Comparative Proteome Analysis of Laboratory Grown Brucella a

Comparative Proteome Analysis of Laboratory Grown Brucella abortus 2308 and Brucella melitensis 16M Michel Eschenbrenner,*,†,‡ Troy A. Horn,†,‡ Mary Ann Wagner,†,‡,§ Cesar V. Mujer,†,| Tabbi L. Miller-Scandle,† and Vito G. DelVecchio*,†,| Institute of Molecular Biology and Medicine, The University of Scranton, Scranton, Pennsylvania 18510 Received March 31, 2006

Brucella species are pathogenic agents that cause brucellosis, a debilitating zoonotic disease that affects a large variety of domesticated animals and humans. Brucella melitensis and Brucella abortus are considered major health threats because of their highly infectious nature and worldwide occurrence. The availability of the annotated genomes for these two species has allowed a comparative proteomics study of laboratory grown B. melitensis 16M and B. abortus 2308 by two-dimensional (2-D) gel electrophoresis and peptide mass fingerprinting. Computer-assisted analysis of the different 2-D gel images of strains 16M and 2308 revealed significant quantitative and qualitative differences in their protein expression patterns. Proteins involved in membrane transport, particularly the high affinity amino acids binding proteins, and those involved in Sec-dependent secretion systems related to type IV and type V secretion systems, were differentially expressed. Differential expression of these proteins may be responsible for conferring specific host preference in the two strains 2308 and 16M. Keywords: Brucella melitensis • Brucella abortus • differential protein expression • two-dimensional gel electrophoresis

Introduction Brucellosis, also called Malta fever, is a zoonotic disease caused by members of the genus Brucella. Six different accepted species have been identified in this genus based on host preferences and pathogenicity: Brucella melitensis, Brucella abortus, Brucella suis, Brucella ovis, Brucella canis, and Brucella neotomae. These bacteria are Gram-negative, nonmotile, facultative intracellular coccobacilli, and belong to the R-2 subgroup of the class Proteobacteria.1 Each Brucella species exhibits host preferences, but can also cross-contaminate other animal species with variable efficiency. B. melitensis is the most infectious to human, but preferentially infects goats and sheep. An attenuated live vaccine strain, B. melitensis Rev 1, was developed against brucellosis in small ruminants and used with high efficiency in many countries.2,3 As for B. abortus that preferentially infects cattle and is also capable of infecting humans,4 two attenuated live vaccines, B. abortus S19 and RB51, are available for preventing brucellosis in cattle. RB51 is the vaccine of choice in the US. However, despite its reported efficacy, this vaccine retains residual pathogenicity that makes * To whom correspondence should be addressed. Institute of Molecular Biology and Medicine, The University of Scranton, 800 Linden Street, Scranton, PA 18510. Tel: (570) 941-6353. Fax: (570) 941-6229. E-mail: [email protected]. or to V.G.D. Tel: (570) 281-2580. Fax: (570) 281-2506. E-mail: [email protected]. † The University of Scranton. ‡ These authors contributed equally to this work. § Present address: Marywood University, Science Department, 2300 Adams Ave, Scranton, PA 18509. | Present address: Vital probes, Inc., 1300 Old Plank Road, Mayfield, PA 18433. 10.1021/pr060135p CCC: $33.50

 2006 American Chemical Society

it unsafe for human use. Brucella is considered a high-risk pathogen because of its potential use as a biological warfare agent and in agroterrorism, since it induces abortion in infected livestock.1 In addition, this organism represents an important human health hazard as it causes persistent chronic infection.5 To date, no vaccine is available for human use to prevent brucellosis. B. melitensis and B. abortus are genetically very similar, each containing two chromosomes of 2.12 and 1.17 Mb,6,7 with more than 90% homology based on DNA-DNA hybridization studies.8 The annotation by Integrated Genomics of the two genomes originally predicted 3198 ORFs, recently updated to 3174 ORFs, for B. melitensis 16M and 3294 ORFs using a 75 contig. version of the genome for B. abortus 9-941 (University of Minnesota, http://www.cbc.umn.edu/ResearchProjects/AGAC/Pub_Brucella/ Brucellahome.html). Recently the genome of B. abortus field isolate 9-941 was completed9 with 3296 predicted ORFs. Despite their significant similarity, each species infects humans with a different degree of infectivity. Understanding the mechanisms leading to host preference might be key to vaccine development against brucellosis. With the availability of the annotated 16M genome,7 our laboratory has initiated the complete proteome analysis of 16M grown under laboratory conditions.10 The final part of the proteomic map of the strain 16M was completed (Wagner et al., unpublished data) and used for comparison with other Brucella species. The annotated B. abortus genome (during this study, only the 75 contig version was available) was also present on our in-house genome database. These two annotated genomes allowed us to perform comparative proteome analyses Journal of Proteome Research 2006, 5, 1731-1740

1731

Published on Web 06/09/2006

research articles of laboratory grown B. abortus 2308 and B. melitensis 16M by two-dimensional (2-D) gel electrophoresis followed by peptide mass fingerprinting (PMF) employing matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS). The results reveal the presence of differentially expressed proteins as well as strain-specific protein spots. Their identification by PMF may shed light on the understanding of host preference between B. abortus and B. melitensis.

Materials and Methods Materials. All reagents and equipment used in this study were described previously10 and listed on our website (http:// www.proteome.scranton.edu). All robotic equipment was purchased from Genomic Solutions. Bacterial Cultures. The virulent strains B. melitensis 16M and B. abortus 2308 were grown by the group of Dr. P. Elzer (Louisiana State University) under laboratory conditions on Schaedler Blood Agar for 3 days at 37 °C in the presence of 5% CO2 as previously described.10 Cells were then collected and killed by chloroform treatment. The chloroform was then removed and the killed cells were stored at -80 °C until use. Protein Extraction. Proteins were extracted using a modified protocol of Rafie-Kolpin et al.11 as described previously.10 All steps were performed on ice. Killed cells (40 µL, sufficient to contain 100 µg total protein) were mixed with an equal volume of 10% TCA, incubated for 5 min and centrifuged. The pellet was washed with 40 µL of 5% TCA, incubated for 5 min and centrifuged. A final wash with 40 µL acetone was performed followed by centrifugation before resuspending the cells in 40 µL of sample buffer 1, and 4 µL of sample buffer 2. After a 10 min incubation period, 160 µL loading buffer and 200 µL rehydration buffer were added. All buffers were purchased from Genomic Solutions. Total Protein Determination. Protein concentration was determined by the method of Bradford12 with the Bio-Rad protein stain using bovine serum albumin as the standard. All quantifications were performed in the presence of 10 µL of a 10:1 (vol:vol) mixture of sample buffers 1 and 2, in both standards and samples. Isoelectric Focusing. Narrow pH range IPG (immobilized pH gradient) strips (Amersham Biosciences), with linear pH gradients (4.0-5.0, 4.5-5.0, 5.0-6.0, 5.5-6.7) were used. At pH 6-11, poor protein resolution obtained with B. abortus 2308 samples prevented any comparative analysis with B. melitensis 16M. The 18 cm strips were rehydrated with 400 µL of the prepared sample overnight at room temperature. IEF was performed for 24 h at 20 °C (maximum of 5000 V; maximum current of 80 µA/gel; 80 000 Vh and end-of-run hold at 125 V). SDS-Polyacrylamide Gel Electrophoresis. All buffers were purchased from Genomic Solutions. After IEF completion, each strip was washed in 15 mL equilibration buffer 1 for 15 min, and then in 15 mL equilibration buffer 2 for 15 min. The strips were loaded on 10% Duracryl gels (22 cm × 23 cm × 1 mm; Tris/Tricine/SDS chemistry). Electrophoresis was completed after an 18 to 19 h run (500 V; 1600 mW/gel) at 4 °C. The next steps were carried out in an automated staining apparatus. All gels were washed in 40% methanol with 10% acetic acid for 30 min, rinsed for 5 min in deionized water, stained for 12 h with SYPRO Ruby (Molecular Probes), rinsed for 5 min in deionized water, destained in 10% methanol with 6% acetic acid for 30 min, and stored in 2% glycerol in the dark at 4 °C. Gel images were recorded with a Fujifilm LAS-1000 Plus imager under 470 nm light. 1732

Journal of Proteome Research • Vol. 5, No. 7, 2006

Eschenbrenner et al.

Gel Image Analysis. Each gel image was analyzed using Investigator HT analyzer software developed by Nonlinear Dynamics. For each pH range, gels were run in triplicate using three independent sample preparations. Spot detection and matching were completed on each of these three independent subgels, and an average gel was then generated. Only protein spots present in at least two subgels were included in the average gel. A base spot, consistent in shape and intensity across all subgels, was chosen for normalization of spot volumes, to compensate for possible variation in staining quality. Normalization proceeded as follows: The volume of each spot was divided by the volume of the base spot and then multiplied by 100. The average standard deviation of the normalized spot volumes ranged from 5% to 49% for 16M and from 7% to 46% for 2308, values similar to those observed in a previously published study.13 Normalized average gels for 16M and 2308 were matched and compared. Electrophoretically unique spots were defined as present exclusively in one strain and therefore missing in all subgels of the second strain. These spots were characterized only by their unique electrophoretic mobility. Differentially expressed protein spots were validated by observation of a consistent pattern (underexpressed or overexpressed) in at least two subgels for each strain. Scrutiny of overlapping spots between the different pH ranges revealed reproducible differential expression patterns (overpression, underexpression and electrophoretically unique spots) between 16M and 2308. Automated In-Gel Trypsin Digestion with O-MU Modification. Protein spots of interest were excised from the gels with a UV box-equipped ProPic robot (Genomic Solutions). Proteins present in the gel plugs were digested with trypsin according to the default long trypsin digestion protocol described by the manufacturer for the ProGest Digestion Station (Genomic Solutions). The protocol was altered to incorporate the modification of the tryptic peptides,10 with O-methylisourea (O-MU), as described by Hale et al.14 During the process, gel plugs were shrunk with acetonitrile and rehydrated with each subsequent reagent solution as described.15 Essentially, proteins were reduced by dithiothreitol, alkylated with iodoacetamide and digested for 7.5 h at 37 °C by trypsin. The resulting tryptic peptides were modified for 1 h at 37 °C with O-MU as described.14 The modified tryptic peptides were recovered in a 7:2 (vol:vol) mixture of 10% formic acid and acetonitrile. Preparation of Tryptic Peptides for MALDI Analysis. The tryptic peptides produced with the ProGest station were dried under vacuum and resuspended in 100 µL 10% formic acid. Samples were desalted and concentrated with Zip Tips (Millipore) in a ProMS workstation (Genomic Solutions). Finally, a 1 µL mixture of peptide and matrix (R-cyanohydroxycinnamic acid, 10 mg ml-1) was spotted onto a 384-well stainless steel Kratos MALDI plate. Mass Spectral Analysis. Spectra were collected with the Kratos Axima CFR mass spectrometer (Shimadzu Biotech) running in reflectron mode using the Kompact software package (Kratos Analytical), as described previously.10 For each spectra, monoisotopic peaks were selected using the Distiller software (Matrix Science) and the peptide mass fingerprints were submitted through Mascot and Mascot Daemon software packages from Matrix Science16 for a search against our inhouse genome database containing the virtual ORFeomes of B. melitensis 16M,7 B. suis 1330,17 and B. abortus 9-941 (75 contig version annotated by Integrated Genomics). To facilitate public accessibility, Integrated Genomics Brucella abortus locus

Brucella abortus and Brucella melitensis Proteomes

research articles

Figure 1. B. abortus 2308 proteome. Composite 2-D gel image generated from images of three overlapping pH ranges (4-5, 4.5-5.5, and 5-6). Overexpressed, underexpressed and strain-specific spots were labeled in red, green, and yellow, respectively. Asterisk (*) indicates spot with tentative identification. Molecular mass scale is indicated in kDa on the left.

tags were converted, based on amino acid sequence homology, to NCBI locus tags of the recently published Brucella abortus 9-941 genome9 and B. abortus 2308 genome18 (see Supporting Information Tables 1, 2, 3, and 4). The search parameters were set for a maximum of one missed cleavage by trypsin, fixed modifications of oxidized methionine and carbamidomethylated cysteine, variable modification of guanidinated lysine, a charge state of +1, and a mass tolerance of ( 0.2-0.8 Da.

Results Protein Spots Detected. For comparison with B. melitensis 16M, B. abortus 2308 protein extracts were run on 2-D gels with narrow pH range IPG strips (i.e., 4.0 to 5.0, 4.5 to 5.5, 5.0 to 6.0). A composite image (Figure 1) was assembled from the gel images recorded. Computer assisted gel image analysis of all pH ranges, followed by removal of overlapping spots between each pH range, resulted in the detection of a total of 549 distinct spots from pH 4-6 characterized by molecular mass ranging from 9.7 to 134 kDa. The total number of spots detected in 2308 is comparable to the 575 distinct spots observed for 16M between pH 4 and 6. Using strain 16M as reference, 15 spots were overexpressed (Table 1) and 70 spots were underexpressed (Table 2) in 2308. More complete information is available in Supporting Information Tables 1 and 2. Proteins Unique to 2308 or 16M. After processing all gel images for each pH range, 111 spots were observed as unique to 2308 based on their distinctive electrophoretic mobility (Table 3, Figure 1). Furthermore, in 16M gels, 87 spots did not

have any corresponding match in 2308 and were therefore classified as electrophoretically unique to 16M (Table 4, Figure 2). Analysis of these strain-specific protein spots by PMF led to the identification of 40 spots in 2308 and 28 spots in 16M. The remaining unique spots were expressed in very small amounts making them extremely difficult to identify by PMF. Between the two strains, the resulting identifications revealed the presence of proteins migrating on 2-D gels at different pIs (very similar molecular mass) but encoded by ORFs with greater than 99% sequence identity and with the same functional assignment. There were 8 different spots of this type, each present in both 16M and 2308, arising from 7 distinct ORFs. These spots were identified as the metal chelate outer membrane receptor (spots 7 and 226), acriflavin resistance protein A (spots 26 and 233), thioredoxin (spots 52 and 241), fumarylacetoacetate hydrolase family protein (spots 59 and 246), ABC transporter substrate binding protein (spots 72 and 248), outer membrane protein (spots 112, 114, and 256) and a hypothetical protein (spots 215, 253, and 308). Protein sequence alignments between the two strains (data not shown) indicated the presence of no more than two to three residue substitutions for each protein. Analysis of their respective experimental and theoretical (deduced from amino acid sequence) pIs showed that, for most, the difference of experimental pI between 16M and 2308 proteins was accounted for by differing ionizable residues present in their sequences. Variation of the experimental pI beyond that due to sequence variation was theorized to be due to variable post-translational modifications (PTMs) Journal of Proteome Research • Vol. 5, No. 7, 2006 1733

research articles


Table 1. Proteins Overexpressed in B. abortus 2308, When Compared to B. melitensis 16M

B. abortus

molecular mass (kDa)

pI

spot no.a

protein nameb

ORFc

exptl

theor

exptl

theor

V2308/V16Md

1 6 12 22 24 91 100 110 164 171 184

ATP-dependent Clp protease, ATP-binding subunit ClpB SSU ribosomal protein S1P trigger factor, ppiase (EC 5.2.1.8) histidinol dehydrogenase (EC 1.1.1.23) succinyl-CoA synthetase beta chain (EC 6.2.1.5) LSU ribosomal protein L12P (L7/L12) D-ribose-binding protein (S) (see spot 260, Table 4) ATP-dependent Clp protease, ATP-binding subunit ClpB malate dehydrogenase (EC 1.1.1.37) (S) alcohol dehydrogenase (EC 1.1.1.1) D-lactate dehydrogenase (cytochrome) (EC 1.1.2.4)e

bruAb1_1843 bruAb1_0027 bruAb1_0910 bruAb1_0280 bruAb1_1902 bruAb1_1249 bruAb1_0566 bruAb1_1843 bruAb1_1903 bruAb1_0198 bruAb1_1405

5.21 5.14 4.86 5.08 4.93 4.66 4.47 5.29 5.29 5.80 5.53

5.41 5.15 4.86 5.10 4.90 4.79 4.61 5.41 5.39 7.66 5.69

107.5 77.5 64.6 55.1 51.1 16.4 36.1 104.2 42.9 41.6 38.6

103.7 63.7 53.9 46.3 42.8 12.6 32.1 103.7 34.8 43.3 37.4

17.2 3.1 6.3 5.3 7.8 3.0 17.2 3.5 3.5 7.7 5.2

a Spot number as marked in Figure 1. b Protein name from B. abortus annotated genome (Integrated Genomics); (S) Protein containing a signal sequence as predicted by SignalP V2.0. c Corresponding ORF from B. abortus 9-941 genome in NCBI database, based on amino acid sequence homology (see “Mass spectral analysis” in “Materials and Methods”). d Each value is the ratio of the volumes of matched spots between strains 2308 and 16M. e Tentative identification. Exptl: experimental; Theor: theoretical.

for thioredoxin and the hypothetical protein (spots 215 and 308). Despite slight sequence variations, identical theoretical pIs were calculated for the outer membrane protein (spots 112 114 and 256), thus suggesting that the difference in migration on 2-D gels appeared to be exclusively due to variable PTMs of these proteins between the two strains. Reciprocal BLAST searches were performed between B. melitensis and B. abortus genomes to determine whether the ORFs coding for these strain-specific spots revealed a corresponding ORF in the genome of the other strain. All identified spots had a matching sequence in the opposite strain (see Supporting Information Tables 3 and 4), except for the histidine-binding protein (BruAb2_0595, spot 68, Table 3) which was missing in 16M. Further analysis of the surrounding sequences revealed a 6.9 kb deletion in 16M of six ORFs starting in B. abortus at BruAb2_0596 and ending at BruAb2_0591. These ORFs are present in B. suis 1330 and encode D-amino acid dehydrogenase small subunit (EC 1.4.99.1), histidinebinding protein, lysine-arginine-ornithine-binding protein, Llysine 6-dehydrogenase (EC 1.4.1.18), transcriptional regulator (AsnC family) and an exoenzymes regulatory protein AepA precursor. This genomic fragment was shown previously as present in B. suis (BRA0630-BRA0636) and missing in B. melitensis17 and was confirmed in the recently published B. abortus genome paper9 as present in B. abortus. All B. abortus 2308 strain-specific proteins (Table 3) were sorted into metabolic pathways based on the classification established by Integrated Genomics: 11 were related to membrane transport [5 for amino acids (spots 40, 43, 58, 68, and 172), 2 for carbohydrates (spots 23 and 156), 1 for metal (spot 23) and 3 for various substrates (spots 48, 52, and 72)]; 9 were associated with information processing related to protein metabolism (spots 4, 9, 11, 60, 81, 86, and 92) and nucleic acid metabolism (spots 30 and 61); 6 were involved in carbohydrate metabolism (spots 52, 92, 139, 148, 160, and 180); 6 in secretion systems (spots 4, 9, 11, 26, 60, and 86); 5 in stress response [oxidative (spots 52, 79, and 80) or heat shock (spots 4 and 60)] and 4 were linked to virulence (spots 7, 26, 112, and 114). As for B. melitensis 16M strain-specific proteins identified (Table 4): 7 were related to membrane transport [3 for amino acids (spots 229, 238 and 243), 2 for carbohydrates (spots 239 and 260), 1 for metal (spot 230) and 1 for an unnamed substrate (spot 248)]; 5 were associated with amino acid metabolism (spots 228, 255, 276, 285, and 295); 5 were involved in 1734


carbohydrate metabolism (spots 235, 241, 255, 295, and 298); 5 were linked to virulence19,20,21,22 (spots 226, 233, 256, 257, and 283) and 3 were associated with information processing (spots 227, 228, and 303). Protein Spots Overexpressed in 2308. Computer-assisted gel image analysis revealed 15 significantly overexpressed proteins in 2308 (from 3-fold to 17-fold increase) when compared to 16M expression levels (Table 1). Ten spots were identified with confidence, one was assigned a tentative identification due to the limited number of peaks detected in the mass spectrum, and 4 spots remained unidentified. The results showed 5 proteins involved in protein metabolism (spots 1, 6, 12, 91, and 110), 4 proteins associated with carbohydrate metabolism (spots 24, 164, 171, and 184), 2 proteins linked to bioenergetics (spots 164 and 171) and also proteins related to membrane transport (spot 100), amino acid metabolism (spot 22) and secretion (spot 12). Protein Spots Underexpressed in 2308. Comparison of the different 2-D gel images between 2308 and 16M indicated 70 protein spots with notably decreased expression levels (from 3-fold to 71-fold) when 16M was used as a reference (Table 2). Analysis of their respective tryptic digests by PMF led to the identification of 38 protein spots with 35 positive identifications and 3 tentative identifications. The protein functions assigned to the different spots of interest were sorted by metabolic pathways based on the classification established by Integrated Genomics. A total of 12 spots were associated with membrane transport [6 for amino acids (spots 16, 17, 37, 38, 39, and 50), 2 for carbohydrates (spots 34 and 42), 1 for metal (spot 186) and 3 for various substrates (spots 37, 49, and 64)]; 8 proteins were linked to information processing [5 related to protein metabolism (spots 14, 85, 97, 143, and 217) and 3 related to nucleic acid metabolism (spots 84, 206, and 208)]; 5 proteins were involved in secretion (spots 14, 82, 85, 97, and 217); 4 proteins were related to carbohydrate metabolism (spots 35, 169, 203, and 211); 2 proteins were associated with virulence22 (spots 189 and 190) and 2 proteins were linked to bioenergetics (spots 63 and 169). A few protein identifications were not correlated to a metabolic pathway according to Integrated Genomics classification.

Discussion General Observations. The different species within the genus Brucella are very similar at the genomic level as detailed in a

research articles

Brucella abortus and Brucella melitensis Proteomes Table 2. Proteins Underexpressed in B. abortus 2308, When Compared to B. melitensis 16M

B. abortus


pI

spot no.a

protein nameb

ORFc

exptl

theor

exptl

theor

V16M/V2308d

14 16 17 21 34 35 37 37 38 39 42 49 50 63 64 70 73 82 84 85 85 90 96 97 143 169 174 176 186 189 190 191 195 203

60 kDa chaperonin GroEL oligopeptide-binding protein OppA oligopeptide-binding protein OppA (S) dihydroorotase (EC 3.5.2.3) glycerol-3-phosphate-binding protein (S) fructokinase (EC 2.7.1.4) ABC transporter substrate-binding protein Leu-, Ile-, Val-, Thr-, and Ala-binding protein precursor (S)e Leu-, Ile-, Val-, Thr-, and Ala-binding protein (S) Leu-, Ile-, Val-, Thr-, and Ala-binding protein (S) glucose-binding protein (see spot 239, Table 4) sulfate-binding protein (S) spermidine/putrescine-binding protein (S) NADH-quinone oxidoreductase chain E (EC 1.6.5.3) D-ribose-binding protein thiG protein Nonsecretory proteine thiol:disulfide interchange protein DsbA (S) DNA protection during starvation protein bacterioferritin protein translocase subunit SecBe conserved Proteine glycoprotein/polysaccharide metabolism (S) 10 kDa chaperonin GroES tail-specific protease (EC 3.4.21.-) alcohol dehydrogenase (EC 1.1.1.1) choloylglycine hydrolase (EC 3.5.1.24) (S) 4-hydroxybutyrate dehydrogenase (EC 1.1.1.61) ferric anguibactin-binding protein immunogenic protein (S) (see spot 283, Table 4) immunogenic protein (S) (see spot 283, Table 4) thiosulfate sulfurtransferase (EC 2.8.1.1) S-formylglutathione hydrolase (EC 3.1.2.12)e probable transaldolase (EC 2.2.1.2) (see spot 298, Table 4) transcription antitermination protein NusG DNA protection during starvation protein peptidyl-prolyl cis-trans isomerase (EC 5.2.1.8) lactoylglutathione lyase (EC 4.4.1.5) 3-hydroxydecanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.60) 10 kDa chaperonin GroES

bruAb2_0190 bruAb2_0684 bruAb2_0685 bruAb2_0624 bruAb2_0571 bruAb1_0165 bruAb2_0422 bruAb2_1129 bruAb2_1129 bruAb2_1129 bruAb2_0537 bruAb1_1330 bruAb1_1599 bruAb1_0820 bruAb1_1340 bruAb1_0210 bruAb1_1585 bruAb1_0518 bruAb1_2123 bruAb2_0659 bruAb1_2047 bruAb2_0845 bruAb1_0462 bruAb2_0191 bruAb1_1816 bruAb1_0198 bruAb1_1463 bruAb2_0141 bruAb2_0553 bruAb1_1199 bruAb1_1199 bruAb1_1060 bruAb1_0124 bruAb1_1785

5.10 5.07 4.94 5.18 5.18 4.83 5.08 5.08 5.14 4.98 4.88 5.11 4.82 5.18 5.06 5.23 5.22 5.22 5.21 4.73 4.73 5.21 4.72 5.32 5.33 5.59 5.45 5.23 5.35 5.30 5.46 5.33 5.35 5.42

5.08 5.13 4.97 5.16 5.31 4.85 5.11 5.20 5.20 5.20 4.97 5.22 4.90 5.23 5.10 6.54 5.17 5.43 5.43 4.74 4.89 5.02 4.65 5.41 6.30 7.66 5.62 5.23 5.36 5.68 5.68 5.39 5.36 5.47

63.0 60.6 59.7 55.7 46.9 46.4 45.8 45.8 45.3 44.9 44.0 42.2 41.7 33.9 33.4 32.0 30.2 26.4 24.3 22.2 22.2 17.6 15.2 12.3 54.2 42.9 40.9 39.7 37.9 36.1 36.0 35.6 32.8 27.8

57.8 56.6 57.1 46.1 44.8 36.0 40.3 41.3 41.3 41.3 43.5 35.1 38.7 26.3 30.7 32.3 22.3 21.2 20.0 20.1 18.0 18.6 10.5 10.4 51.4 43.3 36.8 34.2 31.3 31.5 31.5 31.3 32.3 23.7

4.2 3.1 4.4 4.5 6.2 3.0 3.6 3.6 4.2 4.2 9.2 12.1 4.4 3.0 4.5 71.5 3.7 5.1 43.9 9.3 9.3 4.1 3.4 4.5 4.7 4.5 5.5 5.6 3.2 4.2 9.8 7.0 4.6 3.9

bruAb1_1253 bruAb1_2123 bruAb1_1099 bruAb1_1270 bruAb1_2146

5.73 5.33 5.48 5.20 5.56

6.64 5.43 6.08 5.85 5.93

26.4 23.6 20.3 18.9 19.3

19.7 20.0 19.3 19.9 20.1

46.3 24.3 5.0 4.3 7.7

bruAb2_0191

5.28

5.41

14.4

10.4

3.7

206 208 210 211 212 217

a Spot number as marked in Figure 1. Duplicated spot numbers indicate spots containing a protein mixture. b Protein name from B. abortus annotated genome (Integrated Genomics); (S) Protein containing a signal sequence as predicted by SignalP V2.0. c Corresponding ORF from B. abortus 9-941 genome in NCBI database, based on amino acid sequence homology (see “Mass spectral analysis” in “Materials and Methods”). d Each value is the ratio of the volumes of matched spots between strains 2308 and 16M. e Tentative identification. Exptl: experimental; Theor: theoretical.

recently published paper comparing B. abortus, B. melitensis and B. suis genomes.9 Some authors believe they should be grouped under a single species called B. melitensis. All the other Brucella species would be referred to as biovars of B. melitensis.8 However, each Brucella species has different host preference and fastidious growth requirements, which, in addition to other factors, has greatly influenced their currently accepted classification as distinct species.23 Various methods, including DNA-DNA hybridization and restriction mapping, have been used to compare the six Brucella species, but provided only broad comparative data. More specific information can be generated with other methods such as genomic studies by suppressive subtractive hybridization (SSH), which pinpoints genomic differences of closely related organisms for further study. However, even more valuable information can be gained from proteomic studies which focus upon the protein complement responsible for the physiological needs of the cell. A recent SSH genomic comparison of B. melitensis 16M and B. abortus 2308 has described several deletions in the two strains.24

Unfortunately, none of the proteins identified by PMF in this study correspond to any of the strain-specific ORFs indicated in the SSH study. There are several possibilities which could explain this observation: (1) The proteins may have not been expressed in the current growth conditions, and therefore cannot be visualized on 2-D gels; (2) The proteins may have been expressed, but not visualized on 2-D gels due to the physical limitation inherent to 2-D electrophoresis; (3) The proteins may have been expressed and visualized on the 2-D gels, but not identified by PMF. Despite high genomic similarity, the two strains exhibited extensive differences on the proteomic level supporting their classification as two different species. The metabolic divergence of B. melitensis and B. abortus was evidenced in two areas: comigrating spots, which were identified as different proteins between the two species, and strain-specific spots which, despite their unique electrophoretic mobility, were assigned the same protein identification. The latter phenomenon might arise from amino acid substitutions and differing posttranslational modifications, and Journal of Proteome Research • Vol. 5, No. 7, 2006 1735

research articles


Table 3. Electrophoretically Unique Protein Spots in B. abortus 2308

B. abortus spot no.a

4 7 9 9 11 23 23 26 30 40 43 48 52 52 53 56 58 59 60 61 67 68 72 74 77 79 80 81 86 87 88 92 92 94 112 114 139 139 148 156 160 172 180 193 215


pI

protein nameb

ORFc

exptl

theor

exptl

theor

chaperone protein DnaK metal chelate outer membrane receptor (see spot 226, Table 4) 2-isopropylmalate synthase (EC 4.1.3.12) 60 kDa chaperonin GroELd trigger factor, ppiase (EC 5.2.1.8) low affinity zinc transport membrane protein trehalose/maltose binding protein (S) acriflavin resistance protein A precursor (S) (see spot 233, Table 4) DNA-directed RNA polymerase alpha chain (EC 2.7.7.6) Leu-, Ile-, Val-, Thr-, and Ala-binding protein precursor (S) Leu-, Ile-, Val-, Thr-, and Ala-binding protein precursor (S) ABC transporter substrate-binding protein (S)d thioredoxin (see spot 241, Table 4) ABC transporter substrate-binding proteind nucleoside-binding protein (S) malonyl-CoA-[acyl-carrier-protein] transacylase (EC 2.3.1.39) glycine betaine-binding protein (S) fumarylacetoacetate hydrolase family protein (S) (see spot 246, Table 4) grpE protein DNA polymerase III, delta′ subunit (EC 2.7.7.7)d 2-deoxy-D-gluconate 3-dehydrogenase (EC 1.1.1.125) histidine-binding protein (S) ABC transporter substrate binding protein (S) (see spot 248, Table 4) 3-isopropylmalate dehydratase (EC 4.2.1.33) dihydroxyacetone kinase (EC 2.7.1.29) alkyl hydroperoxide reductase C22 protein (EC 1.6.4.-)d alkyl hydroperoxide reductase C22 protein (EC 1.6.4.-) LSU ribosomal protein L9P protein translocase subunit SecB conserved Protein conserved Protein ribose 5-phosphate isomerase (EC 5.3.1.6)d LSU ribosomal protein L12P (L7/L12)d Nonsecretory protein outer membrane protein (S) (see spot 256, Table 4) outer membrane protein (S) (see spot 256, Table 4) D-Lactate dehydrogenase (cytochrome) (EC 1.1.2.4) adenosylhomocysteinase (EC 3.3.1.1)d dihydrolipoamide succinyltransferase component (E2) of 2-oxoglutarate dehydrogenase complex (EC 2.3.1.61)d glycerol-3-phosphate-binding protein (S)d acetyl-CoA acetyltransferase (EC 2.3.1.9) Leu-, Ile-, Val-, Thr-, and Ala-binding protein (S) succinyl-CoA synthetase alpha chain (EC 6.2.1.5) 3-demethylubiquinone 3-methyltransferase (EC 2.1.1.64) hypothetical protein (S) (see spots 253 and 308, Table 4)

bruAb1_2100 bruAb1_1345 bruAb1_1555 bruAb2_0190 bruAb1_0910 bruAb1_1308 bruAb2_0877 bruAb1_0317 bruAb1_1214 bruAb2_0280 bruAb1_1767 bruAb2_1043 bruAb2_0339 bruAb2_0113 bruAb2_0010 bruAb1_0479 bruAb1_1566 bruAb1_0239

4.91 5.09 5.17 5.17 4.88 4.84 4.84 5.22 4.91 5.11 4.96 4.92 4.70 4.70 4.65 5.01 5.07 5.11

4.86 5.15 5.31 5.08 4.86 4.80 4.77 5.35 4.90 5.15 5.04 5.07 4.88 4.97 4.62 4.94 5.08 6.44

88.7 74.0 69.6 69.6 64.7 52.6 52.6 51.9 49.0 44.1 43.7 43.1 41.5 41.5 41.3 38.0 36.8 34.9

69.0 65.0 65.2 57.8 53.9 38.2 43.5 39.9 37.6 41.5 37.2 37.2 35.4 41.5 36.3 32.7 31.9 35.3

bruAb1_0167 bruAb1_0999 bruAb1_1617 bruAb2_0595 bruAb1_2148

4.64 4.89 4.98 4.62 5.02

4.70 6.44 5.02 4.71 5.03

35.1 34.5 32.6 32.5 30.8

25.0 39.8 26.7 25.4 25.9

bruAb2_0349 bruAb1_1615 bruAb2_0522 bruAb2_0522 bruAb1_0474 bruAb1_2047 bruAb2_0845 bruAb2_0845 bruAb2_0362 bruAb1_1249 bruAb1_0856 bruAb1_1160 bruAb1_1160 bruAb1_1405 bruAb1_2072 bruAb1_1898

5.01 4.79 4.91 5.00 5.00 4.87 5.00 4.85 4.87 4.87 4.89 5.34 5.30 5.35 5.35 5.37

5.13 5.07 5.00 5.00 4.86 4.89 5.02 5.02 5.90 4.79 4.92 5.31 5.31 5.32 5.30 5.50

29.4 28.2 27.7 27.7 26.9 21.5 21.0 20.8 16.4 16.4 15.4 95.3 92.0 56.4 56.4 51.3

23.8 23.0 20.6 20.6 21.0 18.0 18.6 18.6 17.4 12.6 12.2 83.1 83.1 50.9 53.0 43.1

bruAb2_0571 bruAb1_1756 bruAb1_1294 bruAb1_1901 RBV00210e bruAb1_1016

5.29 5.67 5.29 5.44 5.81 5.43

5.31 5.89 5.32 5.81 5.79 5.93

44.7 44.7 41.2 38.9 33.5 17.5

44.8 42.9 35.8 31.6 27.8 16.0

a Spot number as marked in Figure 1. Duplicated spot numbers indicate spots containing a protein mixture. b Protein name from B. abortus annotated genome (Integrated Genomics); (S) Protein containing a signal sequence as predicted by SignalP V2.0. c Corresponding ORF from B. abortus 9-941 genome in NCBI database, based on amino acid sequence homology (see “Mass spectral analysis” in “Materials and Methods”). d Tentative identification. e ORF from B. abortus genome in-house database (Integrated Genomics; ORF not found in NCBI). Exptl: experimental; Theor: theoretical.

may possibly indicate proteins associated with the different host preference of the two species. Comparison of ORF Sequence between B. abortus and B. melitensis. The virtual ORFeomes of each Brucella species referenced by the Mascot search engine are dependent upon the annotations provided by each individual sequencing facility. As each facility used different software packages and parameters for ORF-calling and functional assignment, variation will necessarily be present across the annotated genomes of these highly similar species. In some cases, the protein identifications gained in this study reflected this variation. Most B. abortus proteins identified by PMF arose from an ORF which had a corresponding partner in the 16M genome (see Supporting Information Tables 1, 2, and 3). However, 1736


comparison of the 2308 and 16M ORFs revealed slight differences at the nucleotide level (substitutions, insertions or deletions) which sometimes dictated noticeable differences at the protein level. At times, strain-specific protein spots identified by PMF coincided with strain-specific genomic features. In one example, B. abortus ORF BruAb2_0010 (nucleosidebinding protein, spot 53, Table 3) corresponded to two separate consecutive B. melitensis ORFs (BEMII0083/BMEII0084) (see Supporting Information Table 3), as result of a single nucleotide substitution which yielded a premature stop codon. Similarly, B. abortus 2-deoxy-D-gluconate-3-dehydrogenase (BruAb1_1617, spot 67, Table 3) aligned with ORFs BMEI0394/BMEI0395 (see Supporting Information Table 3) due to a 4 base insertion in B. melitensis. Two 16M strain-specific spots (spots 230 and 276,

research articles

Brucella abortus and Brucella melitensis Proteomes Table 4. Electrophoretically Unique Protein Spots in B. melitensis 16M

B. melitensis spot no.a

protein nameb

ORFc

226 227 228 229 230 233 235 237 238 239 240 241 243 246 248 253 255 256 257 260 263 276 283 285 295 298 303 308

metal chelate outer membrane receptor (see spot 7, Table 3) protease DO (EC 3.4.21.-) histidyl-tRNA synthetase (EC 6.1.1.21) periplasmic dipeptide transport protein precursor (S) nickel-binding periplasmic protein precursor (S) acriflavin resistance protein A precursor (S) (see spot 26, Table 3)d enolase (EC 4.2.1.11)d isovaleryl-CoA dehydrogenase (EC 1.3.99.10) Leu-, Ile-, Val-, Thr-, and Ala-binding protein precursor (S)d glucose-binding protein (see spot 42, Table 2)d periplasmic component of efflux systeme thioredoxin (see spot 52, Table 3)d general L-amino acid-binding periplasmic protein AapJ precursor (S) fumarylacetoacetate hydrolase family protein (see spot 59, Table 3)d ABC transporter substrate-binding protein (S) (see spot 72, Table 3)d hypothetical protein (see spot 215, Table 3) 2-oxoisovalerate dehydrogenase beta subunit (EC 1.2.4.4)d outer membrane protein (S) (see spots 112 and 114, Table 3) outer membrane protein (S)d D-ribose-binding periplasmic protein precursor (S) (see spot 100, Table 1)d 3-oxoacyl-(acyl-carrier protein) reductase (EC 1.1.1.100)d cysteine synthase A (EC 4.2.99.8)d immunogenic protein (S) (see spots 189 and 190, Table 2) acetylglutamate kinase (EC 2.7.2.8) enoyl-CoA hydratase (EC 4.2.1.17) transaldolase (EC 2.2.1.2) (see spot 203, Table 2) single-strand binding protein hypothetical proteine (see spot 215, Table 3)

BMEI0657 BMEI0613 BMEII1056 BMEII0284 BMEII0487 BMEI1630 BMEI0851 BMEI1923 BMEII0633 BMEII0590 BMEI0653 BMEII0401 BMEI1211 BMEI1708 BMEI1954 BMEI0973 BMEII0747 BMEI0830 BMEI1007 BMEI1390 BMEI0032 BMEI0101 BMEI0796 BMEII0273 BMEI1945 BMEI0244 BMEI0880 BMEI0973


pI exptl

theor.

exptl

theor.

4.95 4.79 5.01 5.24 5.33 5.17 4.82 5.26 5.24 4.77 5.18 4.83 5.07 4.93 4.93 5.18 4.79 5.10 4.53 4.45 4.48 5.73 5.50 5.45 5.44 5.64 5.54 5.78

5.00 4.89 4.98 5.20 5.31 5.27 4.99 5.36 5.54 4.97 7.85 4.94 5.09 5.22 4.95 5.74 5.50 5.31 4.56 4.61 4.47 5.72 5.68 5.46 6.05 5.69 5.54 5.74

71.3 62.4 58.7 57.6 57.2 50.1 47.4 44.6 43.8 43.0 42.6 39.6 36.8 35.0 30.4 18.0 16.1 91.9 35.0 36.5 30.7 41.6 36.3 33.6 29.8 28.1 23.2 18.3

65.0 55.4 55.5 51.6 55.7 39.9 45.6 42.1 42.7 43.5 35.1 35.3 35.1 32.5 26.0 16.6 37.3 83.1 23.2 32.1 27.2 35.5 31.5 34.3 32.8 23.7 18.5 16.6

a Spot number as marked in Figure 2. b Protein name from B. melitensis 16M annotated genome (Integrated Genomics); (S) Protein containing a signal sequence as predicted by SignalP V2.0. c ORF from B. melitensis 16M genome from NCBI genomic database. d Spot previously identified by Wagner et al.10 e Tentative identification. Exptl: experimental; Theor: theoretical.

Table 4) could also be traced to two separate ORFs unique to the 16M genome (see Supporting Information Table 4). Here, each genetically unique 16M ORF correlated to two overlapping B. abortus ORFs, arising from a single nucleotide insertion which promoted frameshifts and premature stop codons. Membrane Transport Proteins. Proteins involved in membrane transport play a crucial role in the capture of different nutrients for cell survival. They represent part of the first contact between the bacterial cell and its environment. Interestingly, most strain-specific proteins for either 16M or 2308 belong to the membrane transport protein category, with predominance of the ABC transport system for amino acids transport. They typically consist of a soluble periplasmic solute binding protein and a cytoplasmic membrane associated complex.25 In our study, the periplasmic soluble part was usually detected. Three protein spots associated with branched chain amino acid ABC transport system (spots 40, 43, 172, Table 3) were observed as specific to 2308, with only one corresponding spot in 16M (spot 238, Table 4). The detected Leu-, Ile-, Val-, Thr-, and Ala- (LIVTA) binding proteins were encoded by four different ORFs (three in 2308 and one in 16M). Other spots corresponding to a fifth ORF coding for a LIVTA-binding protein were observed as underexpressed (spots 37, 38, and 39, Table 2). Despite their identical functional assignment, all these proteins display very different protein sequences with only 12 to 18% identity based on Clustal V alignment (DNASTAR software). All belong to the LIV-I high affinity transport system described in Pseudomonas aeruginosa.26 Their different sequences might confer varied affinity for different amino acids. In this class of transport proteins, the differences between 16M and 2308 seemed most prevalent regarding the high affinity

LIV-I transports system, rather than the low affinity LIV-II and LIV-III systems which are specific to branched-chain amino acids alone. These observations suggest that both strains use different sets of proteins with similar functions, possibly with different affinities for their substrates, which may reflect their different ability to infect specific hosts. The need for a high affinity transport system correlates well with the poor nutrient contents described for the Brucellosome, the intramacrophagic replicative niche of Brucella.27 Among all the protein spots identified in this study, only one spot, unique to 2308 (spot 68, Table 3), had no corresponding ORF in 16M (see Supporting Information Table 3), and was part of a large deletion in the latter strain also mentioned by Paulsen et al.17 and Halling et al.9 While these two previous studies only observed this deletion at the genomic level, our data confirm for the first time the reality of this deletion at the proteomic level. The ORF BruAb2_0595, encoding for histidine-binding protein, was also present in the B. suis 1330 genome. This protein has been described in Sinorhizobium meliloti as being transcriptionally induced by histidine, having a high affinity for histidine, and playing a role in nitrogen assimilation.28 This gene was located in Salmonella typhimurium, next to Lys-, Arg-, Ornithine-binding protein,29 similar to that observed in B. abortus (BruAb2_0594). Both genes were considered as paralogs arising from duplication of a common ancestral gene followed by a change of function, illustrated here by a change of substrate specificity. Their transcriptional induction by nitrogen limitation was demonstrated and correlated well with their affinity for nitrogen-rich amino acids.29 This same genomic fragment from B. abortus (missing in B. melitensis) also contains two amino acid dehydrogenases: L-lysine-6-dehydrogenase Journal of Proteome Research • Vol. 5, No. 7, 2006 1737

research articles


Figure 2. B. melitensis 16M proteome. Composite image created from 2-D gel images of four overlapping one-unit pH ranges (4-5, 4.5-5.5, 5-6, and 5.5-6.7). Only strain-specific spots were labeled in this representation. Spots identified in a previous publication10 were labeled in blue, while newly identified spots were labeled in yellow. Spot number labeled with an asterisk (*) designates tentative identification. Molecular mass scale is indicated in kDa on the left.

(BruAb2_0593), an enzyme characterized in Agrobacterium tumefaciens and involved in the catabolism of lysine by catalyzing the oxidative deamination of its -amino group;30 and D-amino acid dehydrogenase small subunit (BruAb2_0596), which catalyzes the oxidative deamination of a broad variety of D-amino acids with D-alanine being its best substrate.31,32 This could participate in energy production through catabolism of alanine with formation of pyruvate and ammonia.33 Ahead of these four ORFs, a gene coding for transcriptional regulatory AsnC family protein was observed. In E. coli, this gene is transcribed divergently from the adjacent gene it controls, asparagine synthase asnA.34 Also in E. coli, AsnC expression was demonstrated as regulated by a nitrogen assimilation control (Nac) protein.35 The similar gene organization observed in B. abortus suggested that all these genes, which correspond to the large deletion in 16M, might afford B. abortus an advantage in nitrogen-rich amino acid capture and catabolism, for the purpose of nitrogen assimilation. This system would be specific to B. abortus 2308 and unavailable in B. melitensis 16M. Paulsen et al.17 also suggested, regarding this fragment, the possibility that B. melitensis might not be able to use certain amino acids when compared to other Brucella strains. Secretion. The original genomic description of Brucella melitensis7 suggested that this organism does not secrete proteins into the extracellular environment. However, 20% of the predicted ORFs have no assigned function to date. Some of these proteins may be secreted and may play a role in the infectious process. Recently, Baldi et al. (University of Buenos Aires, unpublished data) conducted a study on Brucella abortus 2308 secretome and revealed the presence of Brucella-secreted proteins in the extracellular medium. Furthermore, several 1738


ORFs producing specific proteins involved in different secretion systems were predicted in the three different Brucella genomes submitted to GenBank.7,9,17 Several groups are focusing on the VirB operon, which produces elements for a type IV secretion system, and is known to be critical for Brucella virulence.36 Further support for secretion in Brucella is evident in the genome. Several proteins identified in this study contained a leading signal sequence predicted by SignalP V2.0.37 The 89 spots identified in B. abortus were linked to 82 ORFs, and 23 of these were predicted to contain signal sequences. Most 2308 ORFs which contained signal sequences correlated to similar signal-sequence containing ORFs in 16M. In a few cases, signal sequences were predicted in an ORF from one species, but not the other. Although the 5′ untranslated regions of each ORF were identical between the 2 species, differences in ORF-calling requirements lead to selection of alternate start codons. In some cases, the N-terminal protein sequence was so affected that the signal sequence was deleted or abbreviated, and not detected by SignalP. Secretion of proteins by Gram-negative bacteria is a complex process involving numerous proteins, necessitated by the presence of an inner membrane (IM) and an outer membrane (OM) in the cell envelope. In the present study, different elements of the Sec-dependent secretion system were observed as differentially expressed in 2308 as compared to 16M. On the basis of our observation, 2308 may favor the use of the secretion-specific chaperone SecB (spot 86) and also general molecular chaperones such as DnaK (spot 4), GrpE (spot 60) and Trigger Factor Ppiase (spot 11; all in Table 3). In contrast, 16M has higher amounts of another general chaperone, GroES (spots 97 and 217, Table 2). These general molecular chaper-

research articles

Brucella abortus and Brucella melitensis Proteomes

ones are thought to be possible alternative replacements for secretion-specific chaperone SecB.38,39 On the basis of these differentially expressed Sec-dependent secretion accessories, 2308 and 16M may differ in their use of the Sec system, targeting different substrates toward the periplasm, cell surface, or the extracellular milieu. These differences may skew the pathogenicity of each Brucella strain toward different hosts. In addition, a predominant periplasmic chaperone, DsbA (spot 82, Table 2), accumulated less in 2308 compared to 16M. The lower amount of the disulfide bond forming DsbA in 2308 might reflect a reduced capacity toward such bond formation in the periplasmic space. Additionally, the activity of DsbA has been associated with less efficient translocation of the passenger domains of secreted proteins through the β-barrel pore of Type V autotransporters located in the OM, suggesting that Type V substrates are more efficiently exported in a disulfidebond poor/unfolded state.40,41 In support of this observation, a crystal structure of the translocator domain of the NalP autotransporter from Neisseria meningitidis indicated that the secreted protein is passed through the β-barrel pore in an unfolded state.42 Comparatively, DsbA is associated with periplasmic folding of Type IV secretion substrates, as observed for successful export of pertussis toxin from Bordetella pertussis.43,44 Thus, DsbA activity (disulfide bond formation in the periplasm) seems to correlate with Type IV Sec-dependent secretion, while it hinders Type V secretion. The underexpression of DsbA in B. abortus suggests that this strain could favor Type V secretion, while 16M could favor Sec-dependent Type IV secretion. The importance of a type IV secretion system was demonstrated in the literature with the study of two intracellular pathogens, B. abortus45 and Bartonella tribocorum,46 which showed that the presence of an intact functional VirB operon is essential for host infection. The comparative analysis of the three Brucella genomes18 (B. melitensis, B. abortus, and B. suis) revealed genetic differences among the predicted autotransporter genes (type V secretion system). These discrepancies were correlated to a possible role for autotransporters as important contributor toward host specificity.18 Furthermore, the role of DsbA in virulence has been demonstrated in the literature: a Shigella flexneri dsbA mutant showed reduced virulence,47 and a Brucella suis dsbA mutant multiplied poorly in macrophages.27 Thus, there is potential for DsbA to play an indirect role in virulence, possibly through influencing the periplasmic conformation and subsequent secretion of yet-to-be-identified virulence factors which exit via the Sec translocase system. On the basis of this premise, differential expression of DsbA between B. abortus and B. melitensis may be linked to the variable infectivity of these strains toward preferential mammalian hosts.

Conclusion The comparative proteomic study of laboratory grown 16M and 2308 reveals clear differences between the two strains, strengthening their classification as two distinct species. These discrepancies seem to affect various sets of proteins involved in membrane transport as well as secretion systems. A possible distinct amino acid capture combined with divergent amino acid metabolic capabilities could lead to different host preference between 16M and 2308. Furthermore, based on differential expression observed for various Sec-dependent secretion accessories and the periplasmic chaperone DsbA, possible different utilization of their Sec-dependent secretion pathways was hypothesized. This possible behavior disparity may lead

to subsequent variability in the proteins presented on the cell surface or sent into the extracellular environment toward the host, and could reasonably be related to different host preference. These results suggest that the pathogen-associated mechanisms contributing to host preferences between B. abortus and B. melitensis likely arises from the combined action of multiple systems. Abbreviations. 2-D, two-dimensional; IM, inner membrane; IPG, immobilized pH gradient; LIVTA-binding protein, Leu-, Ile-, Val-, Thr- and Ala-binding protein; MALDI, matrix-assisted laser desorption/ionization; O-MU, O-methylisourea; OM, outer membrane; PMF, peptide mass fingerprinting; PTM, posttranslational modification.

Acknowledgment. This work was supported by Grant No. DE-FG02-00ER62773 from the United States Department of Energy.We thank Dr Phil Elzer and Sue Hagius at the Louisiana State University for providing the strains B. melitensis 16M and B. abortus 2308. Supporting Information Available: Supporting Tables 1, 2, 3, and 4. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Corbel, M. J.; Brinley-Morgan, W. J. Genus Brucella Meyer and Shaw 1920, 173AL. In Bergey’s Manual of Systematic Bacteriology, 1984, pp 377-388. Krieg, N. R., Holt, J. G., Eds. Baltimore, MD: Williams & Wilkins. (2) Elberg, S. S.; Faunce, K. Immunization against Brucella infection. VI. Immunity conferred on goats by a nondependent mutant from a streptomycin-dependent mutant strain of Brucella melitensis. J. Bacteriol. 1957, 73, 211-217. (3) Blasco, J. M. A review of the use of B. melitensis Rev 1 vaccine in adult sheep and goats. Prev. Vet. Med. 1997, 31, 275-283. (4) Corbel, M. J. Brucellosis: an overview. Emerg. Infect. Dis. 1997, 3, 213-221. (5) Young, E. J. An overview of human brucellosis. Clin. Infect. Dis. 1995, 21, 283-289. (6) Jumas-Bilak, E.; Michaux-Charachon, S.; Bourg, G.; O’Callaghan, D.; Ramuz, M. Differences in chromosome number and genome rearrangements in the genus Brucella. Mol. Microbiol. 1998, 27, 99-106. (7) DelVecchio, V. G.; Kapatral, V.; Redkar, R. J.; Patra, G.; Mujer, C.; Los, T.; Ivanova, N.; Anderson, I.; Bhattacharyya, A.; Lykidis, A.; Reznik, G.; Jablonski, L.; Larsen, N.; D′Souza, M.; Bernal, A.; Mazur, M.; Goltsman, E.; Selkov, E.; Elzer, P. H.; Hagius, S.; O’Callaghan, D.; Letesson, J. J.; Haselkorn, R.; Kyrpides, N.; Overbeek, R. The genome sequence of the facultative intracellular pathogen Brucella melitensis. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 443-448. (8) Verger, J. M.; Grimont, F.; Grimont, P. A.; Grayon, M. Brucella, a monospecific genus as shown by deoxyribonucleic acid hybridization. Int. J. Syst. Bacteriol. 1985, 35, 292-295. (9) Halling, S. M.; Peterson-Burch, B. D.; Bricker, B. J.; Zuerner, R. L.; Qing, Z.; Li, L. L.; Kapur, V.; Alt, D. P.; Olsen, S. C. Completion of the genome sequence of Brucella abortus and comparison to the highly similar genomes of Brucella melitensis and Brucella suis. J. Bacteriol. 2005, 187, 2715-2726. (10) Wagner, M. A.; Eschenbrenner, M.; Horn, T. A.; Kraycer, J. A.; Mujer, C. V.; DelVecchio, V. G. Global analysis of the Brucella melitensis proteome: Identification of proteins expressed in laboratory-grown culture. Proteomics 2002, 2, 1047-1060. (11) Rafie-Kolpin, M.; Essenberg, R. C.; Wyckoff, J. H. Identification and comparison of macrophage-induced proteins and proteins induced under various stress conditions in Brucella abortus. Infect. Immun. 1996, 64, 5274-5283. (12) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254. (13) Eschenbrenner, M.; Wagner, M. A.; Horn, T. A.; Kraycer, J. A.; Mujer, C. V.; Hagius, S.; Elzer, P.; DelVecchio, V. G. Comparative proteome analysis of Brucella melitensis vaccine strain Rev 1 and a virulent strain, 16M. J. Bacteriol. 2002, 184, 4962-4970.

Journal of Proteome Research • Vol. 5, No. 7, 2006 1739

research articles (14) Hale, J. E.; Butler, J. P.; Knierman, M. D.; Becker, G. W. Increased sensitivity of tryptic peptide detection by MALDI-TOF mass spectrometry is achieved by conversion of lysine to homoarginine. Anal. Biochem. 2000, 287, 110-117. (15) Rosenfeld, J.; Capdevielle, J.; Guillemot, J. C.; Ferrara, P. In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal. Biochem. 1992, 203, 173-179. (16) Perkins, D. N.; Pappin, D. J. C.; Creasy, D.; Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry. Electrophoresis 1999, 20, 3551-3567. (17) Paulsen, I. T.; Seshadri, R.; Nelson, K. E.; Eisen, J. A.; Heidelberg, J. F.; Read, T. D.; Dodson, R. J.; Umayam, L.; Brinkac, L. M.; Beanan, M. J.; Daugherty, S. C.; Deboy, R. T.; Durkin, A. S.; Kolonay, J. F.; Madupu, R.; Nelson, W. C.; Ayodeji, B.; Kraul, M.; Shetty, J.; Malek, J.; Van Aken, S. E.; Riedmuller, S.; Tettelin, H.; Gill, S. R.; White, O.; Salzberg, S. L.; Hoover, D. L.; Lindler, L. E.; Halling, S. M.; Boyle, S. M.; Fraser, C. M. The Brucella suis genome reveals fundamental similarities between animal and plant pathogens and symbionts. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 13148-13153. (18) Chain, P. S.; Comerci, D. J.; Tolmasky, M. E.; Larimer, F. W.; Malfatti, S. A.; Vergez, L. M.; Aguero, F.; Land, M. L.; Ugalde, R. A.; Garcia, E. Whole-genome analyses of speciation events in pathogenic Brucellae. Infect. Immun. 2005, 73, 8353-8361. (19) Reeves, S. A.; Torres, A. G.; Payne, S. M. TonB is required for intracellular growth and virulence of Shigella dysenteriae. Infect. Immun. 2000, 68, 6329-6336. (20) Burse, A.; Weingart, H.; Ullrich, M. S. The phytoalexin-inducible multidrug efflux pump AcrAB contributes to virulence in the fire blight pathogen, Erwinia amylovora. Mol. Plant Microbe Interact. 2004, 17, 43-54. (21) Cloeckaert, A.; Vizcaino, N.; Paquet, J. Y.; Bowden, R. A.; Elzer, P. H. Major outer membrane proteins of Brucella spp.: past, present and future. Vet. Microbiol. 2002, 90, 229-247. (22) Vizcaino, N.; Caro-Hernandez, P.; Cloeckaert, A.; Fernandez-Lago, L. DNA polymorphism in the omp25/omp31 family of Brucella spp.: identification of a 1.7-kb inversion in Brucella cetaceae and of a 15.1-kb genomic island, absent from Brucella ovis, related to the synthesis of smooth lipopolysaccharide. Microbes Infect. 2004, 6, 821-834. (23) Michaux-Charachon, S.; Bourg, G.; Jumas-Bilak, E.; Guigue-Talet, P.; Allardet-Servent, A.; O’Callaghan, D.; Ramuz, M. Genome structure and phylogeny in the genus Brucella. J. Bacteriol. 1997, 179, 3244-3249. (24) Tsoktouridis, G.; Merz, C. A.; Manning, S. P.; Giovagnoli-Kurtz, R.; Williams, L. E.; Mujer, C. V.; Hagius, S.; Elzer, P.; Redkar, R. J.; Patra, G.; DelVecchio, V. G. Molecular characterization of Brucella abortus chromosome II recombination. J. Bacteriol. 2003, 185, 6130-6136. (25) Hosie, A. H.; Poole, P. S. Bacterial ABC transporters of amino acids. Res. Microbiol. 2001, 152, 259-270. (26) Hoshino, T.; Kose-Terai, K.; Sato, K. Solubilization and reconstitution of the Pseudomonas aeruginosa high affinity branched-chain amino acid transport system. J. Biol. Chem. 1992, 267, 2131321318. (27) Kohler, S.; Foulongne, V.; Ouahrani-Bettache, S.; Bourg, G.; Teyssier, J.; Ramuz, M.; Liautard, J. P. The analysis of the intramacrophagic virulome of Brucella suis deciphers the environment encountered by the pathogen inside the macrophage host cell. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15711-15716. (28) Boncompagni, E.; Dupont, L.; Mignot, T.; Osteras, M.; Lambert, A.; Poggi, M. C.; Le Rudulier, D. Characterization of a Sinorhizobium meliloti ATP-binding cassette histidine transporter also involved in betaine and proline uptake. J. Bacteriol. 2000, 182, 3717-3725. (29) Higgins, C. F.; Ames, G. F. Regulatory regions of two transport operons under nitrogen control: nucleotide sequences. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 1083-1087.

1740


Eschenbrenner et al. (30) Misono, H.; Nagasaki, S. Occurrence of L-lysine epsilon-dehydrogenase in Agrobacterium tumefaciens. J. Bacteriol. 1982, 150, 398-401. (31) Olsiewski, P. J.; Kaczorowski, G. J.; Walsh, C. Purification and properties of D-amino acid dehydrogenase, an inducible membrane-bound iron-sulfur flavoenzyme from Escherichia coli B. J. Biol. Chem. 1980, 255, 4487-4494. (32) Janes, B. K.; Bender, R. A. Alanine catabolism in Klebsiella aerogenes: molecular characterization of the dadAB operon and its regulation by the nitrogen assimilation control protein. J. Bacteriol. 1998, 180, 563-570. (33) Reitzer, L. J. Ammonia assimilation and the biosynthesis of glutamine, glutamate, aspartate, asparagine, L-alanine, and Dalanine. In Escherichia coli and Salmonella: Cellular and Molecular Biology; Neidhardt, F. C., Curtiss, R., III, Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, M., Umbarger, H. E., Eds. American Society for Microbiology: Washington, D. C., 1996, pp 391-407. (34) Kolling, R.; Lother, H. AsnC: an autogenously regulated activator of asparagine synthetase A transcription in Escherichia coli. J. Bacteriol. 1985, 164, 310-315. (35) Poggio, S.; Domeinzain, C.; Osorio, A.; Camarena, L. The nitrogen assimilation control (Nac) protein represses asnC and asnA transcription in Escherichia coli. FEMS Microbiol. Lett. 2002, 206, 151-156. (36) Boschiroli, M. L.; Ouahrani-Bettache, S.; Foulongne, V.; MichauxCharachon, S.; Bourg, G.; Allardet-Servent, A.; Cazevieille, C.; Lavigne, J. P.; Liautard, J. P.; Ramuz, M.; O’Callaghan, D. Type IV secretion and Brucella virulence. Vet. Microbiol. 2002, 90, 341348. (37) Nielsen, H.; Krogh, A. Prediction of signal peptides and signal anchors by a hidden Markov model. Proc. Int. Conf. Intell. Syst. Mol. Biol. 1998, 6, 122-130. (38) Stathopoulos, C.; Hendrixson, D. R.; Thanassi, D. G.; Hultgren, S. J.; St Geme, J. W., 3rd; Curtiss, R., 3rd Secretion of virulence determinants by the general secretory pathway in gram-negative pathogens: an evolving story. Microbes Infect. 2000, 2, 10611072. (39) Desvaux, M.; Parham, N. J.; Scott-Tucker, A.; Henderson, I. R. The general secretory pathway: a general misnomer? Trends Microbiol. 2004, 12, 306-309. (40) Jose, J.; Kramer, J.; Klauser, T.; Pohlner, J.; Meyer, T. F. Absence of periplasmic DsbA oxidoreductase facilitates export of cysteinecontaining passenger proteins to the Escherichia coli cell surface via the Iga beta autotransporter pathway. Gene 1996, 178, 107110. (41) Desvaux, M.; Parham, N. J.; Henderson, I. R. Type V protein secretion: simplicity gone awry? Curr. Issues Mol. Biol. 2004, 6, 111-124. (42) Oomen, C. J.; van Ulsen, P.; van Gelder, P.; Feijen, M.; Tommassen, J.; Gros, P. Structure of the translocator domain of a bacterial autotransporter. EMBO J. 2004, 23, 1257-1266. (43) Farizo, K. M.; Huang, T.; Burns, D. L. Importance of holotoxin assembly in Ptl-mediated secretion of pertussis toxin from Bordetella pertussis. Infect. Immun. 2000, 68, 4049-4054. (44) Stenson, T. H.; Weiss, A. A. DsbA and DsbC are required for secretion of pertussis toxin by Bordetella pertussis. Infect. Immun. 2002, 70, 2297-2303. (45) Watarai, M.; Makino, S.; Shirahata, T. An essential virulence protein of Brucella abortus, VirB4, requires an intact nucleosidetriphosphate-binding domain. Microbiology 2002, 148, 14391446. (46) Schulein, R.; Dehio, C. The VirB/VirD4 type IV secretion system of Bartonella is essential for establishing intraerythrocytic infection. Mol. Microbiol. 2002, 46, 1053-1067. (47) Yu, J. Inactivation of DsbA, but not DsbC and DsbD, affects the intracellular survival and virulence of Shigella flexneri. Infect. Immun. 1998, 66, 3909-3917.

PR060135P

Comparative Proteome Analysis of Laboratory Grown Brucella a

Recommend Documents