Proteomic and bioinformatic analysis of outer membrane proteins of the protobacterium Bartonella henselae (Bartonellaceae)

D.M. Li1, Q.Y. Liu2, F. Zhao3, Y. Hu3, D. Xiao3, Y.X. Gu3, X.P. Song1 and J.Z. Zhang3

1Department of Vector Biology and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China 2State Key Laboratory for Infectious Diseases Prevention and Control, Department of Vector Biology and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China 3Diagnosis Department of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China

Corresponding author: J.Z. Zhang E-mail: [email protected]

Genet. Mol. Res. 10 (3): 1789-1818 (2011) Received November 26, 2010 Accepted February 10, 2011 Published August 26, 2011 DOI http://dx.doi.org/10.4238/vol10-3gmr1153

ABSTRACT. Bartonella henselae, an infectious agent causing cat- scratch disease and vasculoproliferative disorders in humans, is a fastidi- ous facultative intracellular pathogen. The outer membrane proteins of B. henselae are key molecules that play a primary role in host-cell interac- tions. We isolated B. henselae outer membrane proteins, using the ionic detergent N-lauroyl sarcosine sodium salt and sodium carbonate, purifi- cation by two-dimensional (2-D) gel electrophoresis, and protein iden- tification using mass spectrometry. Treatment with buffers containing ASB-14 and ZWITTERGENT 3-10 increased solubilization of B. hense- lae proteins, particularly proteins with basic pI. Three hundred and sixty-

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eight spots were detected from the sarcosine-insoluble outer membrane fraction; 94 distinct protein species were identified from 176 spots. In the outer membrane fraction from carbonate incubation, 471 spots were calculated and 259 spots were identified, which included 139 protein en- tries. There were six outer membrane proteins in the sarcosine-insoluble outer membrane fraction compared with nine outer membrane proteins from samples subjected to carbonate incubation. We used bioinformatic analysis to identify 44 outer membrane proteins by prediction of their domains and tertiary structures and documented the potential virulence factors. We established the 2-D reference maps of the outer membrane subproteome of B. henselae using the two different extraction methods, which were partly complementary to each other. Sodium carbonate ex- traction isolated low-abundance and basic proteins better than the lauroyl sarcosine sodium salt extraction, which enriched high-abundance porins.

Key words: Bartonella henselae; Outer membrane protein; 2-D gel electrophoresis; MS; Bioinformatics

INTRODUCTION

Bartonella spp is a genus of aerobic, fastidious Gram-negative, facultative intracellular bacilli. Nine of the 21 species identified cause the various Bartonella diseases in human: B. bacil- liformis, B. elizabethae, B. henselae, B. quintana, B. koehlerae, B. grahamii, B. vinsonii subsp Berkhoffii, and B. vinsonii subsp arupensis. Cat scratch disease (CSD) is a typically benign and self-limiting illness, mostly caused by B. henselae. B. henselae is detected in association with a wide range of wild and domestic feline hosts and has worldwide distribution. Wild and domestic cats serve as the natural reservoir for B. henselae, which establish long-term infections in eryth- rocytes typically without inducing disease symptoms. If transmitted to humans, who are only an incidental host, B. henselae may cause CSD, bacteremia with fever, bacillary angiomatosis and peliosis, endocarditis, and neuroretinitis. In recent years, studies of Bartonella pathogenesis have taken a major step forward. With regard to the pathogenicity factors of Bartonella, studies point to outer membrane proteins (OMPs) of Bartonella as being important, where they are essential for attachment, invasion, and survival within host cells, as seen in other Gram-negative human pathogenic bacteria (Dehio, 2004). Since OMPs are the interface between the bacterium and the host cells, the outer membrane (OM) and individual OMPs are targeted in developments of diagnostic markers and vaccines against many bacterial diseases. The sarcosine-insoluble OM fraction of B. henselae and B. quintana was ex- amined in more detail. By means of two-dimensional gel electrophoresis (2-DE) combined with matrix-assisted laser desorption-ionization time-of-flight-mass spectrometry (MALDI-TOF-MS), seven OMPs of B. henselae and ten OMPs of B. quintana were previously identified (Rhomberg et al., 2004; Boonjakuakul et al., 2007). Two-dimensional gel electrophoresis is the main platform used in proteomics and has been used in studies on profiling of membrane proteins. Although membrane proteins have been underrepresented in 2-DE gels due to difficulties in extracting and solubilizing them in the isoelectrofocusing (IEF) sample buffer, comparatively high amounts of soluble OMPs, which are potential candidates for diagnosis and vaccination, have been represented in 2-DE gels.

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In recent years, advances in the solubilization of intractable proteins have prompted the proteomic analysis of OMPs, especially in various bacteria including Escherichia coli (Molloy et al., 2000), Aeromonas salmonicida (Ebanks et al., 2005) and Dickeya dadantii (Babujee et al., 2007). Because of the importance of OMPs for the molecular basis of pathogenesis and diagnosis and advances of 2-DE technique, we focused on these fractions and developed a technique for 2-DE of Bartonella OMPs using carbonate incubation compared with sodium lauryl sarcosine incubation for evaluat- ing the differentiation of the two OMP extraction methods, followed by improved solubilization conditions for array by 2-DE using the zwitterionic detergent ZWITTERGENT 3-10, ASB-14 and immobilized pH gradients. In addition, by exploiting the genome sequence of B. henselae strain Houston-1, we scanned in silico for proteins predicted to be localized at the cell surface and com- pared predicted and experimentally identifiedB. henselae OMPs.

MATERIAL AND METHODS

Strains and culture conditions

B. henselae strain Houston-1 (ATCC 49882) was routinely grown on tryptic soy agar containing 5% defibrinated sheep blood (TSB agar) in a humidified atmosphere with 5% CO2 at 37°C and harvested for protein preparation after 6 days.

Sample preparation of Bartonella OMPs

Isolation of cell membranes by N-lauroyl sarcosine sodium salt (LSS) method

The isolation of cell membranes using the LSS (Merck, Germany) method was performed according to Rhomberg’s modified procedure (Rhomberg et al., 2004). In brief, 5 × 108-1 × 109 colony-forming units/mL bacteria were harvested from 6-10 TSB agar plates, washed in low-salt phosphate-buffered saline (3 mM KCl, 1.5 mM KH2PO4, 68 mM NaCl, 9 mM NaH2PO4), and pelleted twice by centrifugation at 2500 g for 10 min at 4°C. The pellet was resuspended in 4 mL hyperosmolar buffer (0.2 M Tris, pH 8.0, 0.5 M sucrose, 250 µg/mL lysozyme, 1 mM EDTA) and incubated at 4°C for 1 h. Subsequently, one tablet protease inhibitor cocktail (Roche, Germany) was added, and bacteria were lysed by repeated sonication using a Bandelin HD3200 (single bursts at a 40% amplitude level for no longer than 5 s with cooling in ice-cold water to avoid heat dena- turation), until the solution turned translucent. Cell debris was removed by centrifugation for 30 min at 2500 g at 4°C. The supernatant was then cleared by centrifugation in a HITACHI CP80MX ultracentrifuge with a P55AT-784 rotor for 90 min at 100,000 g at 4°C. The resulting total mem- brane pellet was resuspended in a lysis buffer (10 mM HEPES, pH 7.4, 1% (w/v) lauryl sarcosine), incubated at room temperature for 20 min, and pelleted by ultracentrifugation under the same con- ditions. The lauryl sarcosine-insoluble pellet that contains the outer membrane-peptidoglycan com- plex was washed twice in 10 mM HEPES, pH 7.4, to remove residual detergent and centrifuged at 25,000 g for 30 min at 4°C; the washed pellet was then stored at 70°C.

Isolation of cell membranes by sodium carbonate (Na2CO3 ) method

The isolation of cell membranes using the Na2CO3 method was performed according

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br D.M. Li et al. 1792 to the procedure modified by Molloy et al. (2000). Briefly, harvesting and washing of bacteria were performed as in the preceding procedure. The pellet was resuspended in 10 mL 50 mM Tris-HCl buffer, pH 7.5, supplemented with the protease inhibitor cocktail. Bacteria were lysed by repeated sonication using a Bandelin HD3200 (single bursts at a 40% amplitude level for no longer than 5 s with cooling in ice-cold water to avoid heat denaturation), until the solution turned translucent, and the unbroken cells were removed by centrifugation at 2500 g 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 50 mL and stirred slowly for 1 h at 4°C. The carbonate-treated membranes were col- lected by ultracentrifugation in a HITACHI CP80MX P55AT-784 rotor at 100,000 g for 1 h at 4°C. The supernatant was discarded and the membrane pellet was resuspended and washed in 2 mL 50 mM Tris-HCl buffer, pH 7.5. The pellet was collected by centrifugation at 100,000 g for 20 min (HITACHI CP80MX P55AT-784 rotor) at 4°C.

Protein solubilization and quantitation

Outer membranes were resuspended in the solubilization buffer (5 M urea, 2 M thio- urea, 2% CHAPS, 1% ZWITTERGENT 3-10, 1% ASB-14, 1% DTT). To avoid precipitation of urea, the suspension was then centrifuged at 16,000 g for 15 min at 23°C. The supernatant containing solubilized OMPs was collected, protein content quantified by the 2-D QUANT Kit (Amersham Biosciences, USA), and immediately used or stored at -70°C.

Two-dimensional electrophoresis

Isoelectric focusing electrophoresis

Proteins were separated in the first dimension using the IPGphorTM isoelectric focusing system (Amersham Biosciences) employing immobilized pH gradient strips (Amersham Biosci- ences), according to Amersham Biosciences manuals. The OMP samples were focused to their isoelectric points using 24 cm Immobiline™ DryStrip gels, pH 3-10 (Amersham Bioscience) under the following conditions: 30 V for 5 h, 60 V for 6 h, 100 V for 1 h, 300 V for 1 h, 600 V for 1 h, 1000 V for 1 h, and 8000 V for 11 h. For the sarcosine-insoluble OMP fraction, 600 μg protein was loaded, and for the OMP of sodium carbonate preparations, 800 μg was loaded. Following isoelectric focusing, the proteins were reduced and bound to sodium dodecyl sulfate (SDS) by equilibrating each strip for 15 min in 10 mL SDS equilibration buffer (50 mM Tris- HCl, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.007% (w/v) bromophenol blue) containing 100 mg dithiothreitol (added fresh before use). A second equilibration step in SDS equilibration buffer containing 480 mg iodoacetamide (added fresh before use) instead of dithiothreitol was performed.

SDS-PAGE

After equilibration, the immobilized pH gradient strips were loaded onto 12.5% (w/v) homogeneous acrylamide gels (1 mm thickness × 24 cm width × 19 cm length), sealed with

0.5% (w/v) agarose. Protein samples that contained 600 μg LSS and 800 μg Na2CO3 of the solubilized membrane proteins were separated by electrophoresis with the Ettan DALT II Sys- tem (Amersham Biosciences) at 2.5 W/gel for 30 min at 20°C, and then, the conditions were

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br Proteomic and bioinformatic analysis of Bartonella henselae 1793 changed to 18 W/gel and the gels run until the bromophenol blue dye migrated to about 1 cm from the bottom of the gel. The gels were kept in the fixing solution (dehydrated alcohol:glacial acetic acid:deionized water; 4:1:5) for 30 min, stained with Coomassie brilliant blue G-250 (Neuhoff et al., 1985) (Bio-Rad, USA) overnight, and destained with 1% glacial acetic acid.

Analysis of gels

Individual gels were visualized using an image scanner (Amersham Biosciences) with a transparency adapter using an 8-bit grayscale, 400 dpi. Images were analyzed using the Im- ageMaster™ 2-D Platinum 5.0 software.

Protein enzymolysis

All protein spots of stained gels were excised and gel digestion performed as previ- ously described (Zou et al., 2006). The gel pieces were washed with 100 μL 25 mM ammo- nium bicarbonate, pH 8.0 (Fluka, USA) for 20 min, followed by washing with 50 μL 30% ace- tonitrile (Sigma, USA) containing 0.1 M ammonium bicarbonate until the color disappeared, and vacuum freeze-dried. Lyophilized samples were reconstituted in digestion buffer consist- ing of 25 mM ammonium bicarbonate and 3 μL trypsin solution containing 20 μg/mL trypsin (Sigma) and incubated at 37°C for 16 h. Supernatants were collected and gel pieces extracted twice with a 40-μL extraction buffer containing 50% acetonitrile and 5% trifluoroacetic acid (Fluka) for 60 min each time. The peptide containing supernatant was transferred to a new microtiter plate and pre-frozen at -80°C for 2 h and then dried under vacuum.

MALDI-TOF-MS

Lyophilized samples were dissolved in a 2-μL solution containing deionized water, 50% acetonitrile and 0.5% trifluoroacetic acid, and 0.5 μL of the solubilized proteins was mixed with 0.5 μL matrix (saturated solution of α-cyano-4-hydroxyl-cinnamic acid; Sigma) on a sample plate; the droplet was then left to dry at room temperature. Protein identifications were carried out using a 4700 MALDI-TOF/TOF-MS (Applied Biosystems, Foster City, CA, USA). The mass spectra were obtained using the 4000 Series ExplorerTM software, version 3.0, in the positive ion reflector mode with a mass accuracy of approximately 20 ppm. The MALDI-MS was equipped with a 200- Hz frequency-tripled Nd:YAG laser operating at a wavelength of 355 nm. The MS spectra were acquired in the mass range between 800 and 4000 Da, using 1500 laser shots.

Database searching

The experimental MS data were matched to the corresponding virtual peptide mass database derived from the GPS ExplorerTM v3.6 software (Applied Biosystems) and the Mas- cot v2.1 software (Matrix Science, London, UK) search protein database and NCBInr database (ftp://ftp.ncbi.nih.Gov/blast/db/FAST/nr.gz) to produce an experimental data set consisting of peptide mass fingerprints. One missed tryptic cleavage, variable modified carboxymethyl, and oxidation were selected. Data were searched at 100 ppm peptide mass tolerance. Identifica- tions with a GPS confidence interval greater than 95% were accepted.

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br D.M. Li et al. 1794 In silico analysis

Theoretical mass, pI and grand average of hydropathy (GRAVY) values of OMP can- didates were calculated using the ProtParam tool (http://www.expasy.ch/tools/protparam.html) (Gasteiger et al., 2005). SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP/) was used to hypothesize N-terminal secretory signal peptides of the identified proteins. HMMTOP version 2.0 (Tusnády and Simon, 2001) (http://www.enzim.hu/hmmtop/index.html) was used to predict trans- membrane helices in the identified proteins. Predicted Subcellular Localization of OMPs of B. henselae strain Houston-1 was downloaded from the databases of PSORTdb (http://db.psort.org/) (Rey et al., 2005) and Proteome Analyst PA-GOSUB 2.5 (http://www.cs.ualberta.ca/~bioinfo/PA/ GOSUB/) (Lu et al., 2004). Potential domains were identified by use of InterPro (http://www.ebi. ac.Uk/interpro/) and Pfam 24.0 (http://pfam.sanger.ac.uk/). Secondary and tertiary structure predic- tions were performed using SOPMA (Geourjon and Deleage, 1995), MODBASE (http://modbase. compbio.ucsf.edu/ModWeb20-html/modweb.html) (Pieper et al., 2009) and SWISS-MODEL (http://swissmodel.expasy.org//SWISS-MODEL.html) (Arnold et al., 2006).

RESULTS

2-DE of LSS method and the in silico analysis of protein assignment

Spot pattern and resolution of the two independent gels from two replicate samples were highly reproducible (Appendix Figure 1). In total, about 368 protein spots were exhibited on the 2-DE gels, and 176 spots belonging to B. henselae were verified by MS and searching NCBI nr databases for the B. henselae ATCC 49882 genome sequence (Alsmark et al., 2004) (Figure 1).

Figure 1. Sarcosine insoluble Bartonella henselae Houston-1 outer membrane proteins (OMPs) separated by 2-DE using pH 3-10 IPG and 12.5% SDS-PAGE. Proteins were stained with Coomassie brilliant blue G-250; spots were excised individually and then identified by peptide mass fingerprints (PMF). Each number indicates a protein for which a PMF identity was obtained; these numbers correspond to the protein identities shown in Appendix Table 1 and Appendix Table 2. Proteins in circles are annotated as OMPs.

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The 176 spots (predicted pIs ranging from 4.81 to 9.33 and molecular mass ranging from 15.64 to 80.6 kDa) representing 94 different proteins belonged to 6 OMPs, 6 periplasmic (PP) and inner membrane proteins (IMPs), 59 cytoplasmic proteins (CPs) and 23 proteins with unknown subcel- lular localization. Of the identified proteins, 16 proteins possessed a GRAVY-value >0, indicating that some hydrophobic proteins were also isolated by this approach, including malate dehydroge- nase, a hydrophobic protein with the highest GRAVY value (0.311) (Appendix Table 1). To further characterize the proteins with unknown subcellular localization, in silico pre- dictions by means of SignalP, HMMTOP, Pfam, and PA-SUB were carried out. There were only 7 proteins whose location could be predicted, including 5 CPs, 1 IMP and 1 PP (Appendix Table 2).

2-DE of Na2CO3 method and the in silico analysis of protein assignment

Spot pattern and resolution of the two independent gels from two replicate samples were highly reproducible (Appendix Figure 2). In total, about 471 protein spots were detected and 259 spots belonging to B. henselae were verified by MS and searching NCBI nr databases for the B. henselae ATCC 49882 genome sequence (Alsmark et al., 2004) (Figure 2). The 259 spots (pre- dicted pIs ranging from 4.78 to 9.75 and molecular mass ranging from 15.7 to 89.0 kDa) representing 139 different proteins belonged to 9 OMPs, 8 PP and IMPs, 87 CPs and 35 proteins with unknown subcellular localization. Of the identified proteins, 28 proteins possessed a GRAVY-value >0, in- dicating that some hydrophobic proteins were also isolated by this approach, including malate dehydrogenase, a hydrophobic protein with the highest GRAVY value (0.311) (Appendix Table 3). By using SignalP, HMMTOP, Pfam, and PA-SUB for analysis of the proteins with unknown subcellular localization, there were 8 proteins with signal peptides and 15 proteins

Figure 2. Carbonate insoluble Bartonella henselae Houston-1 outer membrane proteins (OMPs) separated by 2-DE using pH 3-10 IPG and 12.5% SDS-PAGE. Proteins were stained with Coomassie brilliant blue G-250; spots were excised individually and then identified by peptide mass fingerprints (PMF). Each number indicates a protein for which a PMF identity was obtained; these numbers correspond to the protein identities shown in Appendix Table 3 and Appendix Table 4. Proteins in circles are annotated as OMPs.

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br D.M. Li et al. 1796 with 1 to 3 transmembrane helices in these proteins, of which 7 were assigned to CPs, IMP and PP (Appendix Table 4).

Comparison of MS identification results between LSS and Na2CO3 methods

In summary, six OMPs, including HbpA, HbpD, HbpC, Omp43, Pap31, and Plp, were identified using the LSS method. Besides, HbpA, HbpD, Omp43, Pap31, Plp, HutA, Omp89,

BH10650, and BH07600 were extracted by using the Na2CO3 method. The differences be- tween the two OMP purification methods are listed in Table 1.

Table 1. Comparison of mass spectrometry identification results between LSS and Na2CO3 methods.

Subcellular location LSS method specific Na2CO3 method specific Common Total Outer membrane 1 4 5 10 Periplasm and inner membrane 2 5 4 11 Cytoplasm 9 37 49 95 Unknown 9 20 14 43 Total 21 66 72 159

LSS = N-lauroyl sarcosine sodium salt method; Na2CO3 = sodium carbonate method.

Characteristics of B. henselae OMPs identified

We identified a total of 10 OMPs, including HbpA, HbpD, HbpC, Pap31, Omp43, Plp,

HutA, Omp89, BH10650, and BH07600, using the LSS and Na2CO3 methods (Table 2). Many of the OMPs were resolved into more than two protein species, although in some cases only a single-charged species was observed (e.g., HbpC), while for others (e.g., HbpA), multiple- charged protein species existed. Furthermore, 2-DE of LSS showed that two clusters of mass for HbpA and HbpD (25-30 and 68 kDa) existed. To investigate potential functions of these OMPs of B. henselae, the Pfam database was searched for accessory domains. The proteins HbpA, HbpD, HbpC, Pap31, and Omp43 have features of porins. Plp is peptidyl-prolyl cis-trans isomerase, containing isomerase and rotamase domains. HutA, heme receptor, was found to have a tonB-dependent receptor plug domain. Omp89, 798 AA, was an OMP of unknown function of B. henselae. The Pfam da- tabase indicated that there were five surface antigen variable number repeat domains (39- 106AA, 107-184AA, 187-275AA, 278-357AA, 360-433AA) in the N-terminus of the amino acid sequence and one surface antigen domain (463-798AA) in the C-terminus. It exhibited 74-91 and 56-58% identity to the corresponding protein of Bartonella spp and Brucella spp, respectively. The second structure prediction using SOPMA exhibited 24.94% alpha helix, 26.69% extended strand, 6.52% beta turn, and 41.85% random coil. BH07600, 159AA, was a putative uncharacterized protein with no functional anno- tation in Uniprot, and one SmpA_OmlA hit in the Pfam database. It is an OM lipoprotein exhibiting 78-84% amino acid identity to the homologous protein of Bartonella spp (based on the whole genome sequence of B. bacilliformis, B. tribocorum, B. quintana, and B. grahamii, available in the database) and 55-56% to that of Brucella spp. The second structure prediction exhibited 13.84% alpha helix, 30.82% extended strand, 11.95% beta turn, and 43.40% random coil by consensus prediction from multiple alignments.

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br Proteomic and bioinformatic analysis of Bartonella henselae 1797 15/16 22/23 22/23 30/31 20/21 22/23 28/29 36/37 24/25 22/23 28/29 (0.324) (0.953) (0.999) (0.997) (0.994) (0.997) (0.996) (0.560) (0.999) (0.996) (0.994) Cleavage

e analysis (1) (1) (1) (1) (1) (1) (1) Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes ite (P) (0.933) (0.997) (0.987) (0.994) SignalP s Expectation value. Expectation d c in silico Pfam 3.50E-11 4.40E-58 5.10E-91 3.40E-14 1.10E-19 2.50E-17 7.00E-19 2.00E-26 Yes 6.10E-38 9.80E-88 3.70E-171 (P) c (PF07244) Designations of cleavage site of signal Designations of cleavage e variable number (PF02530) lipoprotein Receptor Plug repeat (PF04355) Domain (PF07715) Porin_2 Outer membrane efflux protein (PF02321) Porin_2 Porin_2 Porin_2 Outer membrane Porin_2 TonB-dependent Surface antigen Rotamase Porin_2 Pfam ) (PF00639) ) ) (PF02530) ) ) (PF02530) ) ) (PF02530) ) (PF02530) ) ) ) (PF02530) ％ ％ ％ ％ ％ ％ ％ ％ ％ ％ ％ OM OM OM OM OM OM OM OM OM OM OM model E-value (100 (100 (100 (100 (100 (100 (100 (100 (100 (100 (99.6 PA-SUB

b OM OM OM OM OM OM OM OM OM OM (9.93) (9.94) (9.45) (9.93) 10.00) ( 9.93) (10.00) (10.00) (10.00) PSORTb Unknown

a (P) b

(P) -0.611 -0.254 -0.075 -0.125 -0.205 -0.158 -0.025 -0.064 -0.255 -0.442 -0.207 GRAVY 8.60 I 5.56, 5.71 6.19, 7.10, 8.10, 8.72 8.20-8.60 10.4 4.75 4.80, 5.44, 5.8, 6.53 5.98 7.27, 8.80 7.87, 8.12, p 5.59, 5.73 4.88, 5.37, 5.40 43.5 41.8 30.24 29.5-68.7 31.2-68.7 18.3 35.5 82.5 93.2 27.6 Mass 37.9 32.7, 31.0, (kDa) Experimental I p 8.53 9.71 4.90 5.37 9.06 9.12 8.95 9.34 9.01 5.82 5.37

44224.4 48299.3 25663.7 29916.6 30269.7 17635.4 29956.3 82612.4 88985.9 35596.8 30074.7 Mass Predicted

activity activity activity activity activity Porin Transporter UNA Porin Porin UNA Porin Receptor UNA Rotamase Isomerase, Porin activity Molecular

to to -terminal signal peptides (SignalP), predictions of subcellular localization (PSORTb, PA-SUB), and PA-SUB), -terminal signal peptides (SignalP), predictions of subcellular localization (PSORTb, N membrane, membrane, membrane integral membrane integral membrane membrane membrane membrane membrane Membrane Outer UNK Cell outer Cell outer Outer Cell outer Cell outer Outer UNK Cell outer membrane component function Cellular c Peptidyl-prolyl cis-trans isomerase 31 K major protein, Pap31 - B. henselae phage protein a protein d Omp43 Outer Pap31 Hemin-binding Hemin-binding Putative Hemin-binding Heme Outer Protein UniprotKB annotations Designations of subcellular localization based on PSORT-B. Numbers refer to localization probability (from 0 to 10). probability Numbers refer to localization based on PSORT-B. localization Designations of subcellular b

OMPs identified in this study by PMF using MALDI-TOF-MS and subsequent database query, including query, database subsequent and MALDI-TOF-MS using PMF by study this in identified OMPs

17CA3D Q9L7A9 Q6G2V1 Q6G5U1 UPI0000 Q94M28 Q6G4S7 Q6G487 Q6G3K7 Q6G4S8 Q6G471 Q8VQ24 No. Accession

BH12500 BH10650 BH02000 JC6528 AAK97307 BH02560 BH04810 BH07600 BH02550 BH04970 BH06280 Locus

Bartonella henselae Sequence coverage (%) 44/37/ 38/37/ 37 54/46/ 45/37 receptor 30/39 membrane 56/63/60 42/58/51 57 43/54 39 39/45/ 52/55/ 54 32 23/50/ 37/33/65/ List of Spot ID precursor 254 394 390/344/ 361/338/ 363 379/389/ 577/578 54/567 15/18/19 246/248/ 281 321/322 349/367/ 45/45/44 363 339/357/ 373/375/ 579 232 34/37/ 10/12/14/

Table 2. Table by assignment of Pfam domains (Pfam), predictions calculation of GRAVY. Designations of signal peptide predictions based on SignalP. Numbers Designations refer of to signal probability peptide assigned predictions to based the on identificationSignalP. of a signal peptide (from 0 to 1). Grand average of hydropathy (Kyte/Doolittle). Grand average of hydropathy (Kyte/Doolittle). Gene protein omp43 membrane UNA protein plp pap31 pap31 hbpA hbpD uncharacterized UNA protein protein hbpC a d = unannotated gene; UNK unknown; OM outer membrane. Numbers refer to probability assigned the cleavage site of a signal peptide (from 0 1). UNA peptide based on SignalP. hutA precursor omp89

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br D.M. Li et al. 1798

BH10650, 440AA, was a bona fide OMP of B. henselae, which was identified as a pre- sumptive outer membrane efflux protein (the OEP family) with Pfam (2 OEP domain hits), re- lated to the type I secretion OMP, TolC of Escherichia coli. It exhibited 62-89% and 50% identity to the corresponding protein of Bartonella spp and Brucella spp. The second structure prediction exhibited 69.77% alpha helix, 5.23% extended strand, 2.05% beta turn, and 22.95% random coil.

Analysis of the in silico OM subproteome of B. henselae

PSORTdb (Rey et al., 2005), Proteome Analyst PA-GOSUB 2.5 (Szafron et al., 2004) and UniProt were interrogated to retrieve all B. henselae proteins annotated as OMPs (includ- ing hypothetical proteins) (Table 3). Twenty-nine OMPs were predicted by PSORTdb, except for DNA topoisomerase I, which was a chromosome component, and Plp and BH07600 were not predicted but detected in our study. There were 38 predicted OMPs using PA-GOSUB, and a total of 44 proteins were located on the outer membrane after removing the repetitive pro- teins in the two predicted results and UniProt. Of the 44 OMPs with pI 4.94-10, the predicted molecular mass ranged from 11.70 kDa for VirB3 to 323.67 kDa for BadA1, and there were 7 of these proteins with predicted molecular mass >100 kDa and 19 OMPs with predicted pI >9. Forty-one OMPs were predicted to be hydrophilic using the ProtParam tool, and 29 gene prod- ucts were predicted by SignalP to possess an N-terminal secretion signal. To further substantiate this analysis, in silico predictions by means of Pfam, MODBASE, and SWISS-MODEL were carried out. Table 3 lists the 44 predicted OMPs, whose putative function was predicted based on domain and homology search results, including adhesion-associated proteins (e.g., BadA1 (Riess et al., 2007), filamentous hemagglutinin, etc.), autotransporters in the type V secretion systems (e.g., BH13140, BH13180, etc.), type IV secretion system protein VirB3, tonB-de- pendent receptor (HutA), porins (e.g., HbpA, HbpB, HbpC, and HbpD), hemolysin activation protein Hec, peptidoglycan-associated lipoprotein Pal transferase (BH11350), and others.

DISCUSSION

The bacterial OMPs mediating adhesion and host immune evasion between Bartonella and its hosts are critical targets of the host’s immune response, often having diagnostic relevance, and are potential candidate antigens for vaccine developments. To date, there are only two studies that identified Bartonella surface proteins, including OMPs, using proteomic methods, namely 2-DE and MALDI-TOF-MS (Rhomberg et al., 2004; Boonjakuakul et al., 2007). These two reports mostly focused on the OM compartment prepared with lauryl sarcosine in order to identify poten- tial pathogenicity factors of B. henselae (Rhomberg et al., 2004) and to identify the predominant B. quintana antigens (Boonjakuakul et al., 2007). Seven proteins (of 53 proteins analyzed) and 10 proteins (of 60 proteins analyzed), respectively, were identified as prototypical OMPs; however, PSORTb predicts a significantly higher number of OMPs in B. henselae. Thus, our aims were to develop a better method for the isolation of Bartonella OMPs and to further describe the OM sub- proteome of B. henselae based on 2-DE gels. Molloy et al. (2000) identified 21 of 26 (80%) of the predicted integral OMPs of E. coli that are annotated in SWISS-PROT release 37 using sodium carbonate extraction, which was the more efficient method for the isolation of Bartonella OMPs. We analyzed the OM proteome of B. henselae by using the sarcosine- and carbonate- insoluble OM fraction (Figure 3). We identified six sarcosine-insoluble OMPs including HbpA,

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br Proteomic and bioinformatic analysis of Bartonella henselae 1799 e Collagen-binding, cell adhesion Cell adhesion, type v secretion Cell adhesion, type v secretion Cell adhesion, type v secretion tonB-dependent receptor, ion transport Autotransporter Bacterial cell division protein Autotransporter Autotransporter Protein transport Protein transport Protein transport Protein transport Cell adhesion Autotransporter Protein transport Protein transport UNK Function of homologu Cell adhesion Continued on next page left-handed beta-roll (1p9ha) (PF05860)/beta-helix (1rwr) (PF05860)/beta-helix (1rwr) pertussis beta helix (1daba) pertussis beta helix (1daba) Hep_Hag (PF05658), HIM (PF05662)/ Fil_haemagg (PF05594), Haemagg_act Fil_haemagg (PF05594), Haemagg_act Fil_haemagg (PF05594), Haemagg_act beta-domain (PF03797)/ Surf_Ag_VNR (PF07244)/2qdzA (PF00593), TonB_dep_Rec Plug (PF07715) maltoporin (2fcp) Pertactin (PF03212),Autotransporter (PF03797)/ Autotransporter Pertactin (PF03212), bac_surface_ag (PF01103), bac_surface_ag (PF01103)/UNK UNK (PF05860)/beta-helix (1rwr) Autotransporter beta-domain (PF03797)/ beta-barrels (1uyn) Transmembrane SPOR (PF05036)/transmembrane, inner membrane Autotransporter beta-domain (PF03797/ beta-barrels (1uyn) Transmembrane Autotransporter beta-domain (PF03797)/ beta-barrels (1uyn) Transmembrane beta-barrels (1uyn) Transmembrane domain (PF08479), ShlB (PF03865)/ POTRA beta barrel (2qdz) domain (PF08479), ShlB (PF03865)/ POTRA beta barrel (2qdz) domain (PF08479), ShlB (PF03865)/ POTRA beta barrel (2qdz) domain (PF08479), ShlB (PF03865)/ POTRA beta barrel (2qdz) Pattern (Pfam,ID)/ Tertiary (ModBase,ID) Tertiary Pertactin (PF03212), Autotransporter (PF03797)/ Pertactin (PF03212),

Pathogenesis Binding Binding Binding Receptor UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK Molecular function/ biological process Cell adhesion

-/0.954 -/0.905 -/0.934 -/0.935 0.933/- 1/0.893 score SignalP/ SecretomeP -/0.921 -/0.865 -/0.891 1/0.939 1/0.926 1/0.947 1/0.926 0.732/ 0.474 -/0.582 -/0.715 -/0.605 -/0.614 0.793/0.15

-0.533 -0.28 -0.302 -0.297 -0.255 -0.611 GRAVY -0.332 -0.147 -0.983 -0.413 -0.317 -0.391 -0.383 -0.133 -0.138 -0.131 -0.135 -0.137 -0.171 I

p 5.19 6.22 5.71 5.75 9.01 9.34

5.0 6.34 4.94 8.44 8.71 8.56 7.65 9.47 9.31 9.16 9.16 9.0 9.54

Mass 323674.3 280386.7 278994.6 278069 88985.9 82612.4

138970.7 99331.2 95450.4 95156.1 94913.9 94426.9 93092.5 71579.9 66848.0 66818.9 66788.8 65813.7 62928.0

localization

Subcellular Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Locus BH01510 BH06550 BH07150 BH06670 BH06280 BH04970 BH05490 BH06590 BH09990 BH05510 BH13140 BH13180 BH05500 BH00450 BH07920 BH06660 BH07140 BH06540 BH04050

badA1 fhaB1 fhaB4 fhaB2 omp89 hutA Gene UNA UNA UNA UNA UNA UNA UNA UNA UNA UNA UNA UNA UNA

adhesin OMP list of bioinformatic analysis using PSORTdb, Proteome Analyst PA-GOSUB 2.5 and the proteins annotated as OMPs in UniProt. Analyst PA-GOSUB Proteome list of bioinformatic analysis using PSORTdb, OMP Protein Surface protein / Bartonella hemagglutinin hemagglutinin hemagglutinin Hypothetical protein BH05490 Hypothetical genomic island Hypothetical protein BH09990 Hypothetical protein BH05510 Probable surface protein Hypothetical protein BH13180 Hypothetical protein BH05500 Outer membrane protein Heme receptor precursor Hypothetical Hemolysin activation protein hec Hemolysin activator protein hec Hemolysin activator protein hec Hemolysin activator protein hec Hypothetical protein ins Table 3. Table Orig Psortb Filamentous Filamentous Filamentous protein protein BH04050

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br D.M. Li et al. 1800 e Cell adhesion, type v secretion Peptidyl-prolyl cis-trans isomerase UNK Lipid binding, transporter activity UNK UNK UNK UNK Peptidoglycan binding, tol system UNK Adhesins, type v secretion hemagglutinin activity, isomerase activity activity, Adhesion, pathogenesis UNK UNK UNK Function of homologu Transport UNK Transferase Continued on next page Pal E. coli alpha helical barrel, beta barrel (1ek9) Beta-helix (1rwr) Rotamase (PF00639)/piC-type peptidyl-prolyl OM_YfiO/ UNK tetratricopeptide-like helical (1qsaA) cis-trans isomerase domain (3gpkA) autotransporter (2gr8) Trimeric UNK (PF04355)/2pxg SmpA_OmlA UNK Outer membrane efflux protein (PF02321)/ beta-barrel (1wp1) Porin_2 (PF02530)/beta barrel (1p4t) Porin_2 (PF02530)/beta barrel (1p4t) Porin_2 (PF02530)/beta barrel (1p4t) Porin_2 (PF02530)/beta barrel (1p4t) (PF00691)/ OmpA Protein (1oap) UNK OstA-like protein (PF03968), OstA_C (PF04453) Fil_haemagg (PF05594) beta-helix (1rwr) Pattern (Pfam,ID)/ Tertiary (ModBase,ID) Tertiary Outer membrane efflux protein (PF02321)/ Porin_2 (PF02530)/UNK domain (PF01464)/ SLT Transglycosylase

transport activity Porin activity Porin activity Porin activity Porin activity Binding Lipoprotein Isomerase, substance, outer Binding Lipoprotein Rotamase UNK UNK UNK UNK Molecular function/ biological process Lipid binding, Transporter Porin activity Lipoprotein Response to organic membrane Transmembrane

1/0.949 0.994/0.887 1/0.883 1/0.946 1/- 0.658/- 1/- -/0.952 -/0.752 0.699/0.210 score SignalP/ SecretomeP 0.735/0.283 0.994/0.193 1/0.867 -/0.827 0.910/ 0.068 0.999/0.598 0.987/ 0.090 0.873/0.293 0.964/0.940

-0.304 -0.158 -0.064 -0.205 0.231 -0.047 -0.442 -0.502 -0.07 -0.437 GRAVY -0.198 -0.075 -0.254 -0.437 -0.061 -0.442 -0.025 0.114 -0.397 I

p 9.25 9.06 8.95 5.37 9.36 8.9 5.82 5.79 5.93 9.53

9.77 9.71 8.53 5.17 7.64 9.08 9.12 9.52 6.24

Mass 41403.2 30269.7 29956.3 29916.6 29562.3 19369 35596.8 60592.9 70211.1 34079.1 54338.9 48299.3 44224.4 16819.7 13230.1 90180.5 17635.4 18454.5 89053.5 localization

Subcellular Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Outer membrane Locus BH02570 BH04810 BH02550 BH02560 BH07930 BH14790 BH02000 BH06750 BH07940 BH11160 BH15480 BH10650 BH12500 BH01500 BH01770 BH05430 BH07600 BH11350 BH13020

hbpB hbpD hpbC hbpA fhaB6 pal plp fhaB3 fhaB7 comL Gene UNA UNA UNA UNA UNA UNA UNA UNA UNA Continued. Protein Hypothetical protein BH15480 Outer membrane protein Omp43 precursor Hemin binding protein b Hemin binding protein d Hemin binding protein c Hemin binding protein a associated Peptidyl-prolyl cis-trans isomerase Surface protein Outer membrane lipoprotein Expressed protein Filamentous hemagglutinin Putative uncharacterized protein Filamentous hemagglutinin Competence lipoprotein comL Transmembrane protein Putative uncharacterized protein

ins Table 3. Table Orig Filamentous hemagglutinin Peptidoglycan- lipoprotein PA-GOSUB

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br Proteomic and bioinformatic analysis of Bartonella henselae 1801 e Autotransporter, type V pathway V type Autotransporter, pathway V type Autotransporter, pathway V type Autotransporter, UNK Function of homologu UNK UNK Autotransporter beta-domain (PF03797)/ beta-barrels (1uyn) Transmembrane Autotransporter beta-domain (PF03797)/ beta-barrels (1uyn) Transmembrane Autotransporter beta-domain (PF03797)/ beta-barrels (1uyn) Transmembrane UNK Pattern (Pfam,ID)/ Tertiary (ModBase,ID) Tertiary OMP_19 (PF06604)/UNK

UNK UNK UNK Virulence T4SS determinant, Molecular function/ biological process Outer membrane Lipoprotein

0.951/0.918 0.170/0.100 score 0.963/0.934 SignalP/ SecretomeP 1/0.878 -/0.716 0.747/0.923

-0.455 0.076 -0.239 GRAVY -0.321 -1.091 -0.097 I 4.95 10 p 4.96

8.8 9.64 8.79 155788.2 11704.7

Mass 214343.4 85622.8 42210.9 19595.2 localization Outer membrane Outer membrane Subcellular Outer membrane Outer membrane Outer membrane Outer membrane BH13120 BH13270 Locus BH13030 BH13160 BH14510 BH16590

UNA arp virB3 UNA UNA UNA

Gene

Continued. Putative Putative uncharacterized protein Outer membrane uncharacterized Probable surface protein Acidic repeat protein secretion IV Type system protein virB3 Protein ins Table 3. Table The list was subdivided into 3 sections: the OMPs from Psortdb, the OMPs from Proteome Analyst PA-GOSUB 2.5, the OMPs from UniProt. UNK = unknown; Analyst PA-GOSUB The list was subdivided into 3 sections: the OMPs from Psortdb, the OMPs from Proteome = unannotated gene. UNA protein lipoprotein UniProt Orig

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br D.M. Li et al. 1802

HbpD, HbpC, Omp43, Pap31, and Plp. Sodium carbonate treatment resulted in the isolation of four additional proteins, including Omp89, HutA, BH10650, and BH07600 (Figure 3). Differ- ent 2-DE maps were obtained for OM proteins extracted using the LSS and carbonate methods. LSS OM protein fraction was enriched with the Hbp proteins, which are the products of the hbpCAB/D locus. HbpA and HbpD had the highest abundance among all identified OMPs and were detected as a cluster of proteins with different molecular mass of 25-30 kDa, correspond- ing to studies by Rhomberg et al. (2004) and Chenoweth et al. (2004); however, the higher molecular mass forms of 68 kDa were also expressed. The carbonate method provided bet- ter conditions to detect the alkaline protein fraction, including Omp89, HutA, BH10650, and BH07600, which were alkaline proteins except for the common OMPs with the LSS methods.

Figure 3. Workflow for sample preparation and numbers N( ) of identified proteins and outer membrane proteins (OMPs) of Bartonella henselae.

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br Proteomic and bioinformatic analysis of Bartonella henselae 1803

The hemin-binding proteins, HbpA, HbpD and HutA, cell adhesion-associated protein Omp43 (Burgess et al., 2000), Omp89 and Plp have previously been identified in the sarco- sine-insoluble OM fraction of B. henselae (Rhomberg et al., 2004). Hbp family members, which are homologous to B. henselae Pap31 (Zimmermann et al., 2003), Brucella Omp31, Agrobacterium tumefaciens Omp25 and Neisseria meningitides opacity proteins, contain eight β-barrel transmembrane domains and four extracellular loops (Minnick et al., 2003). HbpA plays an active role in hemin acquisition (Carroll et al., 2000), and the other Hbp family mem- bers constitute a compensatory iron-acquisition system important for B. henselae survival (Burgess et al., 2000). Omp43, which is a porin, and OM antigen and Omp89, which appeared to produce the strongest humoral response (Chenoweth et al., 2004), as the three major puta- tive Fn-binding proteins, may play an important role in B. henselae adherence to and inva- sion of host cells (Dabo et al., 2006). We identified two hypothetical proteins, BH07600 and BH10650. BH07600, an OM lipoprotein, is possibly involved in maintaining the structural integrity of the cell envelope (Ochsner et al., 1999). BH10650 is a putative outer membrane efflux protein, which forms trimeric channels involved in the export of a variety of substrates in Gram-negative bacteria, such as E. coli (Higgins et al., 2004). Outer membrane protein TolC is the prototypical OM channel component involved in multi-drug resistant and type I secretion, which belongs to the resistance-nodulation-cell division protein family involved in the transport of various drugs across the cell envelope in bacteria; it exhibits broad substrate specificity and acts like an efflux pump (Yang et al., 2003; Hernandez-Mendoza et al., 2007). Antibiotic substrates of the drug efflux pump include erythromycin, tetracycline, chloram- phenicol, ampicillin, and rifampin (Okusu et al., 1996; Zgurskaya and Nikaido, 1999), which are the preferred antibiotic treatments for Bartonella infections. Bartonella are susceptible to many antibiotics in vitro (including β-lactams, aminoglycosides, chloramphenicol, tetracy- clines, macrolides, and rifampicin) (Maurin et al., 1995). Conversely, clinical data indicate that the bacteria may be more resistant in vivo. For Bartonella, in which potential mechanisms of drug efflux are not known, the other antibiotic resistance systems such as tetracycline resis- tance protein and macrolide-specific efflux proteins were identified inby silico genome analy- sis of Bartonella (Biswas et al., 2008), suggesting that some drug resistance systems including BH10650 may play an important role in vivo or in overexpressions. One of the major problems encountered in membrane protein studies are the difficulty to solubilize these hydrophobic proteins using solutions compatible with their subsequent separation, digestion, and/or identification by MS analysis (Tan et al., 2008). The application of zwitterionic detergents such as ZWITTERGENT 3-10 and ASB-14 significantly increased the solubilization of the OMPs in the IEF sample buffer and resulted in the identification of more protein spots, especially in the alkaline pI range. Although a higher yield of extracted proteins was detected on the 2-DE gels, their MS identification may be incomplete due to i) the inherent lack of trypsin cleavage sites in these proteins, ii) poor accessibility to proteases in these regions, iii) tendency of the hydrophobic domains to aggregate and precipitate after the removal of SDS, and iv) difficulties in extracting these proteins (Tan et al., 2008). It has been well documented that cytoplasmic and periplasmic components and inner membrane proteins are present as contaminants in the OM preparation. This partially reflects their sur- face localization (e.g., ATPase, chaperon GroEL, FAD, NADH/NADPH-proteins), as well as the tight association between the inner and outer membranes or a technical artifact. The other components from the Bartonella cells besides OMPs were shown on the 2-DE profiles as we

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br D.M. Li et al. 1804 increased the sample amounts compared with the previous studies (Molloy et al., 2000; Rhom- berg et al., 2004) to detect more low-abundance OMPs. Despite all this, the inherent limitation of solubility of membrane proteins in 2-DE (Jungblut et al., 2010) rendered our results with distinct differences relative to the OMPs from the in silico genome analysis. There were 44 OMPs predicted by the two predictor programs along with the Uniprot. There could have been multi-transmembrane alpha helix or β-barrel proteins in the 162 hydrophobic proteins from the predicted 540 proteins with unknown local- ization (data not shown). Therefore, it is possible that more B. henselae OMPs will be identi- fied when optimized methods for their isolation become available. Among the 44 predicted OMPs, 7 OMPs with molecular mass ≥100 kDa, including ad- hesin BadA, filamentous hemagglutinin and autotransporters, and 6 OMPs with Ip ≥9.5, includ- ing VirB3, ComL and hypothetical proteins, which exceeded the resolution capacity of the pH 3-10 IPG strip, were lost from the 2-DE maps. For the other hypothetical proteins, further study is required to understand the lack of their detection by 2-DE. Monitoring of mRNA expression may determine if these proteins are transcribed and processed, and if they could be therefore expected on 2-DE gels. A membrane proteome is the entire complement of membrane proteins present in a cell at a given condition and time. Synthesis of proteins will be induced or repressed in the situation of growing on the artificial culture medium, especially for intracellular bacteria pathogens (Cash, 2006), and therefore, the actual number of proteins, which are the sort of proteins associated with adhesion and invasion observed on a 2-DE gel, is expected to be much lower. For settling the problems of 2-DE in separating membrane proteins, various alternative techniques in gel-based (e.g., blue native-PAGE [BN-PAGE], clear-native-PAGE [CN-PAGE], enzyldimethyl-n-hexadecylammonium chloride [BAC], and SDS/SDS [dSDS]-PAGE) and LC-based (free-flow electrophoresis [FFE] and multidimensional protein identification tech- nology [Mud- PIT]) separation platforms have been employed in membrane proteomics. We established a 2-DE map of the outer membrane subproteome of B. henselae using the sodium carbonate extraction method and the lauryl sarcosine extraction method. We iden- tified a total of 10 proteins present in the OM (HbpA, HbpC, HbpD, Pap31, HutA, OMP89, OMP43, Plp, and two novel proteins BH10650 and BH07600) of B. henselae. To complement the inventory of the OMPs, a bioinformatic approach was performed with two programs, immu- nogenic proteins, adhesion-associated proteins, type I secretion system-associated protein outer membrane efflux protein, type IV secretion system-associated proteins VirB (Schulein et al., 2005), type V secretion system-associated protein autotransporters, type VI secretion-associated protein Pal and hemin-binding surface proteins, thus maximizing the number of protein species in the OM proteome. Although the analysis of detection and identification of membrane proteins remains a difficult task, 2-DE was used to display the soluble and highly abundant OMPs. These findings may help in the further characterization ofB. henselae OMPs immunogenic candidates. Some of these proteins merit further investigations because of their immunodominant properties, conservation across serotypes, structure and functions, and in vivo expression in hosts.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Marina Eeremeeva (Viral and Rickettsial Zoono- ses Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA) for critical read- ing the manuscript. Peijing Li, Fanliang Meng and Peng Wang (National Institute for Commu-

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br Proteomic and bioinformatic analysis of Bartonella henselae 1805 nicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention) are also thanked for supporting techniques. Research supported by grants for major projects of prevention and control technology for major infectious diseases such as AIDS and viral hepatitis (#2008ZX10004-002) and for the study of surveillance strategies and methods and diagnostic techniques of major vectors and vector-borne disease pathogens (#2008ZX10004- 010) from the Ministry of Science and Technology of the People’s Republic of China. The authors have declared no conflict of interest.

REFERENCES

Alsmark CM, Frank AC, Karlberg EO, Legault BA, et al. (2004). The louse-borne human pathogen Bartonella quintana is a genomic derivative of the zoonotic agent Bartonella henselae. Proc. Natl. Acad. Sci. U. S. A. 101: 9716-9721. Arnold K, Bordoli L, Kopp J and Schwede T (2006). The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22: 195-201. Babujee L, Venkatesh B, Yamazaki A and Tsuyumu S (2007). Proteomic analysis of the carbonate insoluble outer membrane fraction of the soft-rot pathogen Dickeya dadantii (syn. Erwinia chrysanthemi) strain 3937. J. Proteome Res. 6: 62-69. Biswas S, Raoult D and Rolain JM (2008). A bioinformatic approach to understanding antibiotic resistance in intracellular bacteria through whole genome analysis. Int. J. Antimicrob. Agents 32: 207-220. Boonjakuakul JK, Gerns HL, Chen YT, Hicks LD, et al. (2007). Proteomic and immunoblot analyses of Bartonella quintana total membrane proteins identify antigens recognized by sera from infected patients. Infect. Immun. 75: 2548-2561. Burgess AW, Paquet JY, Letesson JJ and Anderson BE (2000). Isolation, sequencing and expression of Bartonella henselae omp43 and predicted membrane topology of the deduced protein. Microb. Pathog. 29: 73-80. Carroll JA, Coleman SA, Smitherman LS and Minnick MF (2000). Hemin-binding surface protein from Bartonella quintana. Infect. Immun. 68: 6750-6757. Cash P (2006). Analyzing bacterial pathogenesis at level of proteome. Methods Biochem. Anal. 49: 211-235. Chenoweth MR, Greene CE, Krause DC and Gherardini FC (2004). Predominant outer membrane antigens of Bartonella henselae. Infect. Immun. 72: 3097-3105. Dabo SM, Confer AW, Saliki JT and Anderson BE (2006). Binding of Bartonella henselae to extracellular molecules: identification of potential adhesins.Microb. Pathog. 41: 10-20. Dehio C (2004). Molecular and cellular basis of Bartonella pathogenesis. Annu. Rev. Microbiol. 58: 365-390. Ebanks RO, Goguen M, McKinnon S, Pinto DM, et al. (2005). Identification of the major outer membrane proteins of Aeromonas salmonicida. Dis. Aquat. Organ. 68: 29-38. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, et al. (2005). Protein Identification and Analysis Tools on the ExPASy Server. In: The Proteomics Protocols Handbook (Walker JW, ed). Humana Press, Clifton, 571-607. Geourjon C and Deleage G (1995). SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput. Appl. Biosci. 11: 681-684. Hernandez-Mendoza A, Quinto C, Segovia L and Perez-Rueda E (2007). Ligand-binding prediction in the resistance- nodulation-cell division (RND) proteins. Comput. Biol. Chem. 31: 115-123. Higgins MK, Eswaran J, Edwards P, Schertler GF, et al. (2004). Structure of the ligand-blocked periplasmic entrance of the bacterial multidrug efflux protein TolC.J. Mol. Biol. 342: 697-702. Jungblut PR, Schiele F, Zimny-Arndt U, Ackermann R, et al. (2010). Helicobacter pylori proteomics by 2-DE/MS, 1-DE- LC/MS and functional data mining. Proteomics 10: 182-193. Lu Z, Szafron D, Greiner R, Lu P, et al. (2004). Predicting subcellular localization of proteins using machine-learned classifiers.Bioinformatics 20: 547-556. Maurin M, Gasquet S, Ducco C and Raoult D (1995). MICs of 28 antibiotic compounds for 14 Bartonella (formerly Rochalimaea) isolates. Antimicrob. Agents Chemother. 39: 2387-2391. Minnick MF, Sappington KN, Smitherman LS, Andersson SG, et al. (2003). Five-member gene family of Bartonella quintana. Infect. Immun. 71: 814-821. Molloy MP, Herbert BR, Slade MB, Rabilloud T, et al. (2000). Proteomic analysis of the Escherichia coli outer membrane. Eur. J. Biochem. 267: 2871-2881. Neuhoff V, Stamm R and Eibl H (1985). Clear background and highly sensitive protein staining with Coomassie blue dyes

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br D.M. Li et al. 1806

in polyacrylamide gels: a systematic analysis. Electrophoresis 6: 427-448. Ochsner UA, Vasil AI, Johnson Z and Vasil ML (1999). Pseudomonas aeruginosa fur overlaps with a gene encoding a novel outer membrane lipoprotein, OmlA. J. Bacteriol. 181: 1099-1109. Okusu H, Ma D and Nikaido H (1996). AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants. J. Bacteriol. 178: 306-308. Pieper U, Eswar N, Webb BM, Eramian D, et al. (2009). MODBASE, a database of annotated comparative protein structure models and associated resources. Nucleic Acids Res. 37: D347-D354. Rey S, Acab M, Gardy JL, Laird MR, et al. (2005). PSORTdb: a protein subcellular localization database for bacteria. Nucleic Acids Res. 33: D164-D168. Rhomberg TA, Karlberg O, Mini T, Zimny-Arndt U, et al. (2004). Proteomic analysis of the sarcosine-insoluble outer membrane fraction of the bacterial pathogen Bartonella henselae. Proteomics 4: 3021-3033. Riess T, Raddatz G, Linke D, Schafer A, et al. (2007). Analysis of Bartonella adhesin A expression reveals differences between various B. henselae strains. Infect. Immun. 75: 35-43. Schulein R, Guye P, Rhomberg TA, Schmid MC, et al. (2005). A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells. Proc. Natl. Acad. Sci. U. S. A. 102: 856-861. Szafron D, Lu P, Greiner R, Wishart DS, et al. (2004). Proteome analyst: custom predictions with explanations in a web- based tool for high-throughput proteome annotations. Nucleic Acids Res. 2: W365-W371. Tan S, Tan HT and Chung MC (2008). Membrane proteins and membrane proteomics. Proteomics 8: 3924-3932. Tusnády GE and Simon I (2001). The HMMTOP transmembrane topology prediction server. Bioinformatics 17: 849-850. Yang S, Clayton SR and Zechiedrich EL (2003). Relative contributions of the AcrAB, MdfA and NorE efflux pumps to quinolone resistance in Escherichia coli. J. Antimicrob. Chemother. 51: 545-556. Zgurskaya HI and Nikaido H (1999). Bypassing the periplasm: reconstitution of the AcrAB multidrug efflux pump of Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 96: 7190-7195. Zimmermann R, Kempf VA, Schiltz E, Oberle K, et al. (2003). Hemin binding, functional expression, and complementation analysis of Pap 31 from Bartonella henselae. J. Bacteriol. 185: 1739-1744. Zou Q, Yan X, Li B, Zeng X, et al. (2006). Proteome analysis of sorbitol fermentation specific protein in Vibrio cholerae by 2-DE and MS. Proteomics 6: 1848-1855.

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APPENDIX

Appendix Figure 1. Sarcosine-insoluble Bartonella henselae Houston-1 outer membrane proteins (OMPs) separated by 2-DE, using pH 3-10 IPG and 12.5% SDS-PAGE. Proteins were stained with Coomassie brilliant blue G-250. A and B were performed under the same conditions. Red circles indicate proteins annotated as OMPs.

Appendix Figure 2. Carbonate-insoluble Bartonella henselae Houston-1 outer membrane proteins (OMPs) separated by 2-DE, using pH 3-10 IPG and 12.5% SDS-PAGE. Proteins were stained with Coomassie brilliant blue G-250. A and B were performed under the same conditions. Proteins in red circles are annotated as OMPs.

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br D.M. Li et al. 1808 PSORTb Cytoplasmic (9.97) Cytoplasmic (9.26) Cytoplasmic (8.96) Cytoplasmic (8.96) (Probability) Cytoplasmic (8.96) Cytoplasmic (9.97) Cytoplasmic (9.26) Cytoplasmic (9.97) Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic (9.26) Cytoplasmic (8.96) Cytoplasmic (9.97) Cytoplasmic (9.97) Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic (9.26) Cytoplasmic (9.97) Cytoplasmic (9.97) Cytoplasmic (9.97) Cytoplasmic (9.97) Cytoplasmic (8.96) Cytoplasmic (8.96 ) Cytoplasmic (9.97) Cytoplasmic (8.96) Cytoplasmic (9.97) Cytoplasmic (8.96) Cytoplasmic (9.26) Cytoplasmic (8.96) Cytoplasmic (8.96)

0.143 0.025 0.103 0.249 0.136 -0.611 -0.611 -0.112 -0.145 -0.200 -0.354 -0.294 -0.173 -0.402 -0.225 -0.475 -0.146 -0.296 -0.303 -0.478 -0.024 -0.206 -0.539 -0.356 -0.166 -0.335 -0.358 -0.707 -0.092 -0.102 -0.209 GRAVY Continued on next page I p 4.94 6.05 5.79 5.18 5.30 6.56 5.53 5.36 6.34 9.33 6.54 5.94 6.33 5.46 6.53 5.17 5.61 7.16 5.62 5.14 5.17 5.30 5.61 6.18 4.97 5.56 5.45 5.88 6.34 6.66 Predicted

Mass 38255.5 49371.8 39415.6 62779.6 67181.0 33226.3 40426.0 80601.8 15644.1 33238.3 29349.1 40942.1 50764.8 26068.5 20856.1 22899.8 44693.8 47754.9 50539.9 53189.8 55499.2 42839.2 20152.7 20652.8 25145.2 19073.5 45213.3 49396.3 46509.7 36813.0

Houston-1 proteins identified in this study by PMF using MALDI-TOF-MS. (NADH) activity hydrolase activity synthase activity, regμlator activity other than amino-acyl groups protein binding activity, (glutamine-hydrolyzing) activity Molecular function / Biological process Elongation factor binding, dihydrolipoyl dehydrogenase activity FAD repressor activity Transcription binding, structural constituent of ribosome RNA binding, GTPase activity GTP UTP: glucose-1-phosphate uridylyltransferase activity codon specific release factor activity, Translation binding RNA 3’-5’-exoribonuclease activity, binding Sequence-specific DNA Heat shock protein binding, unfolded binding reductase Binding,enoyl-[acyl-carrier-protein] 3,4-dihydroxy-2-butanone-4-phosphate Peptidase binding, two-component response DNA Magnesium ion binding, orotate phosphoribosyltransferase activity binding, structural constituent of ribosome rRNA transferring acyl groups activity, Transferase Dihydrolipoyllysine-residue acetyltransferase binding, nucleoside-triphosphatase activity, ATP transcription factor activity Peptidyl-prolyl cis-transisomerase activity synthase Glutaminyl-tRNA binding, GTPase activity, GTP translation elongation factor activity elongation regμlator activity Transcription termination Translational Ribose-5-phosphate isomerase activity Response to stress Catalytic activity binding, biotin ATP Cysteine desulfurase activity, pyridoxal phosphate binding ligase activity Acyltransferase activity

Bartonella henselae dehydrogenase Cellular component Intracellular Cytoplasm UNA Ribosome Intracellular UNA Cytoplasm Cytoplasm UNA UNA UNA UNA UNA UNA UNA Ribosome UNA Pyruvate Intracellular UNA UNA Cytoplasm UNA Cytoplasm UNA UNA UNA UNA UNA UniprotKB annotations

UNA complex

repressor hrcA reductase 4-phosphate synthase regμlator synthase II acetyltransferase (E2) amidotransferase subunit B protein nusG Protein name Elongation factor EF-Tu Elongation factor EF-Tu Dihydrolipoyl dehydrogenase Heat-inducible transcription 30S ribosomal protein S1 GTP-binding protein typA UTP-glucose-1-phosphate uridylyltransferase Polynucleotide phosphorylase/ polyadenylase regulator Transcriptional Heat shock protein DnaJ Enoyl-[acyl-carrier-protein] 3,4-Dihydroxy-2-butanone response Two-component Orotate phosphoribosyltransferase 50S ribosomal protein L9 3-oxoacyl-[acyl-carrier-protein] Dihydrolipoamide factor Trigger (Asn/Gln) Aspartyl/glutamyl-tRNA Tu Elongation factor antitermination Transcription A Ribose-5-phosphate isomerase Biotin carboxylase Peptide chain release factor 1 tldD protein Nitrogen regulation protein Ribosome-recycling factor Small heat shock protein 3-oxoacyl-[acyl-carrier-protein] Nitrogenase co-factor glucosamine N-acyltransferase synthase synthesis protein nifS UDP-3-O-[3-hydroxymyristoyl]

Q9F653 Q67B06 Q6G564 Q6G490 Q6G463 Q6G449 Q6G441 Q6G403 Q6G5F8 Q6G5T7 Q6G3Z5 Q6G4C8 Q6G5C6 Q6G5C1 Q6G3C2 Q6G5H4 Q6G4X9 Q6G4X4 Q6G4D6 Q6G4D5 Q6G4D3 Q6G3Y8 Q6G3Y7 Q6G3Y0 Q8VQ23 Q6G3V6 Q6G3G8 Q6G3M3 Q6G4W1 Q8KHX9 Accession No. AAG24621 AAR21288 BH00550 BH00930 BH01700 BH01750 BH01970 BH02100 BH02210 BH04300 BH04310 BH04330 BH04380 BH04780 BH05050 BH05290 BH05370 BH05770 BH05850 BH05940 BH05950 BH06020 BH06050 BH06250 BH06290 BH06410 BH07300 BH07440 BH08050 BH08610 Locus List of the locations and functions sarcosine-insoluble

153/147 90 180 42/21/22 14/15 256 152/155 4 359 233 275/274 176/175 122 269 333 337/334 141 96 101/106 99/85 84 146/148 346 340 192/195/ 193 318 422/349/351/ 183/198/199/ 201/328/ 350 134 120 143 Spot ID

Appendix Table 1. Appendix Table tuf lpdA hrcA rpsA typA galU prfA pnp UNA dnaJ2 fabI2 ribA tldD UNA pyrE rplI fabF1 pdhC ntrX tig gatB tuf1 nusG frr lpxD rpiA ibpA2 fabF2 accC nifS1 Gene

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br Proteomic and bioinformatic analysis of Bartonella henselae 1809 PSORTb Cytoplasmic (9.97) Cytoplasmic (8.96) membrane (8.02) UNK Cytoplasmic membrane (9.46) Cytoplasmic (9.26) (Probability) Cytoplasmic (9.26) Cytoplasmic (8.96) Cytoplasmic (9.26) Cytoplasmic (9.97) Cytoplasmic (9.97) Cytoplasmic (9.97) Cytoplasmic (9.97) Cytoplasmic (9.97) Cytoplasmic (9.97) Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic UNK UNK UNK UNK Cytoplasmic (9.26) Cytoplasmic (9.97) Cytoplasmic (8.96) Cytoplasmic (9.97) Cytoplasmic (8.96)

0.311 0.311 0.010 0.053 0.084 0.303 -0.354 -0.078 -0.314 -0.315 -0.236 -0.193 -0.233 -0.553 -0.058 -0.082 -0.035 -0.206 -0.071 -0.036 -0.256 -0.106 -0.238 -0.139 -0.041 -0.327 -0.099 -0.054 GRAVY Continued on next page I p 6.04 5.53 5.03 5.47 4.85 6.63 5.12 6.18 5.90 6.21 5.44 6.41 5.07 5.75 5.47 5.61 6.20 5.25 6.31 9.56 6.66 6.84 5.14 5.10 6.45 6.61 5.40 Predicted

Mass 30111.3 30111.3 50119.6 50119.6 38009.1 35141.2 18849.5 52640.3 37648.5 21467.4 76284.9 26279.9 40529.1 56559.4 57560.8 42790.3 33642.8 47371.9 27835.7 37069.6 62352.3 50247.4 18154.9 28363.4 24398.8 57589.5 24961.8 52821.6 37515.8

O-methyltransferase activity Pyruvate dehydrogenase Molecular function / Biological process Glycerol metabolic process Peptidyl-prolyl cis-transisomerase activity Glutamate-ammonia ligase activity Transferase Nucleotidyltransferase, binding, adenylate kinase activity ATP binding, GTPase activity, GTP binding, endonuclease activity DNA binding, two-component DNA Aminomethyltransferase activity, Hydrolase Hydrolase, transferase Ligase binding L-malate dehydrogenase activity, Hydrolase activity ATPase binding, ATP or NADPH binding, NADP binding, GTPase activity GTP Aminopeptidase, hydrolase, protease Ribonucleoprotein, ribosomal protein Ribonucleoprotein, ribosomal protein (acetyl-transferring) activity electron carrier activity, oxidoreductase activity electron carrier activity, binding damaged DNA activity, translation elongation factor activity Protein-L-isoaspartate (D-aspartate) transaminase activity binding, unfolded protein binding ATP Fumarate hydratase activity aspartate-semialdehyde dehydrogenase activity NAD or NADH binding, binding, acid-amino acid ligase activity ATP ATPase binding, DNA-dependent ATP

response regμlator activity

Intracellular Cellular component UNA UNA Cytoplasm UNA Cytoplasm Cytoplasm Intracellμlar UNA Cytoplasm Cytoplasm Plasma membrane UNA UNA UNA Proton-transporting UNA Cytoplasm Cytoplasm Intracellular Ribosome Small ribosomal UniprotKB annotations two-sector ATPase two-sector membrane-bounded UNA UNA Cytoplasm acid Tricarboxylic cycle enzyme complex complex, catalytic domain organelle subunit

Cytoplasm

I,E subunit (D-aspartate) O-methyltransferase Pyruvate dehydrogenase Protein name Glycerol-inducible protein Peptidyl-prolyl cis-transisomerase DNA-directed RNA Adenylate kinase Exodeoxyribonuclease III D-glutamate-2,6-diaminopimelate ligase Chaperonin GroEL Fumarate hydratase synthase ATP Bifunctional purine termination Transcription Cell division protein FtsZ Leucyl aminopeptidase 50S ribosomal protein L10 Glutamine synthetase Protein recA Elongation factor G Cell cycle transcriptional Aminomethyltransferase ligase Succinyl-CoA Malate dehydrogenase transporter ATP-dependent Aspartate-semialdehyde 30S ribosomal protein S2 polymerase subunit alpha regulator CtrA subunit beta biosynthesis protein purH [ADP-forming] subunit beta factor rho E1 component alpha subunit dehydrogenase NADH dehydrogenase Protein-L-isoaspartate UDP-N-acetylmuramoylalanyl-

O33963 O69074 Q6G2J6 Q8L1Z6 Q6G392 Q6G312 Q6G306 Q6G294 Q6G5S5 Q6G2Z9 Q6G2Z3 Q6G2E9 Q6G2E5 Q6G3C0 Q6G5C9 Q9FDC7 Q6G2Y6 Q6G2V9 Q6G2V0 Q6G2Q0 Q6G1K7 Q6G5G4 Q6G3X7 Q8KQB3 Q6G1M1 Q6G1M0 Q6G1W9 Accession No. BH08910 BH10020 BH10090 BH10160 BH10230 BH10270 BH10300 BH10540 BH10570 BH10660 BH11290 BH12050 BH12840 BH13530 BH13550 BH15320 BH15970 BH16560 BH16570 BH16700 BH05750 BH08630 BH12890 BH11180 BH02040 BH06080 BH06220 Locus Continued. 302 189/186 348 117 194 187/188 335 6/10/9/151 278 316 110/91 407 168/173 70/72/78/74 130 87/95/ 88/104 60 172 196 126 200 408 184 18 138 356 246 Spot ID

Appendix Table 1. Appendix Table nuoE glpX ppiB1 glnA2 recA rpoA adk fusA xthA1 pcm2 murE ctrA gcvT groEL fumC atpD purH sucC mdh rho pdhA UNA asd ftsZ pepA rplJ rpsB Gene

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br D.M. Li et al. 1810 integral membrane PSORTb Periplasmic ( 9.44) (Probability) protein UNK Periplasmic (9.44) Periplasmic (10.00) UNK OM ( 9.93) OM (9.45) OM (10.00) OM (9.93) Outer membrane UNK UNK OM (9.93)

-0.21 0.026 -0.175 -0.166 -0.157 -0.205 -0.158 -0.064 -0.254 -0.442 -0.061 -0.292 -0.207 GRAVY I p 5.18 8.56 9.02 5.54 6.46 5.37 9.06 8.95 8.53 5.82 5.91 6.27 5.37 Predicted

Mass 54113.3 55511.6 55511.6 22750.9 50562.1 28179.4 28030.2 29916.6 30269.7 29956.3 44224.4 35596.8 34948.9 30074.7

Molecular function / Biological process Ribonucleoprotein, ribosomal protein Hydrolase, protease, serine protease Protein binding, serine-type activity Transporter Hydrolase Porin activity Porin activity Porin activity Porin activity Isomerase Rotamase endopeptidase activity Hydrolase Ligase, transferase Porin activity

cell membrane Cellular component Ribosome Periplasm UNA UNA Inner membrane, Cell outer membrane, Cell outer membrane, Cell outer membrane Membrane UNA UniprotKB annotations Cytoplasm Cell inner membrane, cell membrane Cell outer membrane integral to membrane integral to membrane

phage Bartonella carboxyl transferase alpha subunit protein pstB ATP-binding cis-transisomerase Protein name Probable periplasmic periplasmic ABC transporter, synthase subunit alpha ATP Hemin-binding protein a Hemin-binding protein d Hemin-binding protein c Peptidyl-prolyl 50S ribosomal protein Serine protease Phosphate import Omp43 precursor L25/general stress protein Ctc serine protease DO-like amino acid-binding protein 31 K major protein, Pap31 - henselae Acetyl-CoA carboxylase, Acetyl-CoA

P54925 Q6G487 Q6G1P1 Q6G4S7 Q6G4S8 Q6G2L2 Q6G2Z1 Q6G4T6 Q9L7A9 UPI0000 17CA3D Q6G5U1 Q6G3W0 Q6G1W7 Accession No. BH11850 BH16340 BH04770 BH10250 BH06370 BH15340 BH02460 BH02560 BH04810 BH02550 BH12500 BH02000 Locus Continued. 314 235/242 46/61 103/107 300 93/89 292 17/27/245 JC6528 25/28/30/31/37/ 39/45/208/271/ 277/283/289/306 29/36/47/212/ 218/224/228/ 237/238/239/ 250/262/276/ 279/280/391 232 170 211 Spot ID

Appendix Table 1. Appendix Table rplY accA degP htrA2 UNA atpA pstB Pap31 hbpA hbpD hbpC omp43 plp Gene = unannotated; UNK unknown. UNA

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br Proteomic and bioinformatic analysis of Bartonella henselae 1811 0 0 0 0 0 0 0 0 1 0 0 HMMTOP 0 1 0 0 1 1 0 No No No No No No No No No No No No No No No No No No Signal (P) Continued on next page 0.92 2.3E-225 1.0E-42 1.1E-37 1.8E-100 4.5E-28 3.8E-200 Pfam E-value 0.68 5.9E-22 4.1E-43 5.9E-05 9.8E-38 5.0E-61 4.1E-88 1.1E-32 5.9E-24

DUF1256 (PF06866) IMPDH (PF00478) SapC (PF07277) Redoxin (PF08534) Aminotran_1_2 GSH-S_ATP Peptidase_M29 Pfam model (ID) (PF02955), (PF02629) family (PF09588) (PF02951) (PF02073) Domain (PF07484) GSH-S_N Glyco_hydro_57 Collar (PF07484) CoA_binding GST_N (PF02798) viral YqaJ DUF55 (PF01878 ) Abhydrolase_1 3.3E-15 (PF00561) 0.00016 FAD_binding_6 (PF00970), NAD_binding (PF00175) (PF03065) Tubulin (PF00091) Tubulin (PF01656) CbiA Collar Tail Phage ar ul ) ) ) ), ) ) recombinase ) ) (PF00155) ), ) ％ ％ ％ ％ ％ ％ ％ ％ ％ ％ Cytoplasm (60.9 (99.8 (57.2 No positive Cytoplasm Cytoplasm Cytoplasm No prediction No prediction PA-SUB (P) PA-SUB predictions predictions (100 (98.9 periplasm (99 Inner membrane (100 Cytoplasm (91.7 No prediction No positive No positive Cytoplasm Cytoplasm No prediction (100 No positive No prediction No prediction

Extracell

-0.246 -0.163 -0.090 -0.136 -0.081 -0.467 -0.558 -0.388 -0.828 -0.376 -0.685 -0.258 -0.765 -0.524 0.222 0.052 0.015 0.001 GRAVY I

p 5.81 6.18 6.31 5.97 5.39 5.88 5.96 6.51 6.29 4.97 6.47 6.13 5.75 9.05 8.76 6.98 5.27 5.01 Predicted Houston-1 proteins with subcellular location unknown identified in this study by predictions predictions

Mass 31188.7 31188.7 22269.6 26422.1 28223.5 44066.6 30935.6 35335.5 45251.1 52816.6 30991.7 19696.3 19312.2 27012.8 16494.2 31632.1 41031.7 29426.6 36140.6

Bartonella henselae Bartonella

Cobyrinic acid a, UNA Hydrolase activity Aminotransferase, UNA Aminopeptidase UNA Transferase Oxidoreductase binding, ATP glutathione synthase Molecular function activity c-diamide synthase activity activity activity binding DNA (91.5 transferase nuclease activity activity activity IMP dehydrogenase IMP UNA UNA Ligase Oxidoreductase Single-stranded binding, DNA UNA parA protein parA sapC-related Hydrolase Aspartate Putative Aminopeptidase 35 kDa protein Glutathione Ferredoxin-NADP Glutathione synthetase protein BH14040 subunit alpha DNA-binding protein protein UniProtKB annotations Protein name monophosphate dehydrogenase protein [ADP-forming] S-transferase protein aminotransferase reductase uncharacterized protein Inosine-5’- Succinyl-CoA ligase Succinyl-CoA Phage related Expressed protein Hypothetical Expressed protein Single-stranded Phage related

Q45197 Q6G265 Q6G262 Q6G291 Q6G302 Q6G224 Q93FN9 Q6G5P1 Q6G3E1 Q6G5L6 Q6G4T3 Q6G2C5 Q6G1R7 Q6G1R2 Q6G2G4 Q6G4U0 Q6G1M2 Q6G4W9 Accession No. Analysis of sarcosine-insoluble

Locus BH11640 BH01800 BH14010 BH13200 BH14040 BH15840 BH16550 BH13580 BH12430 BH10130 BH08320 BH12780 BH14450 BH16120 BH02420 BH02500 BH04920 AAL13209 6 71/77/75/ 221 159/160 319 398 236 20 354/352 304 342 290 149 287 295 392 223 136 216 Spot ID 80/76/68

Appendix Table 2. Appendix Table PMF using MALDI-TOF-MS. Gene guaB UNA UNA parA1 UNA UNA sucD UNA UNA ssb4 UNA UNA UNA UNA UNA gshB UNA UNA

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br D.M. Li et al. 1812 0 1 0 3 0 HMMTOP No No No No Yes (1) Yes Signal (P) 2.70E-33 3.4E-16 3.9e-31 Pfam E-value 3.60E-148 3.40E-41 ETF (PF01012) Lactamase_B (PF00089) Trypsin Pfam model (ID) (PF01709) Domain of unknown (PF00753) function DUF28 PfamB PB002926 ) ％ No prediction No prediction Periplasm PA-SUB (P) PA-SUB No prediction No prediction

-0.087 -0.150 -0.401 -0.399 0.138 GRAVY I

p 5.1 6.56 5.57 6.31 6.97 Predicted

Mass 61313.1 32444.2 54858.8 26993.6 10006.2

Hydrolase activity binding, FAD UNA Hydrolase, electron carrier protease (100 Molecular function UNA Putative Electron transfer UPF0082 Serine protease flavoprotein alpha-subunit activity protein BH14810 UniProtKB annotations Protein name uncharacterized protein uncharacterized protein Putative

Q6G271 Q6G5P2 Q6G2T2 Q6G3A3 Q6G2H6 Accession no. Continued.

Locus BH08800 BH12300 BH13950 BH14810 BH10940 50/51 248 379 294 159 Spot ID

Appendix Table 2. Appendix Table Gene UNA etfA UNA UNA htrA3 = unannotated. UNA

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br Proteomic and bioinformatic analysis of Bartonella henselae 1813 PSORTb (Probability) PSORTb Cytoplasmic (9.26) Cytoplasmic (8.96) Cytoplasmic membrane (7.88) Cytoplasmic (9.97) Cytoplasmic (9.97) Cytoplasmic (9.97) Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic (9.26) Cytoplasmic (9.97) Cytoplasmic (9.26) Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic (9.26) Cytoplasmic (9.97) Cytoplasmic (8.96) Cytoplasmic (9.97) Cytoplasmic (8.96) Cytoplasmic (9.97) Cytoplasmic (9.97) Cytoplasmic (9.26) Cytoplasmic membrane (8.02) Cytoplasmic (8.96) Cytoplasmic (8.96)

Continued on next page 0.01 0.303 -0.369 -0.239 -0.038 -0.349 -0.139 -0.099 -0.206 -0.035 -0.071 -0.041 -0.467 -0.233 -0.467 -0.193 -0.236 -0.314 -0.071 -0.209 -0.102 0.311 0.053 0.020 0.069 GRAVY I p 5.1 5.63 6.09 6.35 9.67 6.83 6.61 5.14 5.46 5.75 5.07 5.99 6.41 7.15 6.04 6.21 7.95 5.12 9.75 6.63 4.85 5.03 5.61 6.34 5.88 Predicted Mass 37211.8 21185.6 96059.8 24439.7 50151.2 52854.4 24414.4 47401.6 33664.1 42817.2 43640.3 57597.0 24587.7 36177.2 57625.1 40554.7 23252.4 76333.1 26685.7 21480.9 37672.1 18861.3 27853.3 46539.1 49427.4

Houston-1 proteins identified in this study by PMF using

Chaperone, hydrolase, Hydrolase activity Molecular function binding activity, activity Fumarate hydratase activity Transferase ribosomal protein ribosomal protein ligase activity transferase protease transferase Ligase Methyltransferase, Hydrolase L-malate dehydrogenase Ligase Acyltransferase, Hydrolase, transferase ATPase binding, ATP Oxidoreductase Lyase Chaperone Aminotransferase, transferase Isomerase Elongation factor Ribonucleoprotein, Ribonucleoprotein, Kinase, transferase Nucleotidyltransferase, Isomerase, rotamase activity ATPase binding, ATP Transferase binding, biotin ATP Bartonella henselae Bartonella

Cytoplasm UNA Cellular component catalytic domain ATPase complex, ATPase complex Ttricarboxylic acid cycle enzyme complex Cytoplasm UNA Proton-transporting two-sector UNA UNA Oxoglutarate dehydrogenase UNA UNA UNA UNA Cytoplasm Cytoplasm UNA Cytoplasm Small ribosomal subunit UNA Cytoplasm UNA UNA UNA UNA UNA

UDP-N-acetylmuramoylalanyl- Protein-L-isoaspartate ATP-dependent clp protease, ATP-dependent Putative uncharacterized UniProtKB annotations Protein name D-glutamate-2, 6-diaminopimelate ligase (D-aspartate)O-methyltransferase synthesis protein nifS carboxylase subunit factor Rho [ADP-forming] subunit beta acetyltransferase biosynthesis protein purH dehydrogenase aldolase subunit clpB ATP-binding dependent phosphoglycerate mutase protein subunit alpha Transcription termination Transcription Malate dehydrogenase ligase Succinyl-CoA Dihydrolipoamide Bifunctional purine Cell division protein ftsE Glyceraldehyde-3-phosphate Fructose-bisphosphate Fumarate hydratase 60 kDa chaperonin Aminomethyltransferase 2,3-Bisphosphoglycerate- Elongation factor G 30S ribosomal protein S3 50S ribosomal protein L5 Adenylate kinase polymerase DNA-directed RNA Peptidyl-prolyl cis-transisomerase transporter ATP-dependent Nitrogenase co-factor carboxylase biotin Acetyl-CoA

Q6G255 Q6G2M3 Q6G2Q0 Accession No. Q6G1K7 Q6G1M0 Q6G1M1 Q6G1M4 Q6G5S5 Q6G1V9 Q8L201 Q8L207 Q6G294 O33963 Q6G2E9 Q8L1Z7 Q6G2V0 Q8KQB3 Q6G2X1 Q6G2X7 Q6G2Y6 Q9FDC7 Q6G306 Q6G3C0 Q6G3C2 Q6G3G8

List of the locations and functions of carbonate-insoluble and functions locations of the List Locus BH14110 BH11740 BH11290 BH16700 BH16570 BH16560 BH16530 BH15970 BH15420 BH15080 BH15060 BH13550 BH13530 BH12840 BH12450 BH10660 BH10540 BH10450 BH10390 BH10300 BH10270 BH10090 BH08630 BH08610 BH08050 169/173/573 292/293 224 193 108/107/112 551 302 313/315 9/17/13 181 104/111/129/ 120/140 261/257 441/442 432 156/154/155 407 24/22/25/26 430 575 449 244/251/252 459 402 226 168/166 Spot ID

Appendix Table 3. Appendix Table MALDI-TOF-MS. Gene rho mdh sucC sucB purH ftsE gap fbaB clpB fumC groL gcvT gpmA UNA murE pcm2 fusA rpsC rplE adk rpoA ppiB1 UNA nifS1 accc

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br D.M. Li et al. 1814 PSORTb (Probability) PSORTb Cytoplasmic (8.96) Cytoplasmic (9.26) Cytoplasmic (9.26) Cytoplasmic (8.96) Cytoplasmic (9.26) Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic (9.97) Cytoplasmic (8.96) Cytoplasmic (9.97) Cytoplasmic (9.26) Cytoplasmic (8.96) Cytoplasmic (9.97) Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic (9.97) Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic membrane (9.46) Cytoplasmic (8.96) Cytoplasmic (9.97) Cytoplasmic (9.97) Cytoplasmic (9.97) Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic (9.97) Cytoplasmic (9.26) Cytoplasmic (8.96)

Continued on next page 0 -0.31 -0.15 0.12 -0.358 0.136 0.025 0.249 0.015 0.006 0.103 -0.112 -0.206 -0.154 -0.092 -0.707 -0.056 -0.335 -0.166 -0.356 -0.539 -0.354 -0.065 -0.024 -0.699 -0.478 -0.328 -0.254 -0.303 -0.296 -0.146 GRAVY I p 5.3 6.66 5.62 6.54 6.55 5.45 5.56 4.97 6.18 4.93 5.82 9.63 5.61 5.17 5.14 5.57 7.17 4.81 6.04 6.24 5.61 8.86 5.17 5.42 6.14 6.53 5.46 5.36 6.33 6.86 5.94 Predicted Mass 42866 38033 36836.2 50571.1 29367.7 47780.5 45241.2 19085.2 25161.2 20665.7 32374.8 31272.6 24649.6 20165.1 55534.1 53222.7 46795.7 47785.1 49303.5 30508.9 44721.7 15848.1 22913.7 55335.7 49238.1 20869.1 26084.8 25597.4 50796.4 27053.3 40967.8

Molecular function ribosomal protein factor activity ribosomal protein ribosomal protein response regulator activity reductase (NADH) activity regulator activity Acyltransferase, transferase binding, transcription ATP Binding,enoyl-[acyl-carrier-protein] Transferase Transferase Stress response Isomerase termination Translational Elongation factor UNA ribonucleoprotein, Repressor, elongation Transcription Elongation factor Ligase Chaperone, isomerase, rotamase Chaperone Acyltransferase, transferase Catalytic activity Oxidoreductase Transferase Transferase Ribonucleoprotein, Ribonucleoprotein, Helicase, hydrolase Hydrolase Glycosyltransferase, transferase binding, two-component DNA Catalytic activity UNA binding RNA Hydrolase, lyase

Cellular component

organelle UNA Intracellular UNA Cytoplasm UNA UNA UNA Cytoplasm Cytoplasm UNA ribosomal subunit Large UNA Cytoplasm UNA UNA UNA UNA UNA Intracellular membrane-bounded Cytoplasm UNA UNA Ribosome UNA UNA UNA UNA UNA UNA UNA UNA

lation protein u UDP-3-O-[3-hydroxymyristoyl] Enoyl-(acyl carrier protein) UniProtKB annotations Protein name glucosamine N-acyltransferase synthase II protein nusG amidotransferase subunit B subunit clpX ATP-binding Nitrogen reg acetyltransferase (E2) subunit beta component, alpha subunit aldolase synthase II 4-phosphate synthase reductase Serine hydroxymethyltransferase 3-Oxoacyl-(acyl carrier protein) Small heat shock protein A Ribose-5-phosphate isomerase Ribosome-recycling factor Ts Elongation factor Hypothetical protein BH06210 50S ribosomal protein L1 antitermination Transcription Tu Elongation factor (Asn/Gln) Aspartyl/glutamyl-tRNA factor Trigger Clp protease ATP-dependent Dihydrolipoamide Pyruvate dehydrogenase Pyruvate dehydrogenase E1 2-Dehydro-3-deoxyphosphooctonate 3-Oxoacyl-(acyl carrier protein) 30S ribosomal protein S6 50S ribosomal protein L9 helicase Replicative DNA Dihydroorotase Orotate phosphoribosyltransferase response regulator Two-component Phosphatase tldD protein Hemolysin-like protein 3,4-Dihydroxy-2-butanone

Accession No. Q6G3L3 Q6G3M3 Q6G5C1 Q6G3V6 Q8VQ23 Q6G5C6 Q6G5C8 Q6G5D0 Q6G3X8 Q6G3Y0 Q8KHX9 Q6G3Y7 Q6G3Y8 Q6G3Z2 Q6G3Z5 Q6G403 Q6G404 Q8L1Z6 Q6G409 Q6G441 Q6G446 Q6G449 Q6G450 Q6G5H1 Q6G463 Q6G490 Q6G4B4 Q6G4C8 Q6G4D0 Q6G4D3 Q6G4D5

Continued. Locus BH07540 BH07440 BH07300 BH06410 BH06290 BH06250 BH06230 BH06210 BH06070 BH06050 BH06020 BH05950 BH05940 BH05890 BH05850 BH05770 BH05760 BH05750 BH05710 BH05370 BH05320 BH05290 BH05270 BH05160 BH05050 BH04780 BH04520 BH04380 BH04360 BH04330 BH04310 177/180 186 464/465/463 404/409 295 454 317/318/319 342 399 462 195/202/205/ 216/214/218 566 116 572 146/152 136 569/110/142 304/301 346 199/198 488/489/487 438 106/109 163 450/447 369 431 571/174 552 272 382/384/383 Spot ID

Appendix Table 3. Appendix Table Gene glyA fabF2 ibpA2 rpiA lpxD frr tsf UNA rplA nusG tuf1 gatB tig clpX ntrX pdhC pdhB pdhA kdsA fabF1 rpsF rplI dnaB pyrC1 pyrE UNA UNA tldD tlyA ribA abI2

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br Proteomic and bioinformatic analysis of Bartonella henselae 1815 PSORTb (Probability) PSORTb Cytoplasmic (8.96) Cytoplasmic (9.97) Cytoplasmic (9.26) Cytoplasmic (9.97) UNK UNK UNK UNK UNK Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic (9.97) Cytoplasmic (9.97) Cytoplasmic (8.96) Cytoplasmic (9.26) Cytoplasmic (8.96) Cytoplasmic (9.97) Cytoplasmic (8.96) Cytoplasmic (8.96) Cytoplasmic (9.97) Cytoplasmic (8.96) Cytoplasmic (9.97) Cytoplasmic (10.00) UNK UNK UNK UNK UNK UNK

Continued on next page -0.34 0.143 0.084 0.001 0.284 -0.111 -0.611 -0.212 -0.175 -0.292 -0.089 -0.475 -0.358 -0.225 -0.402 -0.173 -0.294 -0.313 -0.134 -0.108 -0.354 -0.397 -0.200 -0.320 -0.335 -0.036 -0.238 -0.480 -0.141 -0.256 -0.703 GRAVY I p 5.3 5.9 6.2 5.07 6.22 5.18 6.27 8.49 9.33 5.88 6.35 6.54 4.78 5.36 5.53 6.55 8.69 6.19 5.87 5.18 4.93 5.79 6.05 5.74 6.66 9.56 9.66 7.88 6.45 5.25 8.58 Predicted Mass 25147 27099 49403 41024.9 98552.5 22750.9 34948.9 33903.3 33258.7 40664.6 15653.8 24846.6 59077.8 80652.3 40450.9 33247.4 67222.7 37296.4 43504.3 84603.1 62818.1 68261.2 39439.8 59271.6 48034.8 37069.6 28363.4 18154.9 19558.6 42221.9 62313.6

unfolded protein binding polymerase, DNA-directed DNA 4 iron, sulfur cluster binding binding, structural 5S rRNA Molecular function factor activity codon specific Transferase Nucleotidyltransferase, translation elongation factor activity transferase activity dehydrogenase activity constituent of ribosome constituent of ribosome protein binding, transcription aspartate-semialdehyde acetyl-CoA carboxylase activity, carboxylase activity, acetyl-CoA carboxylase activity, Acetyl-CoA transferase activity Heat shock protein binding, UNA binding Sequence-specific DNA UNA binding, nucleotide RNA Nucleotidyltransferase, transferase release factor activity, Translation Nucleotidyltransferase, transferase binding, GTPase activity GTP binding GTP Transferase Oxidoreductase Ribonucleoprotein, ribosomal protein Chaperone Repressor binding, GTPase activity, GTP synthetase, ligase Aminoacyl-tRNA or NADPH binding, NADP Structural constituent of ribosome Structural constituent of ribosome binding, structural rRNA kinase activity binding, UMP ATP Catalytic activity Chaperone Oxidoreductase binding, GTPase activity GTP

Cytoplasm UNA Ribosome Cellular component

Cytoplasm carboxylase Acetyl-CoA complex UNA UNA UNA UNA UNA Cytoplasm Cytoplasm UNA IntracellμLar IntracellμLar UNA UNA Ribosome UNA UNA Cytoplasm Cytoplasm Cytoplasm Small ribosomal subunit Ribosome Ribosome Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm

Acetyl-CoA carboxylase, Acetyl-CoA polymerase III subunit beta DNA Aconitate hydratase 50S ribosomal protein L25 Acetyl-co-enzyme A A carboxylase Acetyl-co-enzyme UniProtKB annotations Protein name nucleotidyltransferase carboxyl transferase alpha subunit carboxyltransferase beta chain 5’-triphosphate deaminase factor NusA uridylyltransferase synthase I repressor hrcA dehydrogenase biosynthetic protein PdxJ Heat shock protein DnaJ 2’-Deoxycytidine regulator Transcriptional UPF0090 protein BH02180 elongation Transcription Polyribonucleotide Peptide chain release factor 1 UTP-glucose-1-phosphate GTP-binding protein typA GTPase ObgE 3-oxoacyl-(acyl carrier protein) Malic enzyme 30S ribosomal protein S1 Chaperone protein dnaK Heat-inducible transcription Peptide chain release factor 3 synthetase Seryl-tRNA Aspartate-semialdehyde 30S ribosomal protein S2 50S ribosomal protein L10 50S ribosomal protein L6 Uridylate kinase Pyridoxal phosphate Chaperone protein dnaJ Dihydrolipoyl dehydrogenase Cell division protein FtsZ

Q6G525 Q6G528 Q6G2L2 Accession No. Q6G4D6 Q6G4R3 Q6G4W1 Q6G4W4 Q6G4W5 Q6G5F8 Q6G5T7 Q6G4X4 Q6G4X9 Q6G4Z2 Q6G516 Q6G541 Q6G5H4 Q6G554 Q6G564 Q6G589 Q6G419 Q6G1P1 Q6G581 Q6G2E5 Q6G5C9 Q6G3X7 Q6G2Y0 Q6G5C7 Q3V7Q9 Q6G553 Q67B06 O69074

Continued. Locus BH11180 BH01190 BH01160 BH11850 BH04300 BH02700 BH02210 BH02180 BH02170 BH02100 BH01970 BH01750 BH01700 BH01560 BH01280 BH01010 BH00930 BH00650 BH00550 BH00250 BH05610 BH16340 BH00340 BH12890 BH06220 BH06080 BH10360 BH06240 BH10900 BH00660 AAR21288 564/352/340/ 297/333 274/228 479 435 79 29/27/28 232 241/368 55/53 288 188/189 236/243 4/5/7/8/6 20 59/69/76/67/ 61/63 44/41 273 89/88 150/151 570 348/343 580 283/284 358/360 468 408/410 574 427 424 192/197 238 Spot ID

Appendix Table 3. Appendix Table Gene dnaJ2 UNA UNA UNA nusA pnp prfA galU typA obgE fabB dnaN acnA maeB rpsA dnaK hrcA prfC lpdA serS accA accD asd rpsB rplJ rplY rplF pyrH pdxJ ftsZ dnaJ1

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br D.M. Li et al. 1816 PSORTb (Probability) PSORTb membrane protein OM (10.00) Periplasmic ( 9.44) UNK OM (9.93) Periplasmic (9.76) Periplasmic (9.76) Periplasmic (9.76) Periplasmic (9.76) Periplasmic (10.00) Cytoplasmic (9.12) OM (10.00) OM (9.93) OM (10.00) Outer membrane integral OM (9.94) OM ( 9.93) OM (9.45) UNK

-0.611 -0.061 -0.207 -0.104 -0.293 -0.328 -0.296 -0.166 -0.004 -0.255 -0.254 -0.075 -0.442 -0.125 -0.205 -0.158 -0.025 0.026 GRAVY I

p 8.6 5.91 5.37 6.18 8.49 7.08 5.54 9.02 6.53 9.34 9.01 8.53 9.71 5.82 4.90 5.37 9.06 9.12 Predicted

Mass 55511.6 41187.3 30074.7 48847.6 50140.9 33815.8 28179.4 50562.1 25552.8 82612.4 88985.9 44224.4 48299.3 35596.8 29916.6 30269.7 17635.4 25663.7

Molecular function

UNA UNA activity Transporter Protein transport, transport activity Transporter activity Transporter Hydrolase, protease Hydrolase Hydrolase Receptor Outer membrane Porin activity activity Transporter Isomerase, rotamase Porin activity Porin activity UNA Porin activity

integral to membrane integral to membrane Cellular component periplasmic space cell membrane periplasmic space cell membrane UNA integral to membrane UNA UNA Periplasm Outer membrane-bounded Outer membrane-bounded UNA Cell inner membrane, Cell inner membrane, Cell outer membrane UNA Membrane Outer membrane UNA Cell outer membrane, Cell outer membrane, Outer membrane Cell outer membrane, phage

UniProtKB annotations Protein name amino acid-binding protein periplasmic ligand-binding protein periplasmic protein binding protein protein BH02760 ATP-binding isomerase 31 K major protein, Pap31 - Bartonella henselae Branched chain aa ABC transporter, ABC transporter, Branched chain aa Glycerol-3-phosphate-binding Protein tolB ABC transporter Amino-acid periplasmic ABC transporter, Hydrolase, protease synthase subunit alpha ATP ABC transporter Putative Heme receptor precursor Outer membrane protein Omp43 precursor Outer membrane protein Peptidyl-prolyl cis-trans Hemin-binding protein a Hemin-binding protein d Putative uncharacterized protein Pap31

Accession No. 17CA3D Q6G3E4 Q6G5J2 Q6G5R6 Q6G2V8 Q6G3W0 Q6G2Z1 Q6G1W7 Q6G4Q8 Q6G471 Q8VQ24 Q9L7A9 Q6G2V1 Q6G5U1 Q6G4S7 Q6G487 Q6G3K7 Q94M28

Continued. Locus JC6528 UPI0000 BH08290 BH01870 BH14840 BH10580 BH06370 BH10250 BH15340 BH02760 BH04970 BH06280 BH12500 BH10650 BH02000 BH02560 BH04810 BH07600 AAK97307 279 269 219/223 576 396 145/148 135/133/137 429 34/37/54/567 10/12/14/15/ 18/19 246/248/254 281 321/322 349/367/394 363 339/357/390/ 344/361/ 338/363 373/375/379/ 389/577/578 579 Spot ID

Appendix Table 3. Appendix Table Gene livJ ugpB tolB ybeJ UNA htrA2 atpA UNA hutA omp89 omp43 UNA plp pap31 pap31 hbpA hbpD UNA = unannotated; UNK unknown. UNA

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br Proteomic and bioinformatic analysis of Bartonella henselae 1817 1 2 1 1 1 0 0 0 1 0 0 0 0 0 0 2 0 1 0 0 HMMTOP No No No No No No 0 No No No No No No No No No No Yes (1) Yes Yes (1) Yes (1) Yes SignalP (P) SignalP Yes (0.625) Yes Yes (0.894) Yes Continued on next page 3.40E-14 2.70E-33 2.60E-48 1.10E-37 3.20E-08 2.60E-42 3.80E-200 1.30E-10 2.10E-97 5.90E-22 1.00E-42 4.10E-98 2.80E-36 2.10E-149 0.8 1.30E-95 2.70E-06 4.10E-43 6.50E-39 Pfam E-value 2.30E-225

(PF04392)

(PF06866) protein Lactamase_B (PF00753) Electron transfer (PF00578) AhpC-TSA Redoxin (PF08534) HATPase_c (PF02518) HATPase_c flavoprotein domain enzyme II C-terminus Peptidase_M20 (PF01546) Peptidase_M29 (PF02073) PDZ (PF00595) Protein of unknown ABC transporter substrate Collar Domain Tail Phage SapC (PF07277) (PF04011) LemA IalB (PF06776) DUF1460 (PF07313) Sorbitol phosphotransferase dehydrogenase (PF02153) Amidohydrolase (PF01979) CoA_binding (PF02629) (PF01230) HIT Pfam model dehydrogenase/ IMP reductase GMP domain (PF00478) (PF01012) function binding (PF07663) (PF07484) ) ) ) ) Prephenate ) ％ ％ ) ) ％ ) ％ ％ ％ ％ ％

Extracellular (92.5 No prediction No prediction Extracellular (63.8 Extracellular (100 Cytoplasm (99.5 Cytoplasm (98.9 No prediction Cytoplasm (100 No prediction No prediction Cytoplasm (95 No prediction No prediction No prediction Periplasm (84.9 No prediction No prediction No prediction No prediction No prediction PA-SUB (P) PA-SUB

-0.193 -0.087 0.015 -0.081 -0.200 0.074 -0.062 0.138 0.001 -0.269 -0.388 -0.185 -0.137 -0.258 0.204 0.000 -0.524 -0.467 -0.198 -0.267 -0.292 GRAVY 5.39 6.56 6.29 5.96 8.91 5.69 5.51 5.57 6.47 6.83 6.13 6.51 pI 6.21 6.98 9.36 6.91 5.01 6.51 8.66 8.99 6.91

Predicted Houston-1 proteins with subcellular location unknown identified in this study by 50692.6 61351.5 52783.6 45279.4 47046.2 33696.5 46866.5 32464.2 30991.7 18228.0 19696.3 15792.3

Mass 66925.5 41031.7 34390.0 37605.1 36163.1 31208.6 24505.2 22444.8 31840.4

) + (NADP

Bartonella henselae Bartonella

Metallopeptidase activity, Metallopeptidase activity, Hydrolase activity dehydrogenase activity IMP Aminopeptidase activity Protein binding, serine-type Binding, prephenate acting on Hydrolase activity, binding, electron FAD UNA UNA UNA UNA UNA Ligase Antioxidant activity, UNA Oxidoreductase activity Catalytic activity UNA UNA Molecular function binding, mismatched ATP binding DNA protein dimerization activity peptidase activity dehydrogenase activity carbon-nitrogen (but not peptide) bonds carrier activity oxidoreductase activity

subunit

Putative uncharacterized Putative uncharacterized Inosine-5’-monophosphate Aminopeptidase Carboxy-terminal Cyclohexadienyl Dihydroorotase Electron transfer Expressed protein Hypothetical protein Periplasmic phosphate Phage related protein sapC-related protein ligase Succinyl-CoA Bacterioferritin Cytoplasmic membrane Expressed protein Hit-like protein involved Putative uncharacterized Putative uncharacterized Protein names mismatch repair DNA protein mutL protein protein processing protease ctpA dehydrogenase flavoprotein alpha-subunit BH16250 binding protein [ADP-forming] alpha co-migratory protein protein in cell-cycle regulation protein protein UniProtKB annotations Q6G4T1 Q6G3A3 Q6G4W9 Q6G4T3 Q6G4Y5 Q6G1Q3 Q6G3F4 Q6G2H6 Q6G265 Q6G1P9 Q6G4T9 Q45197 Q6G1R7 Q6G1M2 Q6G3B5 Q6G3I1 Q6G291 Q6G5D3 Q6G3B0 Q6G5L9 Accession No. Q6G4R4 Analysis of carbonate-insoluble Locus BH02520 BH08800 BH01800 BH02500 BH01630 BH16210 BH08190 BH12300 BH14010 BH16250 BH02430 BH04920 BH15840 BH16550 BH08680 BH07870 BH13580 BH06190 BH08730 BH11610 BH02690

Spot ID 160 84/87 127/128/ 204 212/213/ 308 190 353 234/240 335/337 326 309/310 350/351 306/307 483 544 561/421/ 494 466/514 388 3/48 dehydrogenase 126/124/ 125/121/ 217

Appendix Table 4. Appendix Table PMF using MALDI-TOF-MS. Gene mutL UNA UNA guaB 119 AmpS ctpA tyrC pyrC2 etfA UNA UNA UNA UNA UNA sucD bcp lemA UNA 467/470/ 469 UNA IalB UNA

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br D.M. Li et al. 1818 1 3 0 1 1 1 1 0 0 0 0 0 1 HMMTOP No No No No No No No No No No No 1 Yes (1) Yes Yes (1) Yes SignalP (P) SignalP Yes (0.998) Yes 3.70E-87 1.20E-05 1.60E-174 5.30E-28 1.60E-70 3.50E-57 3.90E-31 3.40E-41 3.60E-146 1.10E-32 7.20E-30 1.00E-45 Pfam E-value 6.30E-35 5.30E-33

(PF00557) Metallopeptidase family SRP54-type protein, helical (PF00890) FAD_binding_2 ABC_tran (PF00005) Extracellular solute-binding ABC transporter (PF00005) Trypsin (PF00089) Trypsin PB002926 DUF28 (PF01709) CbiA (PF01656) CbiA SSB (PF00436) M24 bundle domain (PF02881) Pfam model Peripla_BP_2 (PF01497) (PF01300) Iron/manganese superoxide dismutases (PF00081) proteins (PF00496) ), ) ) ) Sua5_yciO_yrdC ％ ) ％ ％ ％ ％) ％

(100 ) ) ) ％ ％ ％ No prediction Cytoplasm (60.9 Periplasm (100 Cytoplasm (99.8 Cytoplasm (80.1 No prediction Cytoplasm Extracellular (100 Inner membrane Inner membrane Inner membrane No predictions No prediction No prediction (100 (100 PA-SUB (P) PA-SUB (100 periplasm

-0.202 0.222 0.237 -1.233 0.131 -0.484 -0.345 -0.150 -0.236 -0.071 -0.276 -0.405 -0.399 -0.401 GRAVY 5.99 5.81 9.5 5.75 6.73 5.77 8.32 6.31 6.04 5.61 pI 8.84 5.36 6.97 5.1

Predicted 30807.1 22269.6 32546.3 19312.2 34604.0 23119.0 45785.4 54892.7 66972.7 27853.3

Mass 61340.4 60988.0

10012.6 27010.3 activity protease activity activity activity

Aminopeptidase, Cobyrinic acid a, c-diamide Iron ion transmembrane binding Single-stranded DNA UNA YrdC-like domain Contains 1 UNA Metal ion binding, superoxide binding, GTP 7S RNA Protein binding, serine-type binding, electron carrier FAD activity ATPase binding, ATP Molecular hydrolase, synthase transporter activity dismutase binding, nucleoside- triphosphatase peptidase oxidoreductase activity, activity, acting on the CH-CH group of donors function Transporter activity Transporter activity ATPase binding, ATP protein Methionine protein parA Periplasmic hemin- Single-stranded DNA- Putative uncharacterized Putative uncharacterized UPF0082 protein BH14810 Superoxide dismutase Cell division protein ftsY Serine protease Succinate dehydrogenase transporter ATP-dependent Protein names aminopeptidase binding protein protein protein flavoprotein subunit ABC transporter, periplasmic ABC transporter, oligopeptide-binding ABC Putative transporter protein ATP-binding UniProtKB annotations

Q6G3D8 Q6G2C5 Q6G473 Q6G302 Q6G271 Q6G4E2 Q6G5P2 Q6G4A3 Q6G1L5 Q6G2T2 Q6G1S3 Q6G3C0 Q6G2I6 Q6G2P3 Accession No. Continued. Locus BH08350 BH13200 BH04950 BH10130 BH13950 BH04240 BH14810 BH04630 BH16620 BH10940 BH15780 BH08630 BH12180 BH11380

Spot ID 372 426 406 456/533/ 504 332 381 448 175/182 158/159 62 402 98/99/92 81/82/ binding 534 83/86

Appendix Table 4. Appendix Table Gene map parA1 hutB ssb4 UNA UNA UNA SodA ftsY htrA3 sdhA UNA UNA UNA = unannotated. UNA

Genetics and Molecular Research 10 (3): 1789-1818 (2011) ©FUNPEC-RP www.funpecrp.com.br