JOURNAL OF BACTERIOLOGY, Sept. 2007, p. 6580–6586 0021-9193/07/$08.00⫹0 doi:10.1128/JB.00809-07 Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 189, No. 18
MglA Regulates Francisella tularensis subsp. novicida (Francisella novicida) Response to Starvation and Oxidative Stress䌤† Tina Guina,1* Dragan Radulovic,2* Arya J. Bahrami,1 Diana L. Bolton,1 Laurence Rohmer,3 Kendan A. Jones-Isaac,1 Jinzy Chen,4 Larry A. Gallagher,3 Byron Gallis,4 Soyoung Ryu,4 Greg K. Taylor,4 Mitchell J. Brittnacher,3 Colin Manoil,3 and David R. Goodlett4 Department of Pediatrics, Division of Infectious Diseases,1 Department of Medicinal Chemistry,4 and Department of Genome Sciences,3 University of Washington, Seattle, Washington, and Department of Mathematics, Florida Atlantic University, Boca Raton, Florida2 Received 24 May 2007/Accepted 3 July 2007
MglA is a transcriptional regulator of genes that contribute to the virulence of Francisella tularensis, a highly infectious pathogen and the causative agent of tularemia. This study used a label-free shotgun proteomics method to determine the F. tularensis subsp. novicida (F. novicida) proteins that are regulated by MglA. The differences in relative protein amounts between wild-type F. novicida and the mglA mutant were derived directly from the average peptide precursor ion intensity values measured with the mass spectrometer by using a suite of mathematical algorithms. Among the proteins whose relative amounts changed in an F. novicida mglA mutant were homologs of oxidative and general stress response proteins. The F. novicida mglA mutant exhibited decreased survival during stationary-phase growth and increased susceptibility to killing by superoxide generated by the redox-cycling agent paraquat. The F. novicida mglA mutant also showed increased survival upon exposure to hydrogen peroxide, likely due to increased amounts of the catalase KatG. Our results suggested that MglA coordinates the stress response of F. tularensis and is likely essential for bacterial survival in harsh environments.
tularensis lipopolysaccharide component lipid A is a poor stimulator of the human and mouse TLR4 receptors (9, 19). F. tularensis cells also actively suppress innate immune signaling by the infected antigen-presenting cells (3, 4, 45). Intracellular F. tularensis escapes the phagocytic vacuole (16, 41). Once located in the cytoplasm, F. tularensis cells trigger the activation of caspases and the secretion of moderate levels of proinflammatory cytokines (15, 33). The F. tularensis MglA regulator activates the transcription of genes encoded by the Francisella pathogenicity island (FPI) and around 90 other genes (5, 29, 36). MglA and the FPI contribute to F. tularensis virulence in mice, replication in mammalian cells and amoebae (2, 29), and phagosomal escape (16, 41). FPI genes exhibit no significant homology to genes of known function, and their role in disease is not understood. MglA is homologous to the Escherichia coli stringent starvation protein A, SspA (20). Besides MglA, F. tularensis genomes encode a second SspA homolog, which was annotated as SspA. In E. coli, SspA inhibits the stationary-phase accumulation of the DNA-bending H-NS repressor, resulting in the derepression of acid stress and nutrient starvation responses (21). The F. tularensis SspA and MglA amino acid sequences exhibit 21% identity and 44% similarity over the N-terminal two thirds. The N-terminal 70 amino acids of F. tularensis MglA and SspA and the N-terminal domains of other bacterial SspA homologs exhibit similarity to the gluthathione S-transferase active domain in which the gluthathione S-transferase active-site residue Cys is replaced by a Tyr (20). These structural similarities suggested the possible interplay of MglA and SspA in Francisella gene regulation. A recent study demonstrated that F. tularensis MglA and SspA interact with each other, bind RNA polymerase, and regulate FPI genes (7). F. tularensis spp. are
Francisella tularensis is the causative agent of tularemia, a zoonotic disease that is typically transmitted by inhalation, by the bite of an infected arthropod, or by the ingestion of contaminated water. F. tularensis subsp. tularensis, subsp. holarctica, and subsp. mediasiatica are pathogens of humans and various other mammals (12). F. tularensis subsp. novicida (F. novicida) is infectious for immunocompromised humans only (22). All F. tularensis subspecies and F. novicida cause severe disease in mice, though their lethal doses vary in accordance to their propensity to cause human disease (12). Human pathogenic F. tularensis are highly infectious: fewer than 10 bacteria inoculated by the respiratory route can cause the disease in humans and result in a high incidence of mortality if untreated (12). The mechanisms that cause high F. tularensis infectivity and virulence are unknown. One possible mechanism is the remarkable ability of F. tularensis to evade innate immune responses early in infection. In mice infected with F. tularensis, proinflammatory cytokines and chemokines are usually measured only after a significant systemic organ burden and inflammatory granulocyte infiltration occurs (4, 9, 11). F.
* Corresponding author. Mailing address for Tina Guina (correspondent for biological experiments and protein annotation): Department of Pediatrics, Division of Infectious Diseases, University of Washington, Seattle, WA. Phone: (206) 616-3468. Fax: (206) 543-5383. E-mail:
[email protected]. Mailing address for Dragan Radulovic (correspondent for mathematical algorithm for determination of relative peptide abundance): Department of Mathematics, Florida Atlantic University, Boca Raton, FL. Phone: (561) 297-3346. Fax: (561) 297-2436. E-mail:
[email protected]. † Supplemental material for this article may be found at http://www .jb.asm.org/. 䌤 Published ahead of print on 20 July 2007. 6580
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evolutionarily distant from most well-studied bacterial pathogens, and their genomes do not encode obvious toxins or bacterial virulence factor homologs. Therefore, the definition of F. tularensis regulons that contribute to virulence provides a rational approach to the definition of factors that contribute to its pathogenicity. This study utilized a novel proteomics approach and state-of-the-art mass spectrometry (MS) to determine the F. novicida MglA-regulated proteins. Most current MS-based proteomic methods for the determination of differences in the relative protein amounts between two samples involve the use of stable-isotope dilution (e.g., isotope-coded affinity tags) (10). Besides the expense of the reagents, these stable-isotope labeling methods require many hours of labor to prepare and fractionate samples prior to the MS analysis, the latter of which increases the amount of MS analysis time as a direct function of the number of fractions. Instead, this study utilized an alternative method based on label-free shotgun proteomics and novel mathematical and biostatistical approaches for direct measurement of the relative amounts of peptides from the peptide precursor ion intensity values measured in the MS (40). The novel result of this study is that the lack of functional MglA results in reduced amounts of F. novicida stress response proteins. In comparison to the results for the isogenic wild-type strain, an F. novicida mglA mutant showed increased susceptibility to several types of stress. Therefore, this study showed that MglA coordinates the F. tularensis stress response and is likely required for F. tularensis survival in diverse hostile environments.
MATERIALS AND METHODS Growth of F. novicida strains, cellular fractionation, and protein digestion with trypsin. The F. novicida wild-type strain U112 and the mglA mutant strain (2) were obtained from Francis Nano (University of Victoria, Victoria, Canada). Three individual cultures of the F. novicida wild-type strain U112 and the mglA mutant (2) were grown in rich medium—tryptic soy broth (Difco, Detroit, MI) supplemented with 0.1% cysteine (TSB-C)—at 37°C. Both the wild-type and mglA mutant F. novicida cultures grew at similar rates in TSB-C and reached high densities at the stationary phase (optical density at 600 nm [OD600], ⬃3.0). Bacterial cells were collected during the mid-logarithmic phase of growth when the optical absorbance (OD600) of all cultures was 0.6 to 0.7, which corresponds to ⬃6 ⫻ 108 CFU/ml for each strain. The cells were collected by sedimentation at 10,000 ⫻ g for 15 min at 4°C. The cells were then resuspended in a 1:100 volume of ice-cold 50 mM Tris (pH 8.3) and stored at ⫺80°C until further processing. Once thawed, the cells were broken by sonication in an ice water bath. Unbroken cells were removed by sedimentation at 5,000 ⫻ g for 15 min at 4°C, and the whole-cell bacterial extract was saved. A portion of the bacterial extract was sedimented further at 120,000 ⫻ g for 2 h at 4°C to separate the soluble proteins from the insoluble, membrane-enriched protein fraction. The insoluble proteins were homogenized in ice-cold 50 mM Tris buffer (pH 8.3). The protein concentrations of each fraction were determined by using a Bradford protein assay (Pierce, Rockford, IL). Amounts of 300 g each of the whole-cell, soluble, and membrane fractions were dissolved in 6 M urea. The pH of the lysates was raised to 8.8 with the addition of Tris to 100 mM. The lysates were incubated for 1 h at 37°C with 5 mM Tris (2-carboxyethyl) phosphine, followed by incubation with 40 mM iodoacetamide for 1 h in the dark. The proteins were incubated for 1 h with 40 mM dithiothreitol at room temperature and then diluted 10-fold with 50 mM ammonium bicarbonate (pH 7.8) and methanol to 20% (vol/vol). The protein preparations were digested by using sequencing-grade trypsin (Promega, CA) at a 50:1 protein/trypsin ratio overnight at room temperature. The digests were then evaporated to dryness in a speedvac, and the peptides were redissolved in 5% acetonitrile–0.1% trifluoroacetic acid and desalted on a Vydac silica C18 macrospin column (The Nest Group, Southborough, MA). The eluates from the C18 column were evaporated to near dryness, dissolved in 5% acetonitrile–0.1% trifluoroacetic acid, and stored at ⫺80°C until MS analysis.
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MS peptide analysis. Peptide digests of each cellular fraction (triplicates of the whole cell, membrane, and soluble fractions of wild-type F. novicida and the mglA mutant) were analyzed in quadruplicate by microcapillary high-pressure liquid chromatography (LC) electrospray ionization tandem MS on a linear ion trap Fourier transform ion cyclotron resonance MS (LTQ-FT-ICR-MS; Thermo Electron, San Jose, CA). The high-pressure LC system (Michrome Bioresources, CA) was configured as described previously (48) with few modifications. Briefly, 0.05 mg of peptide digest were loaded onto a precolumn (100 mm by 1.5 cm, 5 mm, 200 Å pore Magic C-18 AQ beads; Michrom Bioresources, CA) with solvent A (0.1% formic acid, 5% acetonitrile) at a flow rate of 15 l/min and washed for 5 min. The peptides were then eluted with a gradient of 10 to 45% solvent B (100% acetonitrile) during 60 min on an analytical column (75 mm by 11 cm, 5 mm, 100 Å pore Magic C-18 AQ beads from Michrom Bioresources, CA) at ⬃200 nl/min and further detected by LTQ-FT-ICR-MS. The LTQ-FT-ICR-MS was operated in a data-dependent mode to switch between MS and tandem MS acquisition in full scan mode. Precursor ion scans over a range of 400 to 1,800 m/z were acquired in the FT-ICR, while the five most intense ions were sequentially isolated and subjected to collision-induced dissociation in the LTQ. The general MS conditions were: ESI voltage, 1.5 kV; ion transfer tube temperature, 200°C; collision gas pressure, 1.3 mTorr; and normalized collision energy, 30%. F. novicida protein identification and determination of the peptide and protein expression ratios. The individual peptides from 12 LC-MS data sets for each cellular fraction were identified by matching the tandem mass spectra (generated by quadruplicate analysis of each sample) against the F. novicida genomic database using the SEQUEST algorithm (47) and PeptideProphet (26). Individual proteins were considered identified when more than one unique peptide from a given protein was confirmed by ProteinProphet (37) (cutoff score, ⬎0.95). The average relative amount of each peptide that was identified in either the wildtype F. novicida strain or the mglA mutant was determined by using an improved version of the DRAGON algorithm (40), details of which will be described elsewhere. This study incorporated the high measured-mass accuracy of FTICR-MS data with a series of new mathematical algorithms capable of determining with high confidence the relative expression levels of several thousand peptides. In brief, precursor ion scans measured in quadruplicate in the FTICR-MS for each sample were used to extract the average ion intensity values for each peptide of interest. This method involved the following algorithms for processing raw MS data: (i) filtering to separate peptide signals from the ion current noise and to combine all the MS signals for a given peptide into a composite peptide signal; (ii) normalization to compensate for the fluctuation in total ion current for a given peptide and fluctuations in the amount of total peptide sample loaded between each quadruplicate LC-MS analysis of a given sample; and (iii) alignment of LC-MS ion intensity maps to reliably match a given composite peptide signal between different samples. The output data consisted of a peptide list where each peptide was accompanied by 12 numbers, indicating the total ion current per each MS run (four MS runs for each triplicate sample derived from either wild-type F. novicida or the mglA mutant). We performed a pair-wise, two sided t test between the corresponding quadruplicate runs and selected only those peptides with a P value of ⬍0.01. For these significantly different peptides, we derived the relative ratios by simply computing the fraction of aggregated intensities in the corresponding quadruplicate runs. Since often more than one peptide per protein was detected, we combined these peptide ratios using a P value-based weighted average:
冤冘 冥 k
ratio ⫽ 关R1/共1 ⫺ p1兲 ⫹ …Rk/共1 ⫺ pk兲兴/
共1 ⫺ pi兲
i⫽1
where Ri and pi are the ratios and p values for each of the k peptides that were detected per protein. The final result is the ratio of the relative amounts of protein. Determination of the growth and survival rates of the F. novicida mglA mutant strain during starvation and exposure to oxidative stress. For determination of the bacterial growth and survival rates during starvation, F. novicida was grown in a restrictive medium, Mueller-Hinton broth (MHB; Becton Dickinson, Franklin Lakes, NJ), at 37°C with shaking (250 rpm). Unlike the results with TSB-C (see above), the F. novicida cultures reached an OD600 of only 0.7 during stationary-phase growth. The susceptibility of F. novicida to hydrogen peroxide (Sigma, St. Louis, MO) and paraquat (Sigma, St. Louis, MO) was measured in a disk-diffusion assay. The bacterial cultures were grown until logarithmic phase (OD600, 0.2 to 0.3), diluted 50-fold in MHB, and plated onto TSB-C agar plates. Eight-millimeter filter disks (Remel, Lenexa, KS) were placed onto plates and 15 l of freshly diluted hydrogen peroxide (H2O2) or paraquat was added to each
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disk. The diameters of the bacterial growth inhibition zones were measured after 2 days. Mouse infections. An F. novicida mutant with a transposon insertion in katG (codon 487 of 739) was selected from a defined genomic transposon mutant library (14). The wild-type F. novicida and katG mutant cultures grew at similar rates during in vitro growth in TSB-C. Six- to 8-week-old specific-pathogen-free male BALB/c mice (Charles River Laboratories, Wilmington, MA) were infected intradermally with 5,000 CFU (⬃2⫻ the 50% lethal dose for the wild-type strain) F. novicida in 50 l phosphate-buffered saline as previously described (18). Mouse survival was monitored over the course of 7 days.
RESULTS AND DISCUSSION Definition of F. novicida MglA proteoregulon by a label-free shotgun proteomic method. In this study, the relative amounts of protein in each of the wild-type F. novicida and mglA mutant triplicate cultures were determined by a novel proteomics approach that circumvents the need for stable-isotope dilution. The wild-type F. novicida and mglA mutant strains were grown in the rich TSB-C medium. The OD600 of all six cultures at the mid-logarithmic phase was 0.6 to 0.7 (⬃6 ⫻ 108 CFU/ml). Both the wild-type and mglA mutant F. novicida cultures grew at similar rates in TSB-C and reached high densities (OD, ⬃3.0; ⬃3 ⫻ 108 CFU/ml) at the stationary phase. Whole-cell extracts and the soluble fraction and membrane fraction were prepared from each of the six cultures. The proteins were then denatured and digested with trypsin, and the resulting peptides analyzed in quadruplicate by LC-MS. Mathematical methods based on DRAGON modifications of previously published methods (DRAGON [40]) were used to extract the average peptide ion intensity values from precursor ion scans carried out in the 12 LC-MS runs performed for the analysis of tryptic peptides from each cellular fraction (i.e., quadruplicate LC-MS runs for each of the triplicate samples; see Materials and Methods). Finally, for those proteins (peptides) with amounts that were significantly different by the two-sided t test (P ⬍ 0.01), the relative amounts were derived by comparing the ratios of the aggregated quadruple runs for the wild-type F. novicida and the mglA mutant strains. Using this approach, 732 F. novicida proteins (42.3% of predicted proteins) were identified. Among these, increased relative amounts were determined for 210 proteins (“MglAinduced” proteins), while decreased relative amounts were determined for 132 proteins (“MglA-repressed” proteins) in wild-type F. novicida compared to the amounts in the isogenic mglA mutant (see Tables S1 and S2 in the supplemental material). A previous study utilized DNA microarrays to define 102 MglA-regulated genes (5). Twenty-five of the MglA-induced proteins that were identified by the proteomics approach were encoded by the previously described MglA-induced genes (see Table S1 in the supplemental material). Among these were IglB, IglC, IglD, PdpD, and PdpB, proteins encoded by the FPI (29, 36), and a secreted metalloprotease, PepO (18) (Table 1). Overall, these results confirmed the validity of our analytical approach and indicated that the changes in protein ratios are representative of the F. novicida MglA proteoregulon, which is likely more complex than the MglA transcriptome. One explanation for the large number of protein abundance changes in the MglA mutant background is that they are likely due to global changes in posttranscriptional regulation, often a hallmark of the bacterial stress response (see below).
J. BACTERIOL.
MglA regulates the relative amounts of F. novicida stress response homologs. The novel result of this study was that MglA regulates the amounts of the F. novicida homologs of bacterial stress resistance proteins, some of which are involved in posttranscriptional regulation in other bacteria (Table 1). Among these were the MglA interacting partner SspA, the universal stress protein UspA (28), the cold-shock response protein CspC (39), the activator of osmoprotectant transporter ProQ, and the small RNA chaperone Hfq (Table 1). In E. coli, CspC and CspE upregulate the expression of rpoS, a gene that encodes a global stress response regulator, RpoS, by stabilizing the rpoS message (38). CspC and CspE also regulate the expression of the E. coli stress response genes dps, katG, proP, and uspA (38). The RNA chaperone Hfq is essential for small regulatory RNA stability and binding to its targets. Small RNAs mediate the posttranscriptional regulation of genes that respond to stress and starvation (17). The relative amounts of 25 other F. novicida homologs of the general stress and heat/ cold shock response proteins were also reduced in the mglA mutant (Table 1). MglA-induced were the general stress and heat/cold shock response chaperones GroL (Hsp60), GroS (Hsp10), trigger factor, HtpG (HSP90), and DnaK (HSP70) and the ATP-dependent proteases ClpP, ClpB, HslU, and FtsH (HflB) (Table 1). A recent transcriptional profiling study (5) did not detect MglA-dependent global changes in F. novicida stress-response gene expression. Therefore, in conjunction with evidence that the MglA proteoregulon includes several posttranscriptional regulators (CspC, CspE, and Hfq), our results suggested that MglA participates in F. novicida transcriptional and posttranscriptional stress response networks. The F. novicida mglA mutant exhibits decreased survival during stationary-phase growth. The proteomic analysis results suggested that the F. novicida mglA mutant might be more susceptible than the wild-type strain to starvation-induced stationary-phase stress. To determine the survival during starvation conditions, wild-type F. novicida and the mglA mutant were grown in the restrictive MHB medium in which the wildtype strain culture reaches a maximal OD600 of only 0.7 units. In accordance with the proteomics data, the F. novicida mglA mutant exhibited decreased survival during stationary-phase growth in MHB (Fig. 1). Furthermore, lysis of the F. novicida mglA mutant culture was observed after 24 h of growth (Fig. 1). In contrast, wild-type F. novicida cultures maintained stable optical densities for as long as 55 h at 37°C (Fig. 1). The F. novicida mglA mutant exhibits decreased survival upon exposure to the redox-cycling agent paraquat and increased survival upon exposure to hydrogen peroxide. The generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) by activated immune cells is essential for animal and plant innate immune defenses against invading pathogens (13). Mice defective in inducible nitric oxide synthase (iNOS⫺/⫺) and phagocyte oxidase (p47phox⫺/⫺) are highly susceptible to F. tularensis live vaccine strain (LVS) infection (32), suggesting that the generation of RNS and ROS is essential for host defense against tularemia. Exposure to ROS results in DNA damage, lipid peroxidation, protein modification and misfolding, and other types of molecular oxidation (24, 43). Detoxification of the ROS-exposed cells begins with the conversion of superoxide into H2O2 by action of superoxide dismutases. The conversion of H2O2 into water and
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TABLE 1. MglA regulates F. novicida stress response proteinsa Ratio of amt of protein in wild-type and mglA mutant F. novicida in: Category and F. novicida locus tag
Gene
Description of protein
Whole cell fraction
Soluble fraction
Membrane fraction
MglA induced (previously described) FTN_1186 FTN_1310 FTN_1322 FTN_1323 FTN_1324 FTN_1325
8.2 — 10.0 10.0 10.0 —
14.7 — 44.9 8.9 5.0 —
52.2 10.0 8.0 0.1 13.3 10.0
pepO pdpB iglC iglB iglA pdpD
M13 family metallopeptidase Protein of unknown function Intracellular growth locus protein C Intracellular growth locus protein B Intracellular growth locus protein A Protein of unknown function
Oxidative and nitrosative stress response induced FTN_0804 FTN_0840
9.4 11.3
— 5.0
— —
gshB mdaB
— — — 9.8 —
4.2 5.8 3.1 2.1 6.0
2.9 — — — —
— — grxB trxA1 —
Glutathione synthetase NADPH-quinone reductase (modulator of drug activity B) Peroxiredoxin, AhpC-TSA family protein Peroxiredoxin of the AhpC/TSA family Glutaredoxin 2 Thioredoxin Oxidoreductase
Oxidative and nitrosative stress repressed FTN_0982 FTN_0633
— —
0.4 0.2
— 0.1
grxA katG
Glutaredoxin 1 Peroxidase/catalase
Gene regulation induced FTN_0488 FTN_0549 FTN_0085 FTN_1051 FTN_1465 FTN_0289
— — — — 1.9 —
3.4 10.0 4.7 2.8 8.1 3.6
2.2 — 8.5 — 1.7 —
cspC sspA uspA hfq — proQ
Cold shock protein, DNA-binding Stringent starvation protein A Universal stress protein RNA-binding protein, small RNA chaperone Two-component response regulator Activator of osmoprotectant transporter ProP
Gene regulation repressed FTN_1715
—
—
0.0
kdpD
Two-component regulator, sensor histidine kinase
General stress response induced FTN_0996 FTN_1054 FTN_1057 FTN_1058 FTN_1072 FTN_1074 FTN_1157 FTN_1284 FTN_1285 FTN_1538 FTN_1539 FTN_0266 FTN_0347 FTN_0771 FTN_0668 FTN_1769 FTN_1768 FTN_1743 FTN_0211
— — 10.00 — 10.00 4.89 2.84 — — 3.93 — — — — 4.98 6.77 — 2.54 5.67
5.91 12.74 0.00 11.14 21.98 169.44 1.39 3.39 9.71 0.62 3.47 3.78 10.00 6.12 — — 6.21 3.54 6.51
10.00 4.39 — 6.79 — 4.09 — 1.12 — 4.71 1.73 — 2.78 1.94 6.64 — — 4.67 7.51
hslU hupB clpP tig — — — dnaK grpE groEL groES htpG fkpB — hflB — pepN clpB pcp
ATP-dependent protease HslVU DNA-binding protein HU-beta ATP-dependent Clp protease subunit P Trigger factor -Lactamase X-Prolyl aminopeptidase 2 Translational elongation factor Tu and G Chaperone, heat shock protein (HSP70 family) Chaperone GrpE (Hsp70/Hsc70) Chaperonin GroEL (HSP60 family) Cochaperonin GroES (HSP10) Chaperone Hsp90, heat shock protein HtpG FKBP-type peptidyl-prolyl cis-trans isomerase Protein-disulfide isomerase ATP-dependent metalloprotease Heat shock protein (HSP20 family) Aminopeptidase N Chaperone ClpB Pyrrolidone carboxylylate peptidase
General stress response repressed FTN_1055 FTN_1112 FTN_1209
— 0.52 0.53
0.05 0.59 0.36
0.21 0.32 0.30
lon cphA cphB
DNA-binding, ATP-dependent protease La Cyanophycin synthetase Cyanophycinase
FTN_0958 FTN_0973 FTN_1033 FTN_1415 FTN_0279
a
—, data lacked a statistically significant protein abundance ratio or the peptide was not identified in a given cellular fraction.
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FIG. 1. F. novicida mglA exhibits decreased survival during stationary-phase growth in growth-restrictive MHB medium. Growth rates of wild-type F. novicida (F) and the mglA mutant (Œ) in MHB at 37°C are shown. Lysis of F. novicida mglA cultures was observed after 24 h of growth. Error bars represent standard deviation (n ⫽ 3).
molecular oxygen is mediated by catalase, while peroxidase detoxifies H2O2 by attaching the hydroxyl groups to various organic molecules (24). The results of this study showed that MglA regulates the relative amounts of the F. novicida oxidative stress resistance mediators (Table 1). MglA induced nine F. novicida homologs of oxidative stress resistance proteins that included glutathione synthetase, peroxiredoxins, glutaredoxin, and thioredoxin (Table 1), proteins with redundant functions that participate in resistance to ROS (6). Also induced by MglA was the F. novicida homolog of MdaB (Table 1), an NADPH quinone reductase that protects Helicobacter pilory from ROS formed during electron transfer reactions (46). F. novicida FTN_0279, a homolog of the Yersinia pestis and Salmonella enterica serovar Typhimurium nitrosative stress resistance protein and virulence factor Hmp (1, 42), was also MglA-induced in this study (Table 1). To examine if the mglA mutant is susceptible to oxidative
J. BACTERIOL.
stress, we examined the susceptibilities of F. novicida strains to a redox-cycling compound, paraquat, that induces the generation of superoxide anion. Compared to the results for the wild-type strain, the F. novicida mglA mutant exhibited decreased survival in the presence of paraquat (Fig. 2A), confirming the role of the MglA regulon in the oxidative stress response. In contrast, the F. novicida mglA mutant exhibited increased survival in the presence of H2O2 (Fig. 2B). This result can be explained by the increased amounts of the catalase/peroxidase KatG that are produced in the absence of functional MglA (Table 1). In comparison to the results for the wild-type strain, the F. novicida katG mutant was highly susceptible to both H2O2 and paraquat (Fig. 2A and B). Despite the increased amount of KatG, the F. novicida mglA mutant showed an increased susceptibility to paraquat, suggesting that other oxidative stress resistance effectors contribute significantly to the resistance of F. novicida to superoxide. KatG is a single catalase/peroxidase produced by F. tularensis, and its predicted cellular localization is periplasmic (30). This KatG placement could be advantageous for a quick defensive response upon exposure to moderate amounts of ROS. However, the periplasmic localization of KatG could be disadvantageous in cases when high cytoplasmic ROS concentrations are achieved, such as the secondary effect of exposure to high concentrations of superoxide in activated phagocytes that eventually results in increased generation of ROS inside the bacterial cell. Increased amounts of intracellular ROS could also be a consequence of the fast aerobic growth that results in increased cellular respiration. Therefore, periplasmic catalase would be an effective defense against exogenous ROS but might not be efficient in the detoxification of endogenous ROS (43). An additional explanation for the increased susceptibility of the mglA mutant to paraquat could be the decreased abundance of a yet-unidentified transporter that is involved in the efflux of paraquat. Bacterial multidrug efflux transporters for paraquat and other compounds have been identified (8, 23, 35). A recent study showed that the loss of katG resulted in increased H2O2-mediated killing of human pathogenic F. tularensis subsp. tularensis and subsp. holarctica in vitro, but the
FIG. 2. Susceptibilities of the wild-type F. novicida (black bars), mglA mutant (white bars), and katG mutant (gray bars) strains to paraquat (A) and H2O2 (B) were measured by the disk-diffusion assay. Error bars represent standard deviation (n ⫽ 3). A P value of ⬍0.01 for differences between the strains was determined by t test.
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corresponding katG mutants exhibited the wild-type levels of virulence in mice (31). In contrast, a katG mutant of an attenuated F. tularensis subsp. holarctica LVS was severely attenuated in mice, suggesting that other enzymes and pathways of ROS resistance are affected in the LVS (31). To define the role of katG in F. novicida virulence, groups of five mice were infected intradermally with ⬃5,000 CFU of either wild-type F. novicida or the katG mutant and their survival observed over the course of 7 days. Similar to the results that have been obtained with katG mutants of the human pathogenic F. tularensis (31), F. novicida katG-infected mice became moribund at the same rate as mice that were infected with the wild-type F. novicida (data not shown). All mice were moribund by 7 days postinfection. This result suggested that katG plays no significant role in F. novicida virulence in mice infected via the skin and that the F. novicida ROS resistance pathways are likely more robust than those of the F. tularensis LVS. Previous studies have also shown that F. novicida is more virulent than the LVS in intradermally infected mice (27). The higher resistance to ROS could contribute to the increased virulence of F. novicida compared to that of the LVS. The genes that encode 56 F. novicida MglA-regulated proteins are absent or mutated in human pathogenic F. tularensis. Though they are very similar in their DNA sequence content (⬎95% sequence identity), F. tularensis spp. genomes exhibit significant diversity that is, in part, a result of the insertion sequence-mediated genomic rearrangements (25, 44). Comparison of the annotated F. tularensis spp. genomic sequences revealed that 56 F. novicida genes that encode MglA-regulated proteins were inactivated or absent in either F. tularensis subsp. holarctica or subsp. tularensis or both (see Table S3 in the supplemental material). Among these were the components of the oligopeptide transporter Opp and a secreted metalloprotease, PepO. Previously, we have shown that F. novicida pepO mutants have increased virulence in intradermally infected mice (18). In another study, F. novicida opp and pepO mutants replicated in elicited mouse peritoneal macrophages at faster rates than the wild-type F. novicida (5). These studies suggested that the loss of opp and pepO facilitated F. tularensis infection and survival in the mammalian host. Several studies suggested that gene loss can promote pathoadaptive evolution and adaptation to new hosts and modes of pathogen transmission (34). It is therefore possible that the loss of opp and pepO and other genes also contributed to the pathoadaptive evolution of the highly virulent, human pathogenic F. tularensis. Conclusion. The results of this study showed that the presence of functional MglA is essential for the regulation of a large number of F. novicida proteins, among which are homologs of bacterial stress response effectors and regulators. The global proteome changes in the mglA mutant could be the effect of the changes in the relative amounts of several other transcriptional and posttranscriptional regulators. The pleiotropic phenotype of the mglA mutant suggested that the regulatory activities of MglA, SspA, Hfq, CspC, UspA, and possibly other factors are coordinated in F. tularensis spp. In nonpathogenic, ancestral Francisella, the MglA regulon likely evolved from a starvation response to a complex regulatory network that is likely essential for F. tularensis survival in var-
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ious challenging environments and conditions, such as soil, cold water, protozoa, insects, and animals. ACKNOWLEDGMENTS This study was funded by NIAID award for the Northwest Research Center of Excellence for Biodefense and Emerging Infectious Diseases 5 U54 AI057141-03 to T.G., C.M., M.J.B. and D.R.G. We thank Beth Ramage for excellent technical support and James Charity and Simon Dove for kindly allowing us to mention their results prior to the final publication. REFERENCES 1. Bang, I. S., L. Liu, A. Vazquez-Torres, M. L. Crouch, J. S. Stamler, and F. C. Fang. 2006. Maintenance of nitric oxide and redox homeostasis by the salmonella flavohemoglobin hmp. J. Biol. Chem. 281:28039–28047. 2. Baron, G. S., and F. E. Nano. 1998. MglA and MglB are required for the intramacrophage growth of Francisella novicida. Mol. Microbiol. 29:247– 259. 3. Bosio, C. M., H. Bielefeldt-Ohmann, and J. T. Belisle. 2007. Active suppression of the pulmonary immune response by Francisella tularensis Schu4. J. Immunol. 178:4538–4547. 4. Bosio, C. M., and S. W. Dow. 2005. Francisella tularensis induces aberrant activation of pulmonary dendritic cells J. Immunol. 175:6792–6801. 5. Brotcke, A., D. S. Weiss, C. C. Kim, P. Chain, S. Malfatti, E. Garcia, and D. M. Monack. 2006. Identification of MglA-regulated genes reveals novel virulence factors in Francisella tularensis. Infect. Immun. 74:6642–6655. 6. Carmel-Harel, O., and G. Storz. 2000. Roles of the glutathione- and thioredoxin-dependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress. Annu. Rev. Microbiol. 54: 439–461. 7. Charity, J. C., M. M. Costante-Hamm, E. L. Balon, D. H. Boyd, E. J. Rubin, and S. L. Dove. 2007. Twin RNA polymerase-associated proteins control virulence gene expression in Francisella tularensis. PLoS Pathog. 3:e84. 8. Cho, Y. H., E. J. Kim, H. J. Chung, J. H. Choi, K. F. Chater, B. E. Ahn, J. H. Shin, and J. H. Roe. 2003. The pqrAB operon is responsible for paraquat resistance in Streptomyces coelicolor. J. Bacteriol. 185:6756–6763. 9. Cole, L. E., K. L. Elkins, S. M. Michalek, N. Qureshi, L. J. Eaton, P. Rallabhandi, N. Cuesta, and S. N. Vogel. 2006. Immunologic consequences of Francisella tularensis live vaccine strain infection: role of the innate immune response in infection and immunity. J. Immunol. 176:6888–6899. 10. Domon, B., and R. Aebersold. 2006. Mass spectrometry and protein analysis. Science 312:212–217. 11. Elkins, K. L., S. C. Cowley, and C. M. Bosio. 2003. Innate and adaptive immune responses to an intracellular bacterium, Francisella tularensis live vaccine strain. Microbes Infect. 5:135–142. 12. Ellis, J., P. C. Oyston, M. Green, and R. W. Titball. 2002. Tularemia. Clin. Microbiol. Rev. 15:631–646. 13. Fang, F. C. 2004. Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat. Rev. Microbiol. 2:820–832. 14. Gallagher, L. A., E. Ramage, M. A. Jacobs, R. Kaul, M. Brittnacher, and C. Manoil. 2007. A comprehensive transposon mutant library of Francisella novicida, a bioweapon surrogate. Proc. Natl. Acad. Sci. USA 104:1009–1014. 15. Gavrilin, M. A., I. J. Bouakl, N. L. Knatz, M. D. Duncan, M. W. Hall, J. S. Gunn, and M. D. Wewers. 2006. Internalization and phagosome escape required for Francisella to induce human monocyte IL-1beta processing and release. Proc. Natl. Acad. Sci. USA 103:141–146. 16. Golovliov, I., V. Baranov, Z. Krocova, H. Kovarova, and A. Sjostedt. 2003. An attenuated strain of the facultative intracellular bacterium Francisella tularensis can escape the phagosome of monocytic cells. Infect. Immun. 71:5940–5950. 17. Gottesman, S. 2005. Micros for microbes: non-coding regulatory RNAs in bacteria. Trends Genet. 21:399–404. 18. Hager, A. J., D. L. Bolton, M. R. Pelletier, M. J. Brittnacher, L. A. Gallagher, R. Kaul, S. J. Skerrett, S. I. Miller, and T. Guina. 2006. Type IV pilimediated secretion modulates Francisella virulence. Mol. Microbiol. 62:227– 237. 19. Hajjar, A. M., M. D. Harvey, S. A. Shaffer, D. R. Goodlett, A. Sjostedt, H. Edebro, M. Forsman, M. Bystrom, M. Pelletier, C. B. Wilson, S. I. Miller, S. J. Skerrett, and R. K. Ernst. 2006. Lack of in vitro and in vivo recognition of Francisella tularensis subspecies lipopolysaccharide by Toll-like receptors. Infect. Immun. 74:6730–6738. 20. Hansen, A. M., Y. Gu, M. Li, M. Andrykovitch, D. S. Waugh, D. J. Jin, and X. Ji. 2005. Structural basis for the function of stringent starvation protein a as a transcription factor. J. Biol. Chem. 280:17380–17391. 21. Hansen, A. M., Y. Qiu, N. Yeh, F. R. Blattner, T. Durfee, and D. J. Jin. 2005. SspA is required for acid resistance in stationary phase by downregulation of H-NS in Escherichia coli. Mol. Microbiol. 56:719–734. 22. Hollis, D. G., R. E. Weaver, A. G. Steigerwalt, J. D. Wenger, C. W. Moss, and D. J. Brenner. 1989. Francisella philomiragia comb. nov. (formerly Yersinia
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