cenocepacia-containing vacuoles (BcCV) colocalizing with LysoTracker Red from three independent experiments. The error bars indicate standard deviations.


Figure 6
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  		FIG. 6. mgtC is required for cell envelope integrity of B. cenocepacia within murine macrophages. RAW 264.7 macrophages infected with B. cenocepacia KEM1 (A) or K56-2 (B) expressing mRFP1 were observed 4 h postinfection. Differential interference contrast (DIC), red fluorescence, and merged images are shown in panels A and B. The mgtC mutant bacteria had compromised cell envelope permeability, as shown by the release of mRFP1 into the vacuolar lumen, while the parental bacteria retained mRFP1 within the cytoplasm. The arrows indicate bacterium-containing vacuoles. (C) Average percentages of B. cenocepacia-containing vacuoles (BcCV) containing released mRFP1 from three independent experiments. The error bars indicate standard deviations.

Together, our results demonstrate that B. cenocepacia mgtC is required for intracellular survival of this bacterium in macrophages. Previous studies with S. enterica have suggested that the defect in intracellular survival of mgtC mutants is due to a low-magnesium environment in the vacuole (2). This suggestion was based largely upon the upregulation of PhoP/PhoQ-regulated genes in vivo, as well as evidence suggesting that addition of excess magnesium to cell culture medium improved the growth defect of mgtC-deficient Salmonella mutants within macrophages (2, 47). However, recent studies have demonstrated that the major signal for PhoP/PhoQ within the phagosome is the decrease in pH that occurs with phagosome acidification and that the intraluminal magnesium concentration in phagosomes is approximately 1 mM (40). At this concentration in vitro the B. cenocepacia mgtC mutant can grow to the same extent as the wild type (Fig. 2). In addition, RAW 264.7 cells are naturally devoid of functional NRAMP1, the proton pump believed to be important for creating a vacuolar environment with a low level of divalent cations (18). It is therefore possible that mgtC has a function other than adaptation to a low-magnesium environment in vivo and that in vitro mgtC simply requires magnesium, either directly or indirectly, to perform its critical role. This possibility is consistent with the observation that the B. cenocepacia mgtC mutant is attenuated in vivo (23) despite the presence of genes encoding other magnesium transporter homologues, such as MgtB, MgtA, and CorA, in the B. cenocepacia genome.

mgtC-deficient B. cenocepacia strains are not sensitive to reactive oxygen species, reactive nitrogen species, cationic peptides, or low pH. Several studies have suggested that MgtC is not itself a magnesium transporter and that the survival defect in low-magnesium conditions observed in vitro may be due to a secondary function of the protein (41). In addition, B. cenocepacia mgtC mutants are destroyed as early as 1 h after phagocytosis (data not shown), suggesting that the mgtC gene is involved in more than adaptation to the environment. Since this gene is evidently important for survival in vivo and the mgtC mutant does not survive within macrophages, we tested possible conditions that B. cenocepacia may encounter within the phagosome. The level of resistance to killing by H2O2 was assessed by exposing bacterial cells to concentrations of H2O2 ranging from 2.5 mM to 10 mM. Bacterial susceptibility to intracellular O– was investigated by exposing cells to concentrations of methyl viologen ranging from 0 to 10 mM. The cells accumulate this compound, and its reduction in the cytoplasm causes the formation of O– anions. No differences in the levels of resistance to all of these compounds were detected between mutant KEM1 and the wild-type B. cenocepacia K56-2 strain. In addition, intracellular survival assays after pretreatment of macrophages with DPI, an inhibitor of the enzyme NADPH oxidase, were performed to determine whether reactive oxygen species played a role in killing mgtC mutants in vivo. However, the same proportion of KEM1(pRed-Cm) bacteria leaking mRFP1 into the vacuole was detected with and without DPI pretreatment.

In addition to generating oxidative burst, upon infection macrophages upregulate the production of inducible nitric oxide synthase, which produces reactive nitrogen species in the phagosome. To assess the sensitivity to nitrosative stress in vitro, bacteria were incubated with SIN-1, a nitric oxide donor that spontaneously decomposes, yielding nitric oxide and superoxide anion radicals. Serial dilutions were plated at 30-min intervals up to 120 min after addition of SIN-1 to the culture. The number of CFU recovered after exposure to SIN-1 was the same for both the mgtC mutant and parental isolates. One important mechanism of nonoxidative killing within phagosomes is antimicrobial peptides. We have recently shown that a lipopolysaccharide-defective mutant of B. cenocepacia, which is sensitive to structurally unrelated cationic peptides, has a polymyxin MIC50 of 32 µg ml–1; in comparison, the MIC50 for the parental isolate K56-2 is more than 512 µg ml–1 (33). K56-2 and KEM1 were therefore plated on either LB or minimal medium containing 250 µg ml–1 polymyxin B. The mutant and wild-type cells could grow equally well in the presence of the antimicrobial peptide, demonstrating that they are inherently resistant to its activity. The mgtC mutant was also assayed for sensitivity to low pH. Growth curves in buffered minimal medium at pH 5.4 showed that there was no difference between the wild-type and mutant B. cenocepacia strains. Furthermore, intracellular survival assays after pretreatment of macrophages with bafilomycin, a macrolide antibiotic that acts as a potent and specific inhibitor of vacuolar-type H+-ATPase, demonstrated that acidification of the vacuole is not required for membrane disruption of mgtC-defective bacterial cells within murine macrophages. In addition, blocking vacuolar acidification prevents phagosomal maturation, thus affecting the processing and activity of many endosomal peptides, hydrolases, and proteinases (4, 11, 31, 49, 50). Collectively, these studies demonstrate that the disruption of membrane integrity of the KEM1 mutant bacteria within macrophages does not appear to be due to low pH, oxidative stress, nitrosative stress, or cationic peptides. However, it is difficult to recreate the exact conditions present inside the vacuole in vitro, and the attenuation of the mgtC mutant may be due to a combination of the different components of the macrophage response that awaits future experiments.

Concluding remarks. The results of this study demonstrate that mgtC is important for the pathogenesis of B. cenocepacia. The importance of mgtC for survival of B. cenocepacia and other organisms with an intracellular phase in both animal models of infection and ex vivo models of intracellular survival emphasize the need to elucidate the physiological role of the MgtC protein. To date, the mgtC mutant is the only B. cenocepacia mutant tested that is severely compromised in both the rat agar bead model of lung infection and the RAW 264.7 macrophage infection model. However, the reduced intracellular survival of the mgtC mutant in macrophages is not due to increased sensitivity to low pH, oxidative stress, nitrosative stress, or cationic peptides, nor does it appear to be the consequence of a general membrane defect since mgtC mutants can resist detergents and antimicrobial peptides. Further experiments are under way in our laboratory to elucidate how MgtC may be involved in protecting B. cenocepacia from the intracellular environment of the host cells.


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ACKNOWLEDGMENTS
 
We thank Julie Lamothe for help with the microscopic analysis, the other members of our laboratory for helpful discussions, Roger Y. Tsien for providing the mRFP1, and Julian Parkhill for allowing access to the draft annotation of B. cenocepacia J2315.

This work was supported by a grant from the Canadian Institutes of Health Research. The Bioscreen C automated microbiology growth curve analysis system was obtained with grants from the Natural Sciences and Engineering Research Council and the University of Western Ontario Academic Development Research Fund. M.A.V. holds a Canada Research Chair in Infectious Diseases and Microbial Pathogenesis.


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FOOTNOTES
 
* Corresponding author. Mailing address: Infectious Diseases Research Group, Siebens-Drake Research Institute, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, N6A 5C1, Canada. Phone: (519) 661-3996. Fax: (519) 661-3499. E-mail: mvalvano{at}uwo.ca. Back

Editor: J. N. Weiser


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Infection and Immunity, October 2006, p. 5477-5486, Vol. 74, No. 10
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