Profiling the Bordetella pertussis Proteome during Iron Starvation

Profiling the Bordetella pertussis Proteome during Iron Starvation M. Laura Perez Vidakovics,† Jaime Paba,‡ Yanina Lamberti,† C. Andre´ Ricart,§ Marcelo Valle de Sousa,§ and M. Eugenia Rodriguez*,† CINDEFI, Faculty of Science, La Plata University, La Plata, Argentina, Department of Biochemistry, Federal University of Parana´, Curitiba, Brasil, and Brazilian Center for Protein Research, Department of Cell Biology, Institute of Biology, University of Brasilia, Brazil Received December 19, 2006

Regulation of gene expression in response to local iron concentration is commonly observed in bacterial pathogens that face this nutrient limitation during host infection. In this study, a proteomic approach was used to analyze the differential protein expression of Bordetella pertussis under iron limitation. Whole cell lysates (WCL) and outer membrane fractions of bacteria grown either under iron-starvation or iron-excess conditions were analyzed by two-dimensional (2-D) gel electrophoresis. Statistical analysis revealed 36 proteins displaying differential expression, 9 with higher expression under iron-excess and 27 with increased expression under iron-starvation. These proteins were subjected to tryptic digestion and MALDI-TOF MS. Apart from those previously reported, we identified new low-iron-induced proteins that might help to explain the increased virulence of this phenotype. Additionally, we found evidence that at least one of the identified proteins, solely expressed under iron starvation, is highly immunogenic in infected individuals. Keywords: Bordetella pertussis • Proteome • iron

Introduction Bordetella pertussis is a Gram-negative bacterial pathogen and etiologic agent of whooping cough (pertussis), a highly contagious, acute respiratory illness. B. pertussis is a strictly human pathogen with not known animal or environmental reservoir.1 Although pertussis is relatively well-controlled at present by extensive vaccination programs, it is evident that the circulation of B. pertussis throughout the world continues largely unabated.2 Recent studies suggest a high frequency of yearly cases of pertussis worldwide despite vaccination, with as many as 295 000 deaths.3 To succeed in host colonization, bacterial pathogens must first adhere to target tissues and concomitantly obtain nutrients which are essential for their growth. Iron is usually one of such essential factors, and the ability to scavenge iron is an important virulence trait.4 In mammals, most of the iron content is maintained intracellularly in the form of heme and hemoproteins, while host transferrin and lactoferrin glycoproteins bind the ion in the extracellular milieu.5 Therefore, successful microbial pathogens have developed mechanisms to overcome host iron restriction, including production and utilization of low-molecular-weight iron chelators (siderophores), capture of siderophores produced by other organisms, and direct removal of iron from host proteins via specific bacterial cell surface receptors.6,7 Moreover, the iron * Corresponding author: M. Eugenia Rodriguez, Ph.D. CINDEFI, Faculty of Science, La Plata University, calles 47 y 115, 1900 La Plata, Argentina. E-mail: [email protected]. Phone/fax: +54 221 4833794. † La Plata University. ‡ Federal University of Parana´. § University of Brasilia.

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Journal of Proteome Research 2007, 6, 2518-2528

Published on Web 05/25/2007

content of the surroundings appears to be a signal that triggers the expression of virulence genes in many pathogens.8 Because these virulence factors are essential for bacterial survival, they gain importance as potential targets for the development of vaccines and therapeutic agents. Different approaches have been employed to study Bordetella phenotype under iron starvation conditions. Analyses of the expression of iron-regulated outer membrane proteins (OMP) by SDS-PAGE have been reported before by several groups. Different patterns of protein expression were found with many proteins involved in iron uptake but with several others unidentified or displaying no recognized function.9-12 Identification and purification of transferrin and lactoferrinbinding protein by affinity chromatography was performed by Menozzi et al.10 Genes encoding putative siderophore receptors (bfrD and bfrE) were identified by systematic gene inactivation and generation of transcriptional fusion.13 In other studies, a TnphoA-derived library screened for mutants expressing Fedependent fusion proteins allowed the identification of bfeA (Bordetella ferric enterobactin) and bhuR (Bordetella heme uptake receptor) proteins.14,15 A research group at the Sanger Institute (U.K.) recently sequenced the genomes of B. pertussis strain Tohama I (4.09 Mb; 3816 genes) and the functional annotation of its genes is now available.16 The investigation of bacterial responses to environmental signals has until recently been hindered by a paucity of approaches to gain insights into regulatory networks and pathogenesis. The development of proteomic tools in combination with complete genome sequences now offers the 10.1021/pr060681i CCC: $37.00

 2007 American Chemical Society

B. pertussis Proteome

opportunity of relating genome-wide expression responses to environmental stimuli. In this study, we examined B. pertussis responses to ironstarvation in order to gain a better insight into the infective phenotype and the immunogenicity of the differentially expressed proteins. The results show that iron starvation induces the expression of proteins of different functional classes that may help to explain the virulence of this pathogen and further suggest that at least one of them, belonging to the outer membrane fraction, is highly immunogenic in vivo.

Materials and Methods Bacterial Growth Conditions. B. pertussis strain Tohama I was grown at 37 °C for 3 days on Bordet-Gengou agar (ABG) (Difco Laboratories, Detroit, MI) supplemented with 15% defibrinated sheep blood. Bacteria were then subcultured in Stainer-Scholte (SS) liquid medium at an initial cell density corresponding to an A650 of 0.2. SS cultures were maintained at 37 °C with shaking (150 rpm) for 24 h. Bacterial cells were harvested by centrifugation (10 000g for 15 min at room temperature), washed with sterile iron-free saline solution, and diluted to an estimated concentration of 2 × 108 cfu/mL. Equal volumes of bacterial cell suspensions were used to inoculate 100 mL of iron-replete SS (36 µM iron) (SS) and iron-depleted SS (without addition of FeSO4·7H2O) (SS-Fe). These two cultures were grown at 37 °C with shaking (150 rpm) for 20 h and subcultured twice in the respective culture media. When the growth reached the late exponential phase, cells were harvested by centrifugation (10 000g for 15 min at 4 °C) and stored at -80 °C. Iron-depleted SS medium was prepared using sterile polypropylene tubes (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) or Pyrex glass containers sequentially washed with 3 M nitric acid and deionized water to remove surface-bound iron. Deionized water was used both for solution preparing and washing steps. Iron from the iron-depleted SS medium was further removed by treatment with the cation exchange resin Chelex 100 (Bio-Rad, Hercules, CA) as described by West and Sparling.17 Siderophore Assays. The presence of siderophores in culture supernatants of B. pertussis grown in iron-depleted SS medium was used to confirm iron-limited growth. Briefly, 20 h cultures of B. pertussis in either iron-depleted SS or ironreplete SS (negative control) medium were centrifuged to remove the cells. The supernatants were further filtered through a 0.22 µm-pore-size nylon membrane (Nalgene Co., Rochester, NY) and tested for siderophores by the chrome azurol S (CAS) assay.18 Preparation of the Cell Lysate. For cell lysate preparation, B. pertussis was grown either in iron-depleted or iron-replete SS medium. Triplicate cultures were harvested, and cell lysates were prepared. Briefly, cells from 25 mL of culture were collected by centrifugation at 10 000g for 10 min, washed twice with milliQ water, and resuspended in 0.5 mL of 50 mM Tris/ HCl buffer (pH 7.5), supplemented with 5 mM phenylmethylsulfonyl fluoride (PMSF) protease inhibitor (freshly prepared). The cell suspension was disrupted by sonication (3 pulses with 80% potency during 5 min) using a Soniprep 150 apparatus (Sanyo). The proteins were solubilized during 1 h in 1.5 mL of rehydration buffer (7 M urea, 2 M thiourea, 2% (w/v) Triton X-100, 65 mM DTT, 0.5% (v/v) Pharmalyte pH 4-7 or pH 6-11 (Amersham Biosciences), 1 mM PMSF, 4 mM 4-(2-Aminoethyl)Benzenesulfonyl Fluoride [AEBSF], and 0.002% (w/v) bro-

research articles mophenol blue) and centrifuged at 8000g for 10 min. The protein concentration was determined by using the Ettan 2-D Quant Kit (Amersham Biosciences). Preparation of Outer Membrane Protein Fraction. Membrane protein fractions of B. pertussis grown in either irondepleted or iron-replete SS medium were obtained as previously described by Molloy et al.19 Briefly, the cells were disrupted in an Aminco French press with two cycles at 14 000 psi. Unbroken cells were removed by centrifugation at 8000g for 10 min at 4 °C. The supernatant was diluted with ice-cold 0.1 M sodium carbonate (pH 11) to a final volume of 60 mL and stirred slowly on ice for 1 h. The carbonate-treated samples were submitted to ultracentrifugation in a Beckman 55.2 Ti rotor at 115 000g for 1 h at 4 °C. The supernatant was discarded and the pellet (membrane fraction) resuspended and washed in 2 mL of 50 mM Tris/HCl buffer (pH 7.5). The washed membranes were collected by centrifugation and solubilized during 1 h in 1 mL of TFE-rehydration buffer20 (5 M urea, 2 M thiourea, 50% (v/v) 2,2,2-Trifluoroethanol [TFE, 99.0%, from Fluka, Buchs, Switzerland], 2% (w/v) Triton X-100, 65 mM DTT, 0.5% (v/v) Pharmalyte pH 4-7 or pH 6-11 (Amersham Biosciences), 1 mM PMSF, 4 mM AEBSF, and 0.002% (w/v) bromophenol blue) with periodical vortexing, and further centrifugated at 8000g for 10 min. The protein content of the solubilized membrane fractions was determined by using the Ettan 2-D Quant Kit (Amersham Biosciences). 2-D Gel Electrophoresis. For isoelectric focusing (IEF), precast 18 cm pH 4-7 or pH 6-11 IPG gels (Immobiline DryStrips, Amersham Biosciences, Uppsala, Sweden) were used. Samples were mixed with freshly prepared rehydration buffer or TFE-rehydration buffer resulting in a final protein content of 300 µg in 350 µL. Solubilized sample proteins were applied in the pH 4-7 IPGs by in-gel sample rehydration as previously described.21 IEF was performed in a Multiphor II electrophoresis unit (Amersham Biosciences) using the following program: 500 V for 0.01 h (1 Vh), 3500V for 1.30 h (gradient, 3000 Vh), 3500 V for 5.40 h (20 kVh), resulting in a total voltage of 23 kVh. The alkaline IPG dry strips were rehydrated (350 µL of rehydration buffer or TFE-rehydration buffer) at room temperature overnight using a reswelling tray, and solubilized sample proteins (300 µg in 300 µL) were applied using the paper bridge loading method previously described.22 IEF was performed in a Multiphor II electrophoresis unit (Amersham Biosciences) using the following schedule: 150 V for 1 h (75 Vh), 300 V for 2 h (gradient, 300 Vh), 600 V for 1h (300 Vh), 3500 V for 10 h (17.5 Vh) resulting in a total voltage of 18.2 kVh. Focused strips were either used immediately for the second dimension or were stored at -80 °C until use. After IEF, IPG strips were soaked (15 min) in 10 mL of equilibration buffer (50 mM Tris-Cl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 20 mM DTT, and 0.002% (w/v) bromophenol blue) followed by incubation (15 min) in the same solution but replacing DTT for 20 mM iodoacetamide. After the reduction/ alquilation step, the IPG strips were placed on the top of a 10% SDS-PAGE and sealed with 0.5% agarose in electrophoresis buffer. Electrophoresis was performed at constant current (25 mAmp per gel) at 20 °C until the dye front reached the lower end of the gel, using a PROTEAN II xi 2-D cell electrophoresis unit (Bio-Rad, Hercules, CA) connected to a Multitemp II cooling bath (GE Healthcare). Proteins were routinely visualized by silver staining23 for image analysis and alternatively by Coomasie blue staining24 for spots to be submitted to tryptic digestions and MALDI-TOF-MS. Journal of Proteome Research • Vol. 6, No. 7, 2007 2519

research articles Gel Image Analysis. Silver stained 2-D gels were documented using an Umax PowerLook II scanner, and the image analyses performed with the ImageMaster 2D Platinum 5.0 software (GE Healthcare). Each growth condition assessed (iron-replete or iron-depletion) comprised at least three independent replicate gels. After spot detection and matching, manual editing and filtering were performed. Each group of corresponding spots from different gels belonging to the same growth condition constituted a class. Differences in protein expression among classes were assessed using three parameters: normalized spot volume, Gap value, and Overlap ratio. The Gap value corresponds to the difference between the lower limit of one class interval (class with the highest spot volume mean value) and the upper limit of the other class interval (class with the lowest mean value). The Overlap value is the ratio between the lower limit of one class interval (class with the highest spot volume mean value) and the upper limit of the other class interval (class with the lowest mean value). The Gap and Overlap values allow ascertaining how distant two classes are and if their results are overlapping. Thus, paired spots displaying at least 2-fold changes in normalized spot volumes, a Gap value higher than 0, and an Overlap ratio higher than 2 were assigned as differentially expressed and submitted to MS analysis. Reproducibility of the gels inside a class was determined by comparing the matching of each replicate with the average gel (containing all the spots of all gels for each growth condition). Trypsin Digestion and Gel Extraction of Peptides. Coomasie-stained spots were excised and destained by 15 min incubation with a freshly prepared solution of 15 mM potassium ferricyanide and 50 mM sodium thiosulphate as previously described.25 Gel pieces were then rinsed several times with water to stop the reaction, subjected to alternate 10 min washing cycles with water and acetonitrile, and dried using a Speed Vac evaporator (Savant, Farmingdale, NY). Gels were reswollen in 5 µL of 50 mM NH4CO3 and 5 mM CaCl2 containing 12.5 ng/µL of sequencing grade modified porcine trypsin (Promega, Madison, WI) and incubated at 37 °C overnight. The tryptic peptides were extracted twice with 40 µL of acetonitrile/water/TFA (66:33:0.1 (v/v/v)) solution. The extracts were dried under vacuum, solubilized in 10 µL of 0.1% TFA and desalinized using C18 Zip Tips (Millipore) according to manufacturer’s instructions. MALDI-TOF MS. One microliter of dessalted samples was spotted onto the MALDI target plate with 1 µL of 5 mg/mL R-cyano-4- hydroxycinnamic acid in a 1:1 (v/v) mixture of 50% acetonitrile/ 0.05% TFA. The sample was allowed to dry for approximately 15 min prior to MS analysis. The spectra were collected using the Reflex IV MALDI-TOF mass spectrometer (MS) (Bruker Daltonics, Karlsruhe, Germany) in the reflector mode and internally calibrated using known trypsin autolysis peaks. Samples that could not be identified with certainty by peptide mass fingerprinting were sequenced by tandem mass spectrometry using a MALDI TOF-TOF 4700 Proteomics Analyzer (Applied Biosystem, Framingham, CA) mass spectrometer. Peptide fragmentation was performed using CID at collision energy of 1 keV and a collision gas pressure of 3 × 10-7 bar. External calibration was performed using the b series ions resulting from the fragmentation of Glu-fribrinopeptide B (m/z ) 1570.67). Database Searching. Peptide mass fingerprints were used for database searching using Mascot software (Matrix Science Ltd, U.K.) against the MSDB database downloaded from NCBI. Mass tolerance was set to 100 ppm, and no restrictions were 2520

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imposed on protein molecular mass or phylogenetic lineage. For MS/MS data, uninterpreted tandem mass spectra were first searched by Mascot against the above-mentioned database to identify proteins with tryptic peptides identical to database entries. Precursor mass tolerance was set at 0.1 Da and fragment ion mass tolerance at 0.05 Da. Hits were considered significant if their protein score exceeded the threshold score calculated by Mascot software assuming p < 0.05. Matched MS/ MS spectra were further manually inspected considering the correlation of y-, b-, and a- fragment ions to corresponding m/z values calculated from the peptide sequences as described before.26 All candidate sequences were merged into a single search string and submitted to MS BLAST searches against a nonredundant protein database (nrdb) at http://genetics.bwh.harvard.edu/ msblast/ or http://dove.emblheidelberg.de/ Blast2/msblast.html using default settings. According to the selected MS BLAST threshold scores of statistical confidence, the expected rate of false-positive identification was lower than 1%. Dot Blot Assay. Preparative 2-D gels containing 1000 µg of protein (whole cell lysate or outer membrane fraction) were revealed using Coomasie blue staining, and differentially expressed protein spots were excised and eluted from the gel as previously described by Hunkapiller et al.27 For dot blot assays, equal micrograms of each protein sample were spotted onto a PVDF membrane (Immobilon, Millipore, Bedford, MA) and allowed to dry. Whole cell lysates of B. pertussis grown in iron-replete SS and human holo-transferrin (Sigma, St Louis, MO) were used as positive and negative controls, respectively. Membranes were incubated for 2 h at room temperature in blocking solution (PBS, 0.05% (v/v) Tween 20, 5% (w/v) BSA), and then exposed 2 h at room temperature to IgG fraction purified from pooled sera of pertussis patients with high titers against B. pertussis as measured by ELISA.28 After washing, specific binding of serum IgG was visualized using alkaline phosphatase-conjugated goat anti-human IgG (Jackson, ImmunoResearch, BaltimorePike) as secondary antibody and NBT/BCIP (Bio-Rad) as substrate.

Results and Discussion The ability of a pathogen to integrate and respond appropriately to signals received from multiple iron sources may allow a fast adaptation to a changing host environment leading to a high degree of pathogenic success. In the case of B. pertussis, a number of high affinity iron-transport systems and several outer membrane proteins expressed in response to changes in the iron content of the surroundings have been described.29-32 In this work, we used a proteomic approach to analyze the B. pertussis phenotype induced by iron starvation. Analysis of B. pertussis Whole Cell Proteome. Figure 1 shows the representative 2-D gels (pH 4-7 and 6-11) obtained for whole cell lysates of B. pertussis. The reproducibility (spot number and location) among gels submitted to the same growth condition, ranged from 80 to 90%. The correlation coefficient resulting when spot volumes where compared ranged between 0.893 and 0.961. Representative results are displayed in Figure 2. Image analysis of consensus 2-D maps (pH 4-7) revealed an average of 900 protein spots with molecular masses (MM) ranging from 18 to 123 kDa, regardless of bacterial grown conditions. The average pI/MM ratio of the expressed Proteome was found to be 5.74/53.6 kDa, which is in agreement with the predicted Proteome (5.91/39.6 kDa)


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Figure 1. Representative 2-D gels of whole cell lysates of B. pertussis grown in iron-replete SS (SS) and iron-depleted SS (SS-Fe) medium. A total of 300 µg of whole cell lysate preparations was separated in parallel on 18 cm gels (pI 4-7 or pI 6-11, 10% SDSPAGE) and visualized by silver staining. Proteins identified by MALDI-TOF MS analysis are indicated by black arrows (protein names refer to Table 1). Unidentified proteins are indicated as IIP1-1 and IIP1-5 (Iron Induced Protein), and IRP1-7 and IRP1-8 (Iron Repressed Protein).

derived from the genome sequence. When alkaline IPG strips (pH 6-11) were used, approximately 210 protein spots were detected, displaying molecular masses between 16 and 148 kDa. Once more, the average pI/MM of the expressed Proteome (7/ 39.7 kDa) is in agreement with the predicted values (8.2/36.6 kDa) derived from the bacterial genome. The number of genes coding for proteins with a theoretical pI ranging between 4 and 7 versus the protein spots resolved by 2-D gels showed a ratio of 0.59 protein spots per predicted protein. Similar results have been obtained in other bacterial Proteomes.33,34 To get a real picture of the protein coverage obtained in this study, it would be necessary to identify a higher number of protein spots and to determine the frequency of identical entries for different

spots. This ratio varies among microorganisms. In Pseudomonas putida, 181 spots corresponded to 106 different proteins,34 whereas in Escherichia coli, 2160 spots revealed 575 ORF entries of which 241 were shown to exist in more than one form.33 Comparison of 2-D profiles (pH 4-7 and pH 6-11) of whole cell lysates from bacteria grown in iron-depleted versus ironreplete medium revealed the presence of 23 protein spots displaying differential expression; 8 with higher expression under iron sufficient conditions and 15 with increased expression under iron starvation (Figure 1, and Figure 3). These spots and some major landmark proteins were submitted to tryptic digestions and MALDI-TOF-MS (Table 1). Among spots exhibiting increased expression under iron starvation, five proteins Journal of Proteome Research • Vol. 6, No. 7, 2007 2521

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Figure 2. Scatter plots for groups in B. pertussis whole cell lysates. Normalized volumes of matched spots from one gel member of a class (x-axis) and its corresponding average gel (y-axis), in acid and alkaline 2-D runs, are compared. (SS) Ironreplete medium, (SS-Fe) iron-depleted medium; (count) number of pairs in each assay. The goodness-of-fit is given by the correlation coefficient (corr). This coefficient can vary between -1 and 1, where an absolute value near 1 indicates a good fit.

of known function, namely, BhuS, SodA, RpoA, PpiB, FumC, and GlyA, were identified. The putative heme transport factor (BhuS) is a member of the bhu system,32 required for iron assimilation from heme and hemoproteins by B. pertussis and Bordetella bronchiseptica. BhuS is similar to the so-called hemin-degrading factors from Pseudomonas aeruginosa, Shigella dysenteriae, and Yersinia spp.35-37 Although a hemindegrading activity of these proteins has not been demonstrated, Stojiljkovic and Hantke38 suggested that these proteins have hemin-binding activity and prevent the accumulation of heme to toxic levels in the cell. Since more than 90% of the iron within the human body is associated with heme and hemoproteins,39 bacteria accessing these compounds in vivo and utilizing host heme iron have a significant nutritional advantage.30,40 The SodA protein is an iron-regulated, Mn+2-containing superoxide dismutase41 with its gene being only transcribed under irondepleted conditions. B. pertussis has another superoxide dismutase called SodB.42 Both proteins have been found also in other Gram-negative pathogens.43-46 These enzymes have been associated with a higher microbial resistance to the action of reactive oxygen species produced by phagocytes during the socalled oxidative burst through their enzymatic conversion in a concerted action of SOD, catalase, and other oxidoreductases.47,48 Because phagosomal compartments seem to have only very limited iron content,49 the role of iron-regulated SodA may be relevant to survival of Bordetella. Another protein identified as differentially expressed under iron limitation is the R subunit of RNA polymerase, denominated RpoA. The rpoA gene sequence has 63% identity with those of the rpoA genes of both E. coli and Salmonella typhimurium.50 The R-subunit of RNA polymerase was shown to interact with transcription factors at a number of positively regulated prokaryotic promoters including the BvgA of B. pertussis.51,52 2522



BvgA is the transcription factor of the global regulatory system called Bordetella virulence genes (Bvg). Different gene classes can be differentially regulated by the location and affinity of BvgA binding sites at their promoters and BvgA-RNA polymerase interactions.53 The overexpression of RpoA under iron limitation could be interpreted as a signal of functionality of the Bvg system and the expression of a potentially new phenotypic phase in response to iron availability during infection. Accordingly, a recent report showed pertussis toxin, a virulence factor under the control of the Bvg system, to hold its expression influenced by iron availability.54 The PpiB protein, overexpressed under iron limitation, codes for a periplasmic peptidyl prolyl cis-trans isomerase that showed sequence similarity to Y. pestis PpiB (69% identity), P. putida (68% identity), and E. coli PpiB (66% identity). Protein folding in extracytoplasmic compartments of Gram-negative bacteria occurs in the periplasmic space which is acutely susceptible to changes due to direct contact with the environment. Although the activity of the B. pertussis PpiB protein remains to be demonstrated, it is tempting to speculate that like its homologues, this enzyme facilitates the proper folding of B. pertussis cell envelope constituents within the periplasmic space. The reason for the increase of this enzyme under iron limitation remains to be investigated. FumC is the non-ironcontaining form of the tricarboxylic acid (TCA) enzyme fumarate hydratase, previously described as iron regulated in E. coli, Vibrio cholerae, and P. aeruginosa.55-57 It has been speculated that under iron starvation this enzyme might compensate the decrease of the iron-containing form of the enzyme allowing the normal TCA cycle performance. Finally, GlyA, a serine hydroximethyl transferase, was also found overexpressed under iron limitation. This enzyme catalyzes the conversion of serine into glycine, which is the major source of glycine and C1 units for the cell.58 Under iron limitation, bacterial growth is no longer limited by glutamate as in the iron-replete SS medium. A change in the expression level of enzymes involved in the aminoacid metabolic pathways is consistent with the shift in the relative abundance of glutamate in these two environmental conditions. In addition to the proteins described above, we identified five polypeptides, with either unknown or putative functions and displaying increased expression under iron starvation. These five proteins were IRP1-1, a hypothetical protein; IRP12, a putative lipoprotein; BrfI, a putative ferrisiderophore receptor; AfuA, a putative iron binding protein; MceP, a mcerelated protein; and IRP1-3, a putative exported protein (Table 1). All of them have predicted periplasmic or outer membrane localization and at least three of them are thought to be implicated in iron capture. The 39-kDa protein, designated AfuA, shows a significant homology with periplasmic iron transport proteins such as HitA from Haemophilus influenza, or FbpA from Neisseria meningitidis, both involved in shuttling between outer membrane receptors for host iron binding proteins and the cytosol. Interestingly, sequence analysis further suggested that afuA might be part of an operon containing B and C analogues such as those found in the hitABC and fbpABC loci. BrfI has sequence similarity to P. aeruginosa hydroxamate-type ferrisiderophore receptor nominated FiuA (37% identity) and Salmonella typhimurium ferrichrome iron receptor called FhuA (38% identity). A BLASTP search revealed that the IRP1-1 protein has a high degree of identity to the periplasmic component of an ABC-type transporter of Ralstonia eutropha (58% identity) which was sug-

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Figure 3. Differentially expressed proteins in B. pertussis whole cell extracts. (A) Histograms showing spot volume variation among replicates in iron-replete (a, b, c) and iron-depleted (d, e, f) medium. (B) Corresponding zoom gels of proteins displaying different expression after growing of the bacterium in iron-replete (SS) and in iron-depleted (SS-Fe) medium. Table 1. List of Proteins from Bordetella pertussis Whole Cell Lysates Identified by MALDI-TOF MS

no.

ORF no.

protein name

MS/ MASCOT score

coverage %

growth condition

spot valuea SS/SS-Fe

annotation

putative kDa/pI

experimental kDa/pI

1 2 3 4 5 6 7 8 9 10 12 13 11 14 15 16 17 18 19

Bp0193 Bp0346 Bp1211 Bp2072 Bp1962 Bp3642 Bp2952 Bp3759 Bp1906 Bp1152 Bp0248 Bp1605 Bp3755 Bp3524 Bp2021 Bp3495 Bp2499 Bp2386 Bp0840

SodA BhuS IRP 1-1 IRP1-2 BrfI RpoA GlyA McePb PpiBb IRP1-3 FumCb AfuA IIP1-7b IIP1-9 AcnB GroEl DnaK EnoI OmpP

134 82 124 84 136 74 147 170 117 138 60 60 182 117 131 131 182 94 116

78 35 50 47 24 23 31 10 8 35 30 42 32 68 21 37 38 37 38

iron starvation iron starvation iron starvation iron starvation iron starvation iron starvation iron starvation iron starvation iron starvation iron starvation iron starvation iron starvation iron replete iron replete iron replete iron replete/starvation iron replete/starvation iron replete/starvation iron replete/starvation

0.04/0.32 0.01/0.09 0.05/0.26 0.07/0.4 0.007/0.063 0.005/0.047 0.036/0.26 0.006/0.064 0.003/0.034 0.001/5.1 0.04/0.57 0.002/0.11 0.001/0.20 0.62/0.15 0.086/0.005 7.63/7.14 1.78/1.53 3.67/3.91 8.63/8.93

superoxide dismutase putative hemin transport protein Hypothetical protein Putative Lipoprotein putative ferrisiderophore receptor DNA-directed RNA polymerase alpha subunit serine hydroxymethyl transferase mce related protein peptidyl prolyl cis-trans isomerase B putative exported protein fumarate hydratase class II putative iron binding protein putative outer membrane protein putative acetyltransferase putative aconitate hydratase Chaperonin Molecular Chaperone Enolase. phosphopyruvate hydratase outer membrane porin protein precursor

23.19/5.72 38.47/5.31 28.8/6.13 21.57/6.59 78.37/7.67 36.13/5.6 44.8/6.5 17.9/4.87 18.55/5.58 19.36/6 49.59/6.39 37.5/8.8 23.01/9.22 16.08/5.55 93.11/5.23 57.44/5.13 69.6/4.88 46.09/7.72 41.02/5.51

27.3/5.92 36.3/5.69 31.7/5.8 23.3/5.45 77/6.38 41.3/5.61 50/6.14 21.33/4.75 21.5/5.84 22/5.52 50/6.82 39/7.25 24/7.5 18.5/5.94 97/5.17 63.6/5.02 79.3/4.75 46/4.5 37/5.04

a

Percentage of the sum of all spot volumes in each gel. b Proteins identified by MALDI-TOF MS/MS.

gested to be involved in inorganic ion transport and metabolism. The putative lipoprotein, IRP1-2, showed a predicted outer membrane localization (BOMP program) and sequence similarity to other putative lipoproteins of Gram-negative bacteria. Although considerably shorter than the mammalian cell entry protein (mce) of Mycobacterium tuberculosis, MceP of B. pertussis has sequence similarity (60%) with the mce domain of the above-mentioned virulence factor of Mycobacterium involved in host cell invasion. Finally, the nucleotide sequence of the putative exported protein, IRP1-3, has a conserved domain called Tpd supposedly involved in high-affinity Fe2+ transport. Under iron-repleted conditions, we identified three proteins with increased expression: a putative acetyl transferase (IIP19), a putative outer membrane protein (IIP1-7), and an aconitate hydratase (AcnB). Like E. coli, B. pertussis possesses two distinct aconitases, AcnA and AcnB. The AcnB protein exhibits a remarkable sequence identity to the AcnB of E. coli (72%). Aconitases are iron-sulfur proteins that catalyze the interconversion of citrate and isocitrate in the citric acid and glyoxylate cycles. Several studies indicate that the least stable enzyme AcnB is the major citric acid cycle enzyme, synthesized during exponential growth, whereas AcnA is a stress-induced, stationary-phase enzyme.59 Recently, Tang et al.60 showed that E. coli AcnB forms homodimers and that the monomer-dimer transition is dependent on iron availability. The N-terminal region

of AcnB acts as an Fe2+ sensor that switches AcnB between the catalytic dimeric form under conditions of iron sufficiency and the mRNA binding monomeric form under iron-starvation which eventually acts as a post-transcriptional regulator. In our study, the overexpression of AcnB, which would eventually lead to a dimeric form under iron-excess conditions, is compatible with that model. In the monomeric form, AcnB operates as an apoprotein binding to specific mRNAs exerting post-transcriptional regulation of genes related to the iron metabolism. Analysis of B. pertussis Outer Membrane Fraction Proteome. OMPs of Gram-negative bacteria are involved in diverse cellular functions such as antibiotic resistance, transport of nutrients, cell signaling, attachment to host cells, and virulence in pathogenic strains.61 Because of their location as interfaces between the cell and the environment, OMPs are target antigens for developing strategies to control host infection. 2-D PAGE analysis of membrane extracts is a hard task to be accomplished.19 The hydrophobicity of the proteins turns them refractory to common solubilization protocols, and once in the IPG, they often precipitate at their pI, reducing their transfer to the second-dimension gel. In this work, we used a simple method proposed by Molloy et al.62 for the rapid isolation and separation of integral OMP by 2-D electrophoresis. When this approach was used, the outer membrane fraction was enriched for membrane proteins with an alkaline pH wash, before reconstituting the membrane fraction for 2-D electrophoresis Journal of Proteome Research • Vol. 6, No. 7, 2007 2523

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Figure 4. Representative 2-D gels of outer membrane fraction of B. pertussis grown in iron-replete SS (SS) and iron-depleted SS (SS-Fe) medium. A total of 300 µg of outer membrane preparation was separated in parallel on 18 cm gels (pI 4-7 or pI 6-11, 10% SDS-PAGE) and visualized by silver staining. Proteins identified by MALDI-TOF MS analysis are indicated by black arrows. Protein names are referring to Table 2. Unidentified proteins is indicated as IIP1-6 (Iron Induced Protein).

using strong denaturing conditions. Additionally, we included trifluoroethanol (TFE) in the in-gel sample rehydration buffer to improve membrane protein IEF separation. This procedure was previously described by Deshusses et al.,20 and in agreement with their results, we found a noticeable improvement in the number and resolution of the protein spots. Representative 2-D gels of B. pertussis OMP fraction (pH range 4-7 and 6-11) are shown in Figure 4. The reproducibility (spot number and location) among gels of the same class ranged from 80 to 90%. The correlation coefficient resulting when spot volumes where compared ranged from 0.912 to 0.988. Representative results are displayed in Figure 5. The 2-D gel maps of B. pertussis OMPs (pH range 4-7) displayed 2524


remarkable similarity to those previously reported for E. coli, S. typhimurum, and Klebsiella pneumoniae OMP.19,62 Approximately 200 protein spots were resolved and spread in a molecular mass range of 20-135 kDa, independently of bacterial grown conditions. Furthermore, 300 protein spots were detected in the range of 17-138 kDa when the IEF was performed using alkaline IPG. The average pI/MM of the expressed Proteome was 5.77/42.37 kDa and 7.14/41.6 for pH range 4-7 and 6-11, respectively. Once again, these data resemble the predicted outer membrane subproteome (5.94/ 52.3 kDa and 9.16/39.8). Remarkably, the average pI of the expressed outer membrane subproteome for pH range 6-11 was two units lower than the value derived from the genome

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Figure 6. Differentially expressed proteins in B. pertussis outer membrane fraction. (A) Histograms showing spot volume variation among replicates in iron-replete (a, b, c) and iron-depleted (d, e, f) medium. (B) Corresponding zoom gels of proteins displaying different expression after growing on the bacterium in iron-replete (SS) and iron-depleted (SS-Fe) medium. .

Figure 5. Scatter plots for groups in outer membrane fractions of B. pertussis. Normalized volumes of matched spots from one member of a class (x-axis) and its corresponding average gel (y-axis), in acid and alkaline 2-D runs, are compared. (SS) Ironreplete medium, (SS-Fe) iron-depleted medium; (count) number of pairs in each assay; (corr) correlation coefficient.

sequence. This discrepancy might be explained by the fact that basic proteins are difficult to separate via 2-D electrophoresis and, therefore, are often under-represented during reference mapping analysis. Further studies with narrower basic pH gradient IPG will be necessary to expand our reference map of B. pertussis for these alkaline proteins. To assess variations in OMP expression in B. pertussis grown in media with different iron concentrations, comparative image analysis of 2-D gels (pH range 4-7 and 6-11) were performed. Statistical analysis of spot volumes in paired spots revealed the presence of 13 protein spots displaying differential expression level: 1 with higher expression under iron-replete growth condition and 12 with increased expression under iron starvation (Figures 4 and 6). These spots were submitted to tryptic digestion and MALDI-TOF MS resulting in the identification of PanC, ArgD, PpiB, BrfB, AlcC, FumC, AfuA, IRP1-16, IRP1-3, IRP1-5, and IRP1-6 proteins (Table 2). Among the identified proteins, three are enzymes involved in aminoacid pathways, namely, PanC, ArgD, and PpiB. The pantoate β-alanine ligase protein, nominated PanC, catalyzes the formation of pantoth-

enate from pantoate and β-alanine. The ArgD is a succinylornithine transaminase that participates in arginine and lysine biosynthetic pathways. An overexpression of this enzyme might be linked to an increased amino acid metabolism due to an excess of glutamate under iron limitation. Interestingly, B. pertussis ArgD has a sequence similarity to ArgD of E. coli (40% identity), that displays both N-acetylornithine and diaminopimelate (DAP) aminotransferase activities, and it is involved in DAP formation. Because DAP is an important constituent of the peptidoglycan of many Gram-negative bacteria ArgD overexpression might be a membrane rebuilding response to an iron-restricted environment. Interestingly, DAP is a central constituent of the tracheal cytotoxin, an important B. pertussis virulence factor during host colonization.63 AlcC is involved in alcaligin biosynthesis one of the main high-affinity iron uptake systems of B. pertussis. Five of the 11 identified proteins have predicted periplasmic or outer membrane localization. These proteins were BrfB, a putative ferric siderophore receptor, AfuA, a putative iron binding protein, IRP1-5, a probable zinc binding dehydrogenase, IRP1-6, a putative ABC transport solute binding, and IRP1-3, a putative exported protein. The BrfB of B. pertussis was reported as a putative TonB-dependent siderophore receptor whose relevance in iron uptake is still unclear.64 Nakamura et al.65 recently showed transcriptional up-regulation of brfB, brfC, and other genes involved in iron acquisition during the transition from logarithmic to stationary phase. They suggested that entering in the stationary-phase may induce an iron-starved phenotype, either in direct response to iron restriction or because the stationary phase would simulate an iron-restriction-like state in the bacteria. The IRP1-5 showed similarity with several zinc-containing alcohol dedydrogenases

Table 2. List of Bordetella pertussis OMP Identified by MALDI-TOF MS

no,

ORF no.

protein name

MS/ MASCOT score

coverage %

growth condition

spot valuea SS/SS-Fe

annotation

putative kDa/pI

experimental kDa/pI

1 2 3 4 5 6 7 8 9 10 11 12

Bp2016 Bp3821 Bp0800 Bp2747 Bp1152 Bp0451 Bp1906 Bp2458 Bp0248 Bp1605 Bp2770 Bp3441

BrfB PanC IRP1-5 IRP1-6 IRP1-3 ArgD PpiB AlcC FumC AfuA IRP1-16 BpcP

86 150 213 60 138 116 76 161 137 51 55 80

56 35 87 38 56 54 57 44 55 37 48 37

iron starvation iron starvation iron starvation iron starvation iron starvation iron starvation iron starvation iron starvation iron starvation iron starvation iron starvation iron replete/starvation

0.008/0.318 0/0.06 0/0.141 0.106/0.780 0/4.464 0/0.142 0/0.0480 0/0.11 0.11/.86 0.02/0.56 0.36/1.49 8.74/6.29

putative ferric siderophore receptor pantoate-beta-alanine ligase probable zinc binding dehydrogenase putative ABC transport solute binding putative exported protein succinylornithine Transaminase peptidyl-prolyl cis-trans isomerase alcaligin biosynthesis protein fumarate hydratase class II putative iron binding protein probable short-chain dehydrogenase conserved hypothetical protein

78.66/5.73 31.72/5.93 33.69/5.5 40.7/6.17 19.36/6 43.08/5.79 18.5/5.6 70.2/5.84 49.57/6.39 37.5/8.8 26.3/6.3 19.8/5.1

78.5/5.56 33.5/6.19 39.7/5.68 40/5.94 22.3/5.49 41/6 22/5.44 65/6.28 48/6.86 39/7.41 28/6.7 26.34/5.08

a

Percentage of the sum of all spot volumes in each gel.

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Figure 7. Dot blot analyses of outer membrane fraction proteins of B. pertussis differentially expressed under iron starvation. The proteins were eluted from the gel and probed with IgG fraction purified from pooled sera of pertussis patients. Whole cell lysate of B. pertussis (Bp WC) and human holo-transferrin (ApoT) were used as positive and negative controls, respectively.

of Gram-negative bacteria as Burkholderia sp. A BLAST search for IRP1-6 revealed a high degree of sequence identity to many membrane transport solute binding proteins including a periplasmic amino acid-binding protein of Burkholderia mallei (59% identity) and a ligand-binding receptor of Burkholderia cenocepacia (56% identity). Finally, FumC, AfuA, and PpiB, found in cell lysate fraction analysis as mentioned above, were detected again as iron-regulated proteins. Several proteins of B. pertussis previously described by others as iron-regulated proteins (such as, BfrD, BfrE, BfeA, or BhuR, among others) were not found in this study. This could be due to a number of reasons. Large molecular mass proteins (>80 kDa) are commonly missing from micropreparative 2-D maps. These proteins might be lost in the separation procedure, either at the point of IPG reswelling or during IEF by precipitation at the isoelectric point. Alternatively, these or other iron-regulated proteins may have a low expression level thereby escaping detection analysis. Immunodetection of Iron-Induced OMP. As protective antibodies mostly work through inhibition of bacterial adherence and opsonization, we focused our attention on ironinduced proteins identified in the outer membrane fraction Proteome analysis. The differentially expressed spots were excised and eluted from the gel, and equal quantities of each protein sample were used in dot blot assays with human IgG purified from pooled sera of pertussis-infected individuals. Whole cell lysate was used as a positive control, and human lactoferrin was employed as a negative control. As shown in Figure 7, four proteins, FumC, AfuA, IRP1-16, and IRP1-3, reacted with human antibodies induced by infection. Among them, IRP1-3 showed the strongest reaction, suggesting that this protein is highly immunogenic and expressed during infection. The fact that IRP1-3 is an outer membrane-associated protein solely expressed under iron restriction is consistent with a possible role in the physiologically important iron uptake during host colonization. Therefore, antibodies against this protein might be protective in different ways. They may act either as opsonins, hamper bacterial attachment, or even interfere with essential iron uptake systems, which makes IRP1-3 an attractive vaccine candidate.

Conclusions To our knowledge, this is the first time that the Proteome of the B. pertussis Tohama I was investigated by means of 2-D electrophoresis and MALDI-TOF analysis. We examined the global protein profile of B. pertussis as it varied in response to iron limitation, which is acknowledged to be a critical stressor during infection in the host environment. We found proteins 2526



belonging to different functional classes to have their expression influenced by iron starvation. Among them, we identified new iron-dependent proteins that may help to understand the infectious process. At least four of the differentially expressed proteins proved to specifically react with sera from infected individuals. One of them, a putative exported protein probably involved in iron uptake, seems to be highly immunogenic in vivo. The data presented here provide only a first insight into the physiological changes of B. pertussis in response to drastic changes that take place under physiological conditions. To gain a wider view of the B. pertussis Proteome, it would be necessary the use of narrow-range pH gradients and to evaluate the cellular and humoral response to differentially expressed spots with potential as vaccine component. Studies in this direction are presently being undertaken. Abbreviations: 2-D, two-dimensional; IEF, isoelectric focusing; IPG, immobilized pH gradient; MALDI-TOF, matrixassisted laser desorption/ionization-time of flight; MS, mass spectrometry; PAGE, polyacrylamide gels electrophoresis; OMP, outer membrane protein; WCL, whole cell lysates.

Acknowledgment. This work was partially supported by a SECyT grant (PICT 14 522). We thank Agustin Ure for his technical assistance. M.E.R. is a member of the Scientific Career of CONICET. M.L.P.V. and Y.L. are doctoral fellows of CONICET. References (1) Cotter, P. A.; Miller, J. F. Bordetella. In Principles of Bacterial Pathogenesis; Groisman, E. A., Ed; Academic Press, Ltd.: London, United Kingdom, 2001; pp 619-74. (2) Cherry, J. D.; Heininger U. Pertussis and other Bordetella infections. In Textbook of Pediatric Infectious Diseases, 5th ed.; Feigin, R. D., Cherry, J. D., Demmler, G. J., Kaplan, S., Eds.: The W. B. Saunders Co.,: Philadelphia, PA, 2004; pp 1588-608. (3) Crowcroft, N. S.; Stein, C.; Duclos, P.; Birmingham, M. How best to estimate the global burden of pertussis? Lancet Infect. Dis. 2003, 3, 413-18. (4) Weinberg, E. D. Acquisition of iron and other nutrients in vivo. In Virulence Mechanisms of Bacterial Pathogens, 2nd ed.; Roth, J. A., Bolin, C. A., Brogden, K. A., Minion, F. C., Wannemuehler, M. J., Eds.; American Society for Microbiology: Washington, DC, 1995; pp 81-95. (5) Aisen, P.; Leibman, A. Lactoferrin and transferrin: a comparative study. Biochem. Biophys. Acta 1972, 257, 314-23. (6) Guerinot, M. L. Microbial iron transport. Annu. Rev. Microbiol. 1994, 48, 743-72. (7) Ratledge, C.; Dover, L. G. Iron metabolism in pathogenic bacteria. Annu. Rev. Microbiol. 2000, 54, 881-941. (8) Litwin, C. M.; Calderwood, S. B. Role of iron in regulation of virulence genes. Clin. Microbiol. Rev. 1993, 6, 137-49. (9) Agiato, L. A.; Dyer, D. W. Siderophore production and membrane alterations by Bordetella pertussis in response to iron starvation. Infect. Immun. 1992, 60, 117-23. (10) Menozzi, F. D.; Gantiez, C.; Locht, C. Identification and purification of transferrin- and lactoferrin-binding proteins of Bordetella pertussis and Bordetella bronchiseptica. Infect. Immun. 1991, 59, 3982-88. (11) Passerini de Rossi, B. N.; Friedman, L. E.; Gonzalez Flecha, F. L.; Castello, P. R.; Franco, M. A.; Rossi, J. P. Identification of Bordetella pertussis virulence-associated outer membrane proteins. FEMS Microbiol. Lett. 1999, 172, 9-13. (12) Redhead, K.; Hill, T.; Chart, H. Interaction of lactoferrin and transferrins with the outer membrane of Bordetella pertussis. J. Gen. Microbiol. 1987, 133, 891-98. (13) Antoine, R.; Alonso, S.; Raze, D.; Coutte, L.; Lesjean, S.; Willery, E.; Locht, C.; Jacob-Dubuisson, F. New virulence-activated and virulence-repressed genes identified by systematic gene inactivation and generation of transcriptional fusions in Bordetella pertussis. J. Bacteriol. 2000, 182, 5902-5. (14) Beall, B.; Sanden, G. N. A Bordetella pertussis fepA homologue required for utilization of exogenous ferric enterobactin. Microbiology 1995, 141, 3193-205.

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PR060681I

Profiling the Bordetella pertussis Proteome during Iron Starvation

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