Strain Variation, Virulence, and Carbohydrate ... - ACS Publications

Proteome of Haemophilus ducreyi by 2-D SDS-PAGE and Mass Spectrometry: Strain Variation, Virulence, and Carbohydrate Expression N. Karoline Scheffler,† Arnold M. Falick,‡ Steven C. Hall,‡ William C. Ray,§ Deborah M. Post,† Robert S. Munson, Jr.,§ and Bradford W. Gibson*,† Buck Institute for Age Research, Novato, California 94945, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, Children’s Research Institute and The Ohio State University, Columbus, Ohio 43205-2696 Received January 10, 2003

We have analyzed the proteome of several strains of Haemophilus ducreyi by two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Over 100 spots were analyzed from the soluble and insoluble protein fractions from the prototype strain 35000HP and 122 distinct proteins were identified. Functions of ∼80% of the 122 proteins were deduced by identification with close homologues of Haemophilus influenzae. Four additional wild type and three mutant strains were also analyzed that vary in their virulence and/or outer-membrane lipooligosaccharide structures. Overall, the 2-DE gel maps of the wild type and mutant strains were similar to strain 35000HP, suggesting little proteome diversity in relation to carbohydrate expression and/or virulence. An exception was the Kenyan strain 33921 which contained significant differences in its proteome 2-DE map and also synthesizes an unusual LOS with a trisaccharide branch structure. This African strain may represent a prototype of a second clonal group of H. ducreyi. Keywords: Blast search • carbohydrates • genomes • lipooligosaccharide • Haemophilus ducreyi • Haemophilus influenzae • mass spectrometry • proteome • virulence

Introduction The human pathogen Haemophilus ducreyi is a gramnegative bacterium that causes the sexually transmitted genital ulcer disease “chancroid”.1 The organism is believed to infect the genital epidermis via a portal route such as an abrasion or a small cut of the dermis. This sexually transmitted disease is prevalent in many developing countries and has been linked to the transmission of the human immunodeficiency virus (HIV).2 Although there are few reported cases each year in the United States, outbreaks occur occasionally in urban and regional areas. Furthermore, chancroid may be greatly underreported due to inadequate methods of detection.3,4 A number of bacterial proteins have been identified as potential virulence factors for H. ducreyi (for recent review, see Spinola et al., 2000).5 Such proteins include the outermembrane protein DsrA,6 the peptidoglycan-associated lipoprotein,7 the hemoglobin-binding protein,8,9 and the cytolethal distending toxin.10,11 Additional putative virulence determinants include GroEL,12 the filamentous hemagglutinin-like proteins LspA1 and LspA213 and hemolysin.14-16 * To whom correspondence and reprint requests should be addressed. Telephone: (415) 209-2032. Fax: (415) 209-2231. Email: bgibson@ buckinstitute.org. † Buck Institute. ‡ Applied Biosystems. § Children’s Research Institute and The Ohio State University. 10.1021/pr0340025 CCC: $25.00

 2003 American Chemical Society

In addition to proteins, lipooligosaccharides (LOS) are a major component of the outer-membrane of H. ducreyi and are major surface antigens. Moreover, LOS has been suggested as contributing to the pathobiology of chancroid infection. LOS from various strains of H. ducreyi have been shown to be both cytotoxic17-19 and important in host-pathogen interactions, such as in adherence and invasion of human keratinocytes and/ or fibroblasts.20-23 Given the potential importance of LOS to virulence, our laboratory and others have examined the structures and functions of LOS in the pathobiology of H. ducreyi infection. Like LOS from the related mucosal pathogens Haemophilus influenzae and Neisseria gonorrhoeae, H. ducreyi was found to synthesize LOS consisting of a hexa-acylated lipid A moiety embedded in the outer-membrane and attached to a short but variable oligosaccharide.24-26 The majority of H. ducreyi strains, including the prototype strain 35000 and its human passaged analogue 35000HP, contain a dominant LOS glycoform that consists of a linear pentasaccharide branch emanating from a conserved tri-heptose core linked to a lipid A moiety through 2-keto-deoxy-octulosonic acid (Kdo) (see Figure 1). This pentasaccharide branch terminates in the disaccharide N-acetyllactosamine that is partially substituted with sialic acid (Nacetyl-neuraminic acid, NeuAc).24,25 A smaller group of strains make a much smaller lactose disaccharide branch oligosaccharide that has also been shown to undergo partial sialylaJournal of Proteome Research 2003, 2, 523-533

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Figure 1. Lipooligosaccharide structures of all strains investigated in this study. (A) The wild type H. ducreyi strains 35000 and O41 contain a dominant LOS glycoform that consists of a linear pentasaccharide branch emanating from a conserved tri-heptose core linked to a lipid A moiety through Kdo. This pentasaccharide branch terminates in the disaccharide N-acetyllactosamine that is partially substituted with sialic acid. The wild type strains A77 and BG411 are human isolates and synthesize truncated LOS structures that either contain no branch structure (BG411) or terminate prior to the formation of the N-acetyllactosamine (A77). The prematurely truncated LOS glycoforms produced by strain A77 is the result of a mutation in the galactosyltransferase II gene61 and by strain BG411 is the result of a defect in the galU gene encoding UDP-glucose pyrophosphorylase, consistent with a structure absent of glucose and galactose sugars (Munson and Gibson, unpublished data). Strain 33921 has an oligosaccharide branch that bypasses the addition of the branch D-glycero-D-manno-heptose (DDHep), but like A77, stops short of forming a terminal N-acetyllactosamine, the main acceptor for sialic acid. (B) The gene knockout strains are laboratory constructs in the strain 35000 or 35000HP backgrounds. These strains synthesize specific truncated LOS structures due to the lack of the corresponding glycosyltransferase genes lbgA (35000.4) or lbgA and lbgB (35000.3), or the CMP-NeuAc synthetase gene neuA (35000HP-RSM202). The polar mutation in the neuA gene also results in the loss of expression of the downstream sialyltransferase gene (lst).42

tion.26 Sialic acid has been identified as a component of the LOS of several other related bacterial pathogens including N. gonorrhoeae and H. influenzae and been shown to contribute to resistance to phagocytosis and neutrophil attack.27,28 Recently, we have become interested in how protein expression may be related to differences in virulence and/or to differences in the expression of outer-membrane carbohydrates (or LOS). To approach this question, we chose to first compare the difference in protein expression among a few wild-type strains where carbohydrate expression is either similar to those expressed by the prototype strain 35000HP, or where a different set of LOS-glycoforms are known to exist that lack sialic acid. Although there are several approaches one could use to examine the proteome of H. ducreyi, two-dimensional electrophoresis (2-DE) is the most widely used and readily available technique capable of separating and visualizing several hundred to over a thousand proteins. Indeed, 2-DE has been used successfully for resolving and visualizing complex protein mixtures from such bacteria as Escherichia coli29-33 and the related pathogen Haemophilus influenzae,34,35 among others. In the present study, we present a detailed analysis of the whole cell lysate of H. ducreyi strain 35000HP, a prototype human-passaged organism that is capable of causing disease.16 Strain 35000HP synthesizes a typical LOS structure25 and was the prototype strain chosen for the now completed genome sequencing effort for this pathogen.36 In addition to this prototype strain, the proteomes of four other wild type strains were investigated; one of which has a similar LOS glycoform 524

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profile to strain 35000HP (strain O41) and three of which have been previously found to synthesize LOS-glycoforms lacking both N-acetyllactosamine and sialic acid (strains A77, BG411 and 33921) (see Figure 1). These latter three “atypical” strains have been reported to have altered or reduced virulence in various animal and tissue assays of the disease.21,37-40 In addition to these five wild-type strains, three mutant strains were also examined (35000.3, 35000.4, and 35000HP-RSM202) that are isogenic constructs deficient in LOS-oligosaccharide branch biosynthesis. Strains 35000.3 (lbgA-), and 35000.4 (lbgAB-) contain mutations in genes encoding glycosyltransferases needed for complete LOS assembly.41 Strain 35000HPRSM202 has a mutation in the neuA gene preventing activation of NeuAc to CMP-NeuAc, and therefore lacks sialic acidcontaining LOS glycoforms.42 LOS from both the wild type and isogenic mutant strains examined in this study have been previously isolated and subjected to structural characterization using mass spectrometry and NMR methods24,25,41 (Melaugh and Gibson, unpublished data).

Experimental Section Materials and Reagents. For culturing bacteria, GC Medium base (dehydrated) and hemoglobin were obtained from Difco (Detroit, MI) and the growth supplement BBL IsoVitaleX was purchased from Becton-Dickinson (Cockeysville, MD). For electrophoresis, IPG strips (5 mm wide, 18 cm long, pH 3-10 linear), urea, Pharmalyte pH 3-10, and glycerol (87% w/w) were

Proteome of Haemophilus ducreyi

all obtained from Pharmacia Biotech (Uppsala, Sweden). Similarly, Tris (electrophoresis grade), TEMED (electrophoresis purity reagent), and acrylamide (40% solution, acrylamide-tobisacrylamide ratio 37.5:1), and a Bradford protein assay kit were all obtained from Bio-Rad (Hercules, CA), whereas CHAPS (electrophoresis grade), thiourea (ACS grade), DTT (electrophoresis grade), iodoacetamide (electrophoresis grade), DNAase, ammonium bicarbonate and heavy mineral oil (for in vitro diagnostic use) were obtained from Sigma (St. Louis, MO). The protease inhibitor cocktail tablets were obtained from Roche (Indianapolis, IN). For protein staining of gels, bromphenol blue (Millipore, Bedford, MA), Coomassie Fast stain (Zoion; Newton, MA) and silver staining reagents (sodium thiosulfate, silver nitrate, formaldehyde (Sigma, St. Louis, MO), and sodium carbonate (Fisher Scientific, Fair Lawn, NJ) were employed. Glacial acetic acid (ACS grade), glycine (tissue culture grade), methanol (HPLC grade), SDS (electrophoresis grade) were all purchased from Fisher Scientific (Fair Lawn, NJ). For MALDI analysis of peptides, R-cyano-4-hydroxycinnamic acid matrix solution was purchased from Hewlett-Packard and used without further purification. Trifluoroacetic acid employed in the extraction of peptides from the destained gel spots and bovine serum albumin (albumin standard), were obtained from Pierce (Rockford, IL). C18 Zip-Tips were purchased from Millipore (Bedford, MA) and nanoelectrospray tips (medium length) were obtained from Protana (Odense, DK). High purity water (18.2 MΩ/cm) was prepared from a Millipore gradient water purification system and was used for all protocols in this study. Culture Conditions. All bacterial strains were grown on chocolate agar plates43 from skim milk stock solutions. Plates were inoculated with a disposable sterile loop and grown for 2 days in a candle jar at 34 °C. Bacteria were harvested with a loop and transferred into an Eppendorf tube containing 400 µL phosphate buffered saline (PBS). Bacteria were pelleted by spinning for 5 min at 12 000 ×g and resuspended twice in PBS followed by centrifugation. A typical yield of bacteria from one plate was 60-100 mg of wet cells corresponding to 6-10 mg of dried bacteria. This is referred to as the bacterial pellet. Preparation of Soluble Protein Fraction for 2-DE. Cell lysis was performed by adding lysis buffer consisting of 7 M urea/2 M thiourea/4% CHAPS/65 mM DTT to the bacterial pellets. The cell suspension was vortexed for 2 h followed by centrifugation for 10 min at 12 000 ×g to remove precipitated DNA and insoluble cell debris. To determine the protein content of this whole cell lysate, a protein assay (Bio-Rad protein assay Kit II) was performed using bovine serum albumin as a standard. Two-Dimensional SDS-PAGE (2-DE) Analysis. The soluble protein fractions obtained from the whole cell lysate were diluted in rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT, Pharmalyte pH 3-10 (1:50)) containing traces of bromophenol blue for a final volume of 0.4 mL containing 0.8 mg of protein. Centrifugation for 10 min at 12 000 ×g (room temperature) separated out insoluble debris that was often observed upon dilution of the cell lysate. The soluble fraction was removed and applied to the IPG strip as described below. Strips were rehydrated for 16 h in a rehydration tray (Pharmacia) following isoelectric focusing in a Multiphor II (Pharmacia) unit at 20 °C. The following gradient program was used: step 1: 0.01 h, 500V, 1 mA, 7 W; step 2: 5 h, 500 V, 1 mA, 7 W; step 3: 5 h, 3500 V, 1 mA, 7 W; step 4: 14 h, 3500V, 1 mA, 7 W. Total focusing time was 24 h.

research articles After the isoelectric focusing run was complete, the strips were removed from the Multiphor unit, the oil was allowed to drip off, and strips were equilibrated with gentle shaking in two steps of 20 min each in 5 mL equilibration buffer (0.05 M Tris-HCl pH 6.0, 35% glycerol, 1% SDS, traces of bromphenol blue) containing additionally 0.1 mg DTT in the first and 0.14 mg iodoacetamide in the second step. Strips removed from the second equilibration solution were blotted on a wet paper towel and placed on top of a 20 × 20 × 0.1 cm 12% acrylamide nongradient gel (vertical systems: BioRad Protean II Cell with 2-D conversion kit). The strips were sealed with 0.75% agarose/ 125 mM Tris pH 6.8/1% SDS/trace of bromphenol blue. Gels were run at constant current for ∼16 h at 10 °C and 15 mA per gel until the blue dye front reached the bottom of the gel. Gels were washed and fixed three times in 45% MeOH/10% acetic acid and then stained with colloidal Coomassie blue (CCB) (Fast Stain; Zoion; Newton, MA) for 2 h followed by destaining in 10% acetic acid. Washing, staining, and destaining of the gels was performed while gently shaking. Alternatively, gels were stained with silver.44 Gels were scanned and analyzed using an Image Master 2D (Pharmacia) system with a Sharp Color Image scanner JX-330. Preparation of an Insoluble Membrane Fraction (Pellet). The bacterial pellet (0.5 g wet weight) was resuspended in 5 mL of Tris/sucrose buffer (10 mM Tris, 0.25 M sucrose, 1 mM EDTA, pH 7.5). A protease inhibitor cocktail and DNAse were added. The cell suspension was cooled on ice, and sonicated 3 times for 15 s with a 15 W sonicator fitted with a microtip probe (Sonics & Material, Danbury, CT) and operating at 50% max. Unbroken cells and cell debris were removed by centrifuging at 12 000 ×g for 10 min at 4 °C. The pellet was discarded, and the supernatant was spun in an ultracentrifuge for 90 min at 150 000 ×g at 4 °C. The supernatant was removed and the remaining pellet was analyzed by PAGE using a two-detergent system as described below. Gel Electrophoresis Using a Two-Detergent System. The membrane-enriched insoluble protein fraction obtained as a pellet from the Tris/sucrose sonication method was solubilized and electrophoretically separated as described by Hartinger and MacFarlane.45,46 Briefly, separation in the first dimension was achieved by an inverse, discontinuous electrophoresis system using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and a separating gel at pH 2.1. The sample was dissolved in sample buffer containing urea, 16BAC and pyronin Y as a tracking dye. After staining of the gel the whole lane was cut from the gel and placed horizontally on top of a standard SDS gel with a wide slot to accommodate the gel strip. In both dimensions, the proteins were separated nominally according to their molecular weights. In-Gel Digestion. Protein spots of interest were cut out of the gel and diced into small pieces (∼1 mm2) with a stainless steel scalpel and placed into siliconized microcentrifuge tubes. The gel was destained and dehydrated by washing two times for 10-15 min with 150 µL of 25 mM NH4HCO3 in 50% acetonitrile until the gel pieces shrank in size and showed a white appearance. If very dark spots were still not destained after a third wash, then 100 µL of water was added to reswell the gel pieces and facilitate the release of the stain. One more rinse with 25 mM NH4HCO3 in 50% acetonitrile removed residual stain. The destained gel particles were then dried for 30 min under vacuum in a Speed-Vac. Forty µL of trypsin solution (0.025 mg/mL) was added to each tube. If necessary, 25 mM NH4HCO3 solution was added until gel pieces were fully Journal of Proteome Research • Vol. 2, No. 5, 2003 525

research articles covered with liquid. Tubes were sealed with Parafilm and incubated for 16 h at 37 °C in a water bath. When the digest was completed, 100 µL of water was added and tubes were sonicated for 10 min. The supernatant was removed from each tube and transferred into fresh tubes. Further recovery of the peptides was accomplished by extracting twice with 50% acetonitrile/ 5% TFA acid. All supernatants were pooled and placed in a Speed-Vac to minimize volatile salts by reducing the volume to ∼5 µL. Extracts were taken up in 100 µL of water and dried down again three times until no salt crusts were visible on the tube walls. After the last drying step 15 µL of 50% acetonitrile/5% TFA acid was added. Control digests were performed on gel slices that did not contain any protein, typically found at the sides of the gel. Trypsin autoproteolysis products and CCB contaminants could be identified in the subsequent mass spectrometric analyses of these controls. Mass Spectrometry of Protein Digests. As described in Matsui et al.,47 0.5 µL of the unseparated tryptic digest mixture was mixed with an equal volume of R-cyano-4-hydroxycinnamic acid solution and analyzed on an Applied Biosystems Voyager DE STR MALDI-TOF mass spectrometer operating in delayed-extraction reflector mode.48 A standard peptide mixture, Calmix I and II (Applied Biosystems), was used for external calibration of MALDI-TOF spectra and consisted of Des-Arg1bradykinin, angiotensin-1, Glu1-fibrinopeptide B, neurotensin, angiotensin1, ACTH (1-11), ACTH (18-39), ACTH (7-38) and bovine insulin. To obtain sequence data for individual peptides, peptides were either subjected to post-source decay (PSD) analysis49 or nanoelectrospray MS/MS analysis. Calibration of PSD spectra was carried out by a one-point correction of the parent ion obtained in the segment. For MS/MS analysis using nanoelectrospray, samples obtained from in-gel digests containing peptide mixtures were purified on C18 Zip-Tips (Millipore, Bedford, MA). Peptides were eluted with 70% methanol, 3% formic acid. Spectra were acquired on a Q-Star (AB Sciex, Toronto, Canada) quadrupole/time-of-flight mass spectrometer in nanospray mode. Chromatographic Separation of Tryptic Digests. Separation of the tryptic peptide mixture was performed on an Eldex MicroPro HPLC using a MagicMS (15 mm × 200 µm i.d.) capillary column at a flow rate of 1 µL/min. Typically, 2-3 µL of the digest solution were injected corresponding to 0.5-1 pmol per peptide. One µL fractions were collected directly on a MALDI target and analyzed after adding 1 µL of matrix. Database Searches. A program available on the Internet (http://prospector.ucsf.edu/) and developed at the University of California, San Francisco (UCSF) was used to search the protein databases SwissProt and NCBI as well as the genome of H. ducreyi (www.microbial-pathogenesis.org/). For the genomic database searches, the genome was translated in all six reading frames and then added as a database into the Protein Prospector search engine. The program MS-Fit uses peptide mass fingerprints to search databases for matching peptides from known proteins. The following parameters were used in the searches: protein molecular weight range from 1000 to 100 000 Da, trypsin digest (one missed cleavage allowed), 50 ppm mass accuracy, minimum number of peptides matched set at 4, and searching all species. The program MS-Tag uses fragment ion masses (generated by MALDI-PSD and MS/MS from a given precursor) to search the databases. The following parameters were used in the searches: parent ion mass tolerance of 1 Da, fragment ion mass tolerance of 1 Da, allowed 526


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Figure 2. 2-D SDS-PAGE obtained from a whole cell lysate of Haemophilus ducreyi wild-type strain 35000HP. 0.8 mg of total soluble protein was applied to a linear IPG strip pH 3-10 (from left to right) in the first dimension and separated on a linear 12% SDS-PAGE in the second dimension. The gel was stained with CCB. The gel shown here is highly reproducible and representative of more than five independent experiments and over 20 gels. The numbers correlate with the proteins identified and listed in Table 1. Spot #50 (in bold) has been identified as an 18 kDa outermembrane protein (see Table 1) and appears to be unique to this strain.

fragment ion types a, b, y, a-NH3, b-NH3, y-NH3, b-H2O, and internal. Open reading frames obtained from genome searches were submitted to BLAST (NCBI blastp) searches against the nonredundant NCBI protein database.

Results Analysis of the Soluble Protein Fraction of H. ducreyi strain 35 000 by 2-DE. For proteome mapping, we used a whole cell lysate to prepare a soluble protein fraction of H. ducreyi which was separated by 2-DE and stained with either CCB or silver. The 2-DE protein spot patterns obtained on over 20 gels from 5 separate protein preparations were highly reproducible in terms of both the total number of proteins detected and their relative positions and intensities. A typical 2-DE gel is shown in Figure 2. The reproducibility of the 2-DE gels was observed whether they were simple replicates or originated from separate bacterial preparations. Silver stained gels that were loaded with a tenth of the amount of material of the CCB stained gels (∼80 µg) showed only a relatively small number (∼20) of additional faint spots (data not shown). Identification of Proteins Separated by 2-DE by Mass Spectrometry. To identify the major proteins separated by 2-DE, 120 of the more abundant spots as visualized by CCB staining (see Figure 2) were excised and subjected to tryptic in-gel digestion. The peptide mixtures obtained were analyzed on a MALDI-TOF mass spectrometer and the peptide masses obtained were used for database searches. Initially, the NCBI and Swiss-Prot databases (species: Haemophilus) were used. However, in most cases (>90%) no protein in the databases


matched the submitted mass fingerprints using this constraint. Only 12 proteins were identified using publicly available databases. This is due to the fact that only a very small percentage of H. ducreyi proteins are currently available in public databases (174 proteins in NCBI compared to an estimated total of 1719 proteins for H. ducreyi strain 35000HP) and the homology to other bacterial proteins from species whose genomes are available (i.e., Haemophilus influenzae) was insufficient to determine identity solely from peptide mass fingerprint data. To identify more proteins, we used the newly (and only partially annotated) sequenced genome of H. ducreyi (www.microbial-pathogenesis.org/) that was implemented as a translated database in Protein Prospector. MS-Fit searches were performed, attempting to match the submitted peptide mass fingerprints against the unannotated ORFs of the genome. In nearly all of the cases (>98%), the searches obtained at least one matching ORF. The identified ORFs were subjected to BLAST searches to assign a putative protein name to the ORF. Proteins were identified based on the homology (∼80%) to orthologous H. influenzae proteins. Out of the 120 analyzed spots, 116 distinct proteins were identified (Table 1). Analysis of the Insoluble Protein Fraction Using a Twodetergent Gel System. Because of known difficulties in solubilizing and separating hydrophobic proteins by conventional 2-DE techniques, we analyzed spots from a gel obtained from a membrane fraction of H. ducreyi separated using a twodimensional detergent system.45,46 The advantage of this latter method is that solubilization of the sample is more likely to yield hydrophobic and membrane proteins. However, the properties of the acidic 16-BAC system used in this twodetergent separation are different from those of the basic SDSsystem resulting in a substantial separation. As shown in Figure 3, the gel map obtained yielded a significant but smaller number of proteins than were detected by the conventional 2-DE SDS-PAGE gels. To determine the types of protein that constituted this “insoluble membrane fraction”, nine of the most abundant spots from the two-detergent gel were analyzed. Several of these proteins were also present (but generally in lower abundance) in the “soluble protein fraction” run shown in Figure 2; the major outer-membrane protein (MOMP) (spots 125 and 127; and spot 9), EF-Tu (spot 124; and spot 3), outer membrane protein P2 (spot 125; and spots 9 and 5), CdtB (spot 127; and spot 86), periplasmic serine protease DO (spot 123; and spots 21 and 64) and pyruvate and 2-oxoglutarate dehydrogenase complexes E3 component (spot 122; and spot 27). The remaining proteins that were only found in the “insoluble protein fraction” were all outer membrane proteins, including Hlp, DsrA, cytolethal distending toxin, P4 lipoprotein, spermidine/putrescine-binding protein and HgbA. Analysis of Other Strains of H. ducreyi using 2-DE SDSPAGE. As shown in Figure 4, the soluble protein fraction obtained from the whole cell lysates of three wild-type strains (O41, A77, and BG411) and three LOS-deficient mutant strains were separated by 2-DE and visualized by CCB staining. Our objectives at this point were 2-fold: one, to determine whether wild-type strains that synthesize different LOS structures and/ or are less virulent show significant differences in protein expression and two, to assess whether the knockout of LOS biosynthesis genes has a significant effect on protein expression? Comparison of the 2-DE gels from extracts of strain 35000HP and three other wild type strains shown in Figure 4 (i.e., O41,

research articles A77 and BG411) show that the overall spot pattern is nearly identical with respect to both the location and size of the protein spots. Only a few distinct and reproducible differences could be detected. Similarly, the isogenic strains containing defects in sialic acid biosynthesis (35000HP-RSM202; Figure 4D) or glycosyltransferases (35000.3 and 35000.4; Figure 4, parts E and F) showed little variation; both among them as well as in comparison to the other wild-type strains (Figure 4, parts A-C), including the prototype 35000HP strain shown in Figure 2. The single exception to this similarity was found in the 2-DE gel map of strain 33921 as shown in more detail in Figure 5. In this latter African strain, the overall 2-DE spot pattern was found to be significantly different from all other strains studied, including some of the most abundant protein spots (as will be discussed later). Differences in the Expression of the 18 kDa OuterMembrane Protein. In comparing the 2-DE gels among different strains, the main differences were found in the basic region. Although this region appears to contain significant differences, it is difficult to say whether they represent true protein differences or are artifacts of the separation that is influenced by other components of the outer membranes of these organisms. Spot #50 (Figure 2) was the most noticeable and reproducible protein difference found between strain 35000HP and all other strains tested. Gels obtained from strain 35000HP extracts were the only ones that contained this protein spot, even when these same gels were stained with the more sensitive silver staining reagent. On the basis of the MALDI spectrum for this trypsindigested spot (Figure 6A), the protein was identified as an 18 kDa outer membrane protein with homology to peptidoglycanassociated lipoprotein or PAL.7 This result was at first surprising given the molecular weight and pI of this protein was predicted to be 10-15 kDa with a pI of 5. The identified protein as found in the database has a listed molecular weight of 18 kDa and a pI of 7.4. However, these apparent discrepancies are likely due to the combined effects of cleavage of a highly basic 19 amino acid signal sequence and the subsequent posttranslational lipidation of this protein as suggested in the original report by Spinola and colleagues 7. To obtain additional information on this protein, the peptide mixture was separated by HPLC and fractions were directly collected onto a MALDI target. All fractions were screened by MALDI analysis and a peptide MH+ of m/z 1318.7 was selected for MALDI-PSD analysis. The identity of the outer membrane protein was unambiguously confirmed based on the fragment masses obtained for this peptide (Figure 6B). Comparison of 2-DE Gel Maps from Strains 35000HP and 33921. On first comparison, one can see many similarities between the 2-DE maps of strain 35000HP (Figure 2) and 33921 (Figure 5). For one, the three most abundant spots, #1-3 (HSP 70, chaperonin and EF-Tu, respectively), are strikingly similar between the two gels. These spots and others are also shared among the other wild-type strains and mutants shown in Figure 3. Despite these and other similarities, several groups of smaller spots appear to have disappeared or shifted in the first dimension (pI) in the 2-DE map of strain 33921. For a more detailed comparison of the 2-DE gel maps obtained from both strains, 50 spots each from a 12% pH 3-10 gel were excised and subjected to mass spectrometric analysis in an attempt to find spots that represented the same proteins. Figure 5 shows spots identified from strain 33921, which are numbered in accordance with the proteins found on the 2-DE gel from strain Journal of Proteome Research • Vol. 2, No. 5, 2003 527

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Table 1. Proteins Assigned from the 2-DE Gel in Figure 1 (spots #1-120) and from the Two-Detergent 2D Gel in Figure 3 (spots #121-129) of the Wild-Type H. ducreyi Strain 35000HPa spot no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 528

accession no.

H.d. no.

MW (kDa)

pI

P44865 P45111 P43713

HD0189 HD1784 HD0658 HD0663 HD0010 HD0477 HD1588 HD0657 HD0007 HD0045 HD1435 HD1900 HD1951 HD0700 HD0612 HD1625 HD0990 HD1191 HD1723 HD1624 HD0008 HD0838 HD0418 HD0260 HD0768 HD1073 HD0198 HD1190 HD1331 HD1623 HD1716 HD1686 HD0700 HD0564 HD0833 HD1456 HD0446 HD0084 HD1435 HD1266 HD1600 HD0680 HD1659 HD1340 HD0708

68.5 57.8 45.5 48.4 49.7 45.8 77.3 77.1 60.4 44.2 44.6 26.6 36.4 26.6 42.4 99 86.4 88.7 73.1 55.6 55.3 56.3 61.4 50.7 35.4 22 34.3 29 59.5 50.8 39.2 35.5 26.6 51.6 53.3 43.8 51.6 41.7 44.6 28.1 26.2 27.5 26.5 25.5 25

4.7 4.8 5.4 4.9 5 5 5.1 5 5.1 9.2 9 9.3 5.1 5.3 5.4 5.5 5.6 6.6 5.8 5.7 5.6 6 6 8.4 5.6 4.6 6.3 8.7 5.5 6.1 8.4 4.6 5.3 5.1 5 5.3 6.7 8.4 9 5.6 7 8.7 6.8 7.8 6.6

H.i. H.d. H.d. H.i. E.c. V.c. H.i. H.i. H.d. H.d. H.i. H.i. H.i.

P31810 Q47953 Q47953 P43771 P17288 A82403 P44853 P43709 1150840 399239 P44348 P44894 P45107

HD0638 HD0068 HD0068 HD1407 HD0169 HD0620 HD0661 HD1931 HD1772 HD1785 HD1879 HD0030 HD1457

23.7 21.1 21.1 20.7 19.4 19.7 19.2 11.5 17.1 10.2 12.2 66.2 76.8

7.7 5.8 5.8 4.9 5 5.3 4.2 4.6 6.3 5 4.7 6.1 5.3

H.i. H.i. N.m. H.i. H.i. H.i. M.p. H.i. H.i. N.m. H.i. H.i. H.i. H.i. H.i. H.i. H.i.

P43820 P45055 7227111 P43822 P44910 P43829 P75067 P43833 P45129 7379352 P43889 Q57337 P45040 P44304 P45048 P44338 P44429

HD1483 HD0310 HD0233 HD1942 HD0299 HD1411 HD1445 HD1133 HD0260 HD1282 HD1511 HD1082 HD0896 HD1291 HD1890 HD1913 HD0864

87.4 34.9 118.3 75.6 68.3 52.6 55.6 48.8 51.1 51.6 49.3 45.6 33.9 35.8 35.3 37.3 39.2

5.2 5.1 5.2 5.3 5.4 5.5 5.6 5.8 8.7 6.7 7.7 6 6.5 6.1 5.7 5.5 5.3

protein

speciesb

DnaK (HSP 70) 60 kDa chaperonin elongation factor Tu (EF-Tu) trigger factor ATP-synthase beta chain enolase polyribonucleotide nucleotidyltransferase elongation factor G (EF-G) ATP-synthase delta chain major outer membrane protein (MOMP) outer membrane protein P2 FKBP-type peptidyl-prolyl cis-trans isomerase DNA directed RNA polymerase alpha chain peroxiredoxin/glutaredoxin family protein 3-oxoacyl-(acyl-carrier protein) synthase I pyruvate dehydrogenase complex E1 component formate acetyltransferase protective surface antigen D15 transketolase pyruvate dehydrogenase complex E2 component ATP-synthase alpha chain glucose-6-phosphate 1-dehydrogenase glucose-6-phosphate isomerase periplasmic serine protease DO mannose specific PTS IIAB component GrpE protein (HSP-70 cofactor) periplasmic zinc-binding ABC transport protein unknown (no homology found) phosphoenolpyruvate carboxykinase pyruvate dehydrogenase complex e3 component putative ATPase putative adenine-specific methylase peroxiredoxin/glutaredoxin family protein (see spot #12) aspartate ammonia lyase 6-phosphogluconate dehydrogenase acetate kinase pyruvate kinase L-lactate dehydrogenase outer membrane protein P2 (see spot #9b) enoyl-(acyl carrier protein) reductase (NADH) 30S ribosomal protein S2 unknown (no homology found) phosphoglycerate mutase thiol disulfide interchange protein DSBC 3-ketoacyl-acyl carrier protein reductase no protein matched with PMF thiol:disulfide interchange protein fine tangled pili major subunit fine tangled pili major subunit elongation factor P (EF-P) inorganic pyrophosphatase conserved hypothetical protein VCA0907 protein-export protein SecB acyl carrier protein (ACP) 18 kDa outer membrane protein 10 kDa chaperonin, groES 50 S ribosomal protein L7/L12 fumarate reductase flavoprotein subunit phosphate acetyltransferase no protein matched with PMF phenylalanyl-tRNA synthetase beta-chain transaldolase carbamoyl-phosphate synthase large subunit glycyl-TRNA synthase beta chain GTP binding protein Typ A asparaginyl tRNA synthase hypothetical protein homolog seryl-tRNA synthase periplasmic serine protease DO formate-tetrahydrofolate ligase bifunctional GlmU protein Fe-S cofactor synthase protein cysteine synthase glyceraldehyde 3-phosphate dehydrogenase ADP-glycero-D-manno-heptose-6-epimerase Aspartate-ammonia ligase fructose-bisphosphate aldolase

H.d. H.d. H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.d. H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.i. E.c. H.i. H.d. H.d. H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.i.

P48209 P31294 P43926 P44837 P43715 P43806 P44584 P43925 P48082 P96773 Q48221 P44760 P43737 P44758 P43710 P45119 P43753 F64102 P43757 P45118 P43714 P44311 P44312 P45129 P08186 P43732 6002738

H.i. H.i. H.i.


P43923 P43784 Q57184 P45106 P44758 P44324 P43774 P44406 P43924 P46454 Q48221 P44432 P44371

PMFc MS/MS

% coverage

12/0 12/0 17/0 5/0 5/0 13/1 17/0 14/0 13/3 10/0 11/0 10/0 12/0 8/0 6/0 24/0 18/0 19/0 16/0 10/0 16/0 8/0 7/0 9/0 9/4 9/0 9/0 10/1 12/0 11/0 5/0 7/0 8/0 5/1 5/0 12/4 12/0 9/0 11/5 8/3 11/0 13/0 16/0 7/1 6/0 7/ 7/0 12/0 6/0 5/0 7/0 12/0 4/2 4/1 3/1 6/0 6/0 13/0 18/0 6/ 26/0 5/0 7/0 12/0 16/0 12/0 9/0 6/0 6/0 8/0 7/0 13/0 8/0 7/3 10/0 10/0 6/0

20 26 38 14 30 42 28 24 32 33 29 43 42 30 18 29 25 28 24 17 37 16 16 25 36 42 35 31 34 28 15 22 32 13 9 62 26 58 28 31 45 40 61 25 27

% Identity to H.i.d

84 65 91 90 83 86 74 32 45 90 78 80 88 82 49 84 70 85 78 79 62 34 59 80 85 82 64 78 84 88 73 70 83 32 93 81 83 57 76

29 72 38 28 40 54 45 13 14 51 71 18 32

60

43 21 11 19 30 24 16 16 20 22 19 37 33 24 32 32 23

67 79 77 79 85 83 39 77 62 73 69 83 76 81 79 87 86

84 83 57 76 76 69 80 78

research articles

Proteome of Haemophilus ducreyi Table 1 (Continued) spot no.

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 127 128 129

protein

speciesb

accession no.

H.d. no.

MW (kDa)

pI

PMFc MS/MS

% coverage

% Identity to H.i.d

conserved hypothetical protein transcription antitermination protein NusG uracil phosphoribosyltransferase hypothetical 40.1 kDa protein RecA protein adenylosuccinate synthetase thioredoxin reductase putative pyridoxine biosynthesis protein acetyl-CoA carboxylase alpha subunit mannose specific phosphotransferase IIAB component (see spot #22) aerobic respiration control protein ArcA uridylate kinase adenylate kinase cytolethal distending toxin protein B acetyl-CoA carboxylase beta subunit unknown (no homology found) conserved hypothetical protein single stranded DNA binding protein ribose-phosphate pyrophosphokinase adenosine deaminase THP succinyltransferase periplasmic iron-binding ABC transport protein glycine cleavage system transcriptional activator rod shape-determining protein MreB no protein matched with PMF conserved hypothetical protein glyceraldehyde 3-phosphate dehydrogenase lysyl-tRNA synthase transcription termination factor Rho histidyl-tRNA synthetase glutamyl-tRNA synthetase elongation factor Ts (EF-Ts) elongation factor Ts (EF-Ts) malonyl-CoA acyl carrier protein transacylase putative hexulose-6-phosphate isomerase major outer membrane protein (MOMP) transcriptional regulatory protein CpxR putative peroxiredoxin/glutaredoxin family protein (see spots #12 and 30) NADH-ubiquinone oxidoreductase alpha subunit pyruvate and 2-oxoglutarate dehydrogenase complexes E3 component (see spot #27) unknown (no homology found) 30 S ribosomal protein S6 nucleoside diphosphate kinase 50 S ribosomal protein L10 50 S ribosomal protein L9 conserved hypothetical protein universal stress protein A ribosome recycling factor shikimate kinase conserved hypothetical protein B45 peptidyl-prolyl cis-trans isomerase B (PPIase B) no protein matched with PMF hemoglobin binding protein (HgbA) pyruvate and 2-oxoglutarate dehydrogenase complexes E3 component (see spot #27) periplasmic serine protease DO elongation factor Tu (EF-Tu) major outer membrane protein (MOMP) outer membrane protein P2 spermidine/putrescine-binding protein major outer membrane protein (MOMP) outer membrane P4 lipoprotein cytolethal distending toxin protein B serum resistance protein DsrA lipoprotein Hlp

H.i. H.i. H.i. E.c. H.i. H.i. H.i. H.i. H.i. E.c.

P44058 P43916 P43857 P39300 P43705 P45283 P43788 P45293 P43872 P08186

HD0600 HD1885 HD1178 HD1061 HD0410 HD1807 HD0333 HD1593 HD0051 HD0768

39.4 21.5 22.5 41.2 40.2 47.3 35.5 31.9 35.2 35.4

5.5 5.9 5.6 5.8 4.9 5.6 5.9 5.4 5.4 5.6

14/0 9/0 9/0 13/0 14/4 16/0 4/2 10/0 9/0 9/3

30 52 31 28 46 39 18 43 39 36

80 86 83 61 66 82 71 54 82 34

H.i. H.i. H.i. H.d. H.i.

P44918 P43890 P24323 2102681 P43778 P44634 P44409 P44328 Q9AK25 P45284 G64063 P45099 P44474

30.5 25.7 23.6 31.5 32.7 33.5 26.2 19.6 34.3 37.8 29.7 33.3 33.9 37.3

5.6 5.7 5.6 8.8 8.2 8.5 4.6 5.7 5.3 5.3 5.6 6 5.9 5.5

14/0 11/4 8/0 13/0 6/0 8/0 5/2 11/0 6/0 4/0 6/0 11/0 6/0 11/0

58 59 50 59 19 24 21 42 20 11 19 51 21 42

77 80 83 / 77

H.i. H.i. H.i. S.c. H.i. H.i. H.i. H.i.

HD0278 HD1597 HD0826 HD0903 HD1466 HD1920 HD0596 HD1285 HD1627 HD0377 HD0630 HD1816 HD1028 HD1292

H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.d. H.i. H.i.

P44726 P44304 P43825 P44619 P43823 P43818 P43894 P43894 P43712 P44990 U60646 P44895 P44758

HD0302 HD1291 HD1318 HD0895 HD1039 HD0320 HD1599 HD1599 HD0707 HD1864 HD0046 HD1469 HD0700

33 35.8 56.1 46.9 46.4 54.9 30.3 30.3 33 33 44.7 27.3 26.6

5.2 6.1 5 6.8 6.6 5.8 5.1 5.1 5 5.1 9.4 5 5.3

8/4 4/6 22/1 11/0 9/4 13/0 12/0 12/0 6/0 9/0 11/0 7/0 8/0

28 13 38 27 24 29 62 62 23 28 36 24 34

56 72

H.i. H.i.

P43955 P43784

HD0379 HD1623

47.9 50.8

6.3 6.1

15/0 8/0

40 18

71 85

H.i. H.i. H.i. H.i. H.i. H.i. H.i. H.i. V.c. H.i.

P44375 P43802 P44350 P44349 P45028 P44880 P44307 P43880 A82403 P44499

HD0665 HD1045 HD1053 HD1881 HD1048 HD0256 HD1428 HD1596 HD0423 HD0620 HD1092

15.8 14.1 15.5 15.4 17.7 14.6 15.7 20.8 19.5 19.7 19.5

5.4 6.1 5.9 6.5 6.6 9.5 4.7 6.7 5.1 5.3 5.1

40 60 61 67 49 27 56 56 51 32 54

86 56 91 51 52 69 86 81 57 76

H.d. H.i.

Q47952 P43784

HD2025 HD1623

110.9 50.8

9.1 6.1

7/5 8/4 8/0 10/0 10/0 4/0 7/0 9/0 7/0 8/0 6/0 12/6 23/0 7/0

22 18

85

H.i. H.i. H.d. H.i. H.i. H.d. H.i. H.d. H.d. H.d.

P45129 P43926 P96773 Q48221 P45168 P96773 M68502 2102683 7188587 6969432

HD0260 HD0658 HD0045 HD1435 HD1074 HD0045 HD1170 HD0903 HD0769 HD0580

51.1 45.4 44.2 44.6 39.8 44.2 31.5 31.5 28.5 23

8.7 5.4 9.2 9 5 9.2 9.2 8.8 9.2 4.6

12/0 11/0 10/0 8/0 7/0 6/0 6/0 4/0 5/0 6/0

35 31 36 21 31 27 23 22 24 45

94 71 91 34 89 75 51 81 70 81 78 93 77 79 86 86 71 72

62 84 32 80 58

a Summary of proteins identified from a whole cell lysate of H. ducreyi separated by 2D SDS-PAGE using mass fingerprints (MFP) and sequence information (MS/MS or PSD) obtained by mass spectrometry. b The column ”species” contains H. ducreyi as well as other species. In cases where a protein entry for H. ducreyi was available in a public database, H. ducreyi is listed. In those cases where the protein’s name was obtained by homology searches (BLAST searches) the other species and the database accession number is listed. Accession numbers are SwissProt or NCBI. Abbreviation used for bacterial species are: H.i., Haemophilus influenzae; H.d., Haemophilus ducreyi; E.c., Escherchia coli; M.p., Mycoplasma pneumonia; N.m., Neisseria meningitidis; S.c., Streptomyces coelicolor; V.c., Vibrio cholerae. c PMF/MS-MS refers to the number of peptides whose masses (PMF or peptide mass fingerprints) were used to identify the protein and the number of peptides that provided limited sequence information after being subjected to PSD or MS/MS analysis. d % identity to H.i. refers to the sequence identity of the H. ducreyi protein to the homologous protein in H. influenzae based on the those predicted from translating their respective gene sequences.

Journal of Proteome Research • Vol. 2, No. 5, 2003 529

research articles

Scheffler et al.

Figure 3. Acrylamide gel obtained from the insoluble fraction (pellet) of strain 35000HP using a two-detergent system. The pellet was resuspended in buffer containing the cationic detergent 16-BAC and electrophoretically separated on a polyacrylamide gel. The gel was fixed, stained with CCB, and the whole lane was excised and placed on a 12% SDS gel bearing one big well to accommodate the gel slice. The proteins were again electrophoretically separated on an SDS polyacrylamide gel and subsequently visualized by staining the gel with CCB. The gel shown is representative of two independent experiments.

35000HP. Out of these 50 proteins, 42 were identified to be the same as proteins found in strain 35000HP, although their positions on the gel in the pI dimension were in some cases slightly different.

Discussion In the analysis of wild-type strains of H. ducreyi, our major purpose in this study was not to provide a high-coverage analysis of their proteomes, but rather to see if any global changes were evident that may relate to their geographic origin, virulence and/or carbohydrate expression. In fact, very few differences were found among the proteomes of the prototype strain 35000HP and the three of the four wild-type strains (i.e., O41, BG411 and A77) suggesting a limited genetic diversity. Isogenic mutants strains that were defective in LOS synthesis, i.e., 35000.3, 35000.4, and 35000HP-RSM202, also showed very little, if any, differences in their overall protein-spot patterns and relative intensities in comparison to the prototype strain 35000HP or to each other. The latter result is consistent with previous reports from our group of nearly identical 1D SDSPAGE gel profiles of outer-membrane proteins obtained from mutant organisms of H. ducreyi defective in LOS-oligosaccharide branch synthesis and their parental wild-type strains.50,51 Some small differences, however, were found in the highly basic region as noted for the 18 kDa outer-membrane protein, a peptidoglycan-associated lipoprotein (PAL). The significance of these variations has not yet been determined, but immunoreactivity to the PAL-like protein has been reported to be largely conserved among H. ducreyi strains.7 The absence of PAL in all but the prototype 35000HP strain, therefore, may be an artifact of protein solubilization or related to some as yet unknown modification of the protein. A more significant difference among the 2-DE gel profiles was found between the wild type strain 33921 and all other strains (both mutant and wild type) examined in this study. It is interesting to note that strain 33921 was found in a previous 530


Figure 4. 2-D SDS-PAGE obtained from 0.8 mg total protein obtained from whole cell lysate of three wild type strains (A) O41, (B) A77, and (C) BG411 (formerly Hd9), and the three LOSdeficient isogenic mutant strains, (D) 35000HP-RSM202, (E) 35000. 3, and (F) 35000.4.

study to synthesize an atypical oligosaccharide branch structure that did not contain N-acetyllactosamine or lactose, terminal structures found on the vast majority of wild type strains that are also partially modified by sialic acid 25,26,52 (Gibson and Melaugh, unpublished data). In addition, this African strain lacks the DD-heptosyltransferase gene responsible for the incorporation of the unusual branch DD-heptose that is present in most wild type H. ducreyi strains (Munson and Gibson, unpublished data). Differences in the spot pattern on the 2-DE gel profiles of strain 33921 and the other strains could be caused by small changes in the amino acid composition that result in a change in the pI of the proteins and thus alter their positions on the 2-DE gel. In support of this possibility, 42 of the 50 spots from strain 33921 were found to be the same protein or a close homologue of ones found in strain 35000HP. However, many of the proteins from strain 33921 were shifted in their absolute position. Spot #7, for example, in strain 35000HP (see Figure 2) contains both PNPase and elongation factor EF-G, whereas there are two closely separated spots for these proteins in strain 33921 (Figure 5, spots #7a and #7b). Another example is spot #32 (acetate kinase), which was found to be decreased in size and significantly shifted toward the basic side in strain 33921. Eight proteins on the 2-DE gel from strain 33921 could not


Figure 5. 2-D SDS-PAGE analysis of 0.8 mg total protein obtained from a whole cell lysate of Haemophilus ducreyi strain 33921, a recent isolate from Kenya. The soluble protein extract was separated on a linear IPG strip pH 3-10 (from left to right) in the first dimension and by linear 12% SDS-PAGE in the second dimension. The gel was stained with CCB. The arrows indicate spots that were analyzed by MALDI-MS but where no protein identifications could be made. The peptide mass fingerprints obtained from these indicated spots also failed to match data obtained from the analysis of any of the spots obtained from the prototype strain 35000HP. Gel spots with boxed numbers (spots #4, 5, 7b, 16, 25, 32, 34, 36, 43/44, and 69/98) correspond to proteins that have shifted in pI relative to the same protein match in strain 35000HP. The gel spots with descending arrow to the right (spots #15,17,21, 25, 34, 37) indicate proteins that have significantly decreased in relative abundance compared to the corresponding protein spot in strain 35000HP. Gel spots labeled A (HD1250, UDP-sugar hydrolase) and B (HD889, purine nucleoside phosphorylase) were not identified in the 2D gel from 35000HP and are therefore presumed to be upregulated in strain 33921 (ascending arrow). The gel shown here is highly reproducible and representative of more than 3 independent experiments and over 10 gels.

readily be identified using the H. ducreyi genome. These proteins may have so many alterations in primary sequence that the database search cannot match them with any sequence in the genome, or they may represent new proteins that are not present in the wild type 35000HP genome. It should be noted, however, that approximately 10% of a diverse collection of H. ducreyi strains also lack the DD-heptosyl transferase gene (Munson, unpublished data). Should these isolates also have a proteome more similar to strain 33921, this African strain may well be the prototype of a second clonal group of H. ducreyi strains. With regard to the size and complexity of the H. ducreyi proteome, analysis of the prototype strain 35000HP (and other wild-type strains) visualized around 500 to 800 distinct spots by 2-DE gel analysis. Assuming that all protein spots were unique proteins, this is still well short of the estimated ∼1700 proteins that the H. ducreyi genome predicts. There are several possible reasons for this discrepancy. First, some proteins, such as integral membrane proteins of low solubility, are incompat-

research articles

Figure 6. (A) MALDI-MS spectrum obtained from the peptide mixture obtained after in-gel digest of spot #50. (B) MALDI-PSD spectrum of a peptide with a monoisotopic mass of m/z 1318.73 obtained after HPLC separation. Peaks labeled “T” in the MALDIMS spectrum (panel A) are trypsin autolysis fragments.

ible with the 2-DE gel separation system. Other proteins may be too small or large, too basic, or perhaps overly hydrophobic to be properly visualized or separated by 2-DE. Second, some proteins are likely to be present at concentrations too low to be detected in an unfractionated whole cell lysate. Third, some proteins may not be expressed under the conditions used in these studies to grow the organisms. Among the 129 proteins identified by mass spectrometry in this study, there was a clear bias toward soluble cytosolic proteins. These proteins include numerous ribosomal proteins and tRNA synthases, chaperones, and proteins involved in energy production, nucleotide transport, carbohydrate metabolism, and transcription (see Table 1). This bias was due in large part to having the spot picking limited to the most abundant proteins, or somewhat less than 10% of the total expected proteins predicted from the genome (129 spots for ∼1700 proteins). Taken as a group, these proteins are analogous to the most abundant proteins observed in the more extensive 2-DE gel study reported for H. influenzae.34,35 As expected, the more hydrophobic or membrane proteins known to be associated with the bacteria were distinctly underrepresented. The absence of many membrane proteins may be attributed, in part, to precipitation and adsorption processes, such as through protein adsorption to the IPG matrix.53,54 Journal of Proteome Research • Vol. 2, No. 5, 2003 531

research articles Although several protocols have been proposed to improve coverage of membrane proteins during 2-DE gel analysis (see for example,55-57), we used a two-detergent system based on 16-BAC and SDS to increase their coverage.45 Indeed, a number of membrane proteins were identified in this two-detergent system that were not seen by 2-DE SDS-PAGE, such as DsrA,6 hemoglobin binding protein,9 major outer-membrane protein or MOMP,58 and a novel lipoprotein, Hlp.59 It is worth noting that many of these membrane proteins have been implicated as virulence factors in H. ducreyi (for review, see Spinola et al., 2002).5 The sensitivity of protein spot detection, and therefore the visualization of the H. ducreyi proteome, depends to large extent on the amount of total protein loaded and staining procedure. In our studies, we used silver and CCB staining techniques that are known to be compatible with mass spectrometric identification. Given the large variation in copy numbers among proteins, some proteins will be present at levels below the limits of detection of CCB staining (∼100 ng/ spot), or even silver staining (∼10 ng/spot). However, for comparative expression studies, such as those carried out on the different wild type and glycosyltransferase knockout mutant strains, we found silver staining to be less reproducible with a narrow dynamic range of detection. It is possible that this limitation can be reduced if fluorescence-staining techniques are employed such as SYPRO Red, which has been reported to increase both dynamic range and reproducibility.60 In addition to problems involving protein separation and visualization, protein identification using mass spectrometry can be problematic. Even if good quality mass spectra are obtained, protein identification will likely fail if the database does not contain the protein or a very close homolog. In the case of organisms whose genomes have not yet been sequenced, the best option appears to be to search using homology instead of identity. But even this option does not often lead to a successful identification. At the beginning of this study, no genome-wide sequence data for H. ducreyi was available. To circumvent this limitation, we tried to identify proteins based on homology with the closely related organism H. influenzae. Despite the availability of high quality mass spectra for many of the H. ducreyi protein spots, only 12 proteins were identified by homology. A similar problem can occur even when only strain-to-strain differences are involved, as in the case of strains 35000HP and 33921. Some of the proteins found on the gel map of strain 33921 that were not present on the gel map of strain 35000HP could not be identified by any means because the genome that was searched was obtained from strain 35000HP. In retrospect, these results are not surprising. Although close homology can, in some cases, produce a protein identification by the methods described above, even minor, conservative amino acid changes can easily cause enough changes in the tryptic peptide masses to make identification difficult or impossible. One can easily calculate that, if an unknown protein is 90% identical to a known one, the probability that a 15 residue peptide from the unknown protein will have at least one substitution compared to the known one is >75%. In this case, both the PMF and the MS/MS methods are more likely to fail than to succeed, and therefore, direct de novo peptide sequencing is essential. 532


Scheffler et al.

Conclusion In this study, we have begun to establish a database of the proteome of H. ducreyi. Both a soluble and an insoluble protein fraction were analyzed by 2-DE SDS-PAGE and a two-detergent system, respectively. Currently, 122 distinct proteins were identified, most of which are sufficiently analogous to proteins in H. influenzae that their functions were assigned (see Table 1). This database is the first step toward an annotated proteome of H. ducreyi and will be a solid basis for further proteomics studies. In addition to the prototype strain 35000HP, several other strains were also investigated by 2-DE, including strains whose LOS-glycoforms expression patterns are atypical and show a reduction in virulence in one or more models of the disease. The lack of any substantial differences in the 2-DE gels in both the wild type and “atypical” (lower virulence) strains suggests little diversity exists in the more abundant soluble protein fraction. Nonetheless, these studies do not preclude the possibility that significant variations exist among their proteomes, particularly in outer-membrane proteins that were not well represented in this 2-DE study. Last, a significant variation in the proteome was noted for the African strain 33921 that has been shown to be less virulent and to display a variant LOS structure. These differences, along with previously reported differences in outer-membrane carbohydrate expression, suggests that this latter strain may represent a second clonal group. A continued, more in-depth analysis of the proteomes of strains 35000HP and 33921 will likely be needed to provide some functional insight into the nature of the virulence, carbohydrate and protein differences observed among these (and other) H. ducreyi strains.

Acknowledgment. We thank Richard Jacobs for his help in implementing the unannotated H. ducreyi genome into the database search engine. This work was supported by Grants to B.W.G. (AI 31254) and RSM (AI 38444 and AI45091). We also thank A. Campagnari (SUNY at Buffalo, NY) and E. Hansen (Southwestern University Medical Center, TX) for the bacterial strains used in this project. References (1) Trees, D. L.; Morse, S. A. Clin. Microbiol. Rev. 1995, 8, 357-375. (2) Mertz, K. J.; Trees, D.; Levine, W. C.; Lewis, J. S.; Litchfield, B.; Pettus, K. S.; Morse, S. A.; St Louis, M. E.; Weiss, J. B.; Schwebke, J.; Dickes, J.; Kee, R.; Reynolds, J.; Hutcheson, D.; Green, D.; Dyer, I.; Richwald, G. A.; Novotny, J.; Weisfuse, I.; Goldberg, M.; O’Donnell, J. A.; Knaup, R. J. Infect. Dis. 1998, 178, 17951798. (3) Flood, J. M.; Sarafian, S. K.; Bolan, G. A.; Lammel, C.; Engelman, J.; Greenblatt, R. M.; Brooks, G. F.; Back, A.; Morse, S. A. J. Infect. Dis. 1993, 167, 1106-1111. (4) Schulte, J. M.; Martich, F. A.; Schmid, G. P. Mor. Mortal Wkly. Rep. CDC Surveill. Summ. 1992, 41, 57-61. (5) Spinola, S. M.; Bauer, M. E.; Munson, Jr., R. S. Infect. Immun. 2002, 70, 1667-1676. (6) Elkins, C.; Morrow, K. J., Jr.; Olsen, B. Infect. Immun. 2000, 68, 1608-1619. (7) Spinola, S. M.; Hiltke, T. J.; Fortney, K.; Shanks, K. L. Infect. Immun. 1996, 64, 1950-1955. (8) Elkins, C. Infect. Immun. 1995, 63, 1241-1245. (9) Stevens, M. K.; Porcella, S.; Klesney, T. J.; Lumbley, S.; Thomas, S. E.; Norgard, M. V.; Radolf, J. D.; Hansen, E. J. Infect. Immun. 1996, 64, 1724-1735. (10) Cope, L. D.; Lumbley, S.; Latimer, J. L.; Klesney-Tait, J.; Stevens, M. K.; Johnson, L. S.; Purven, M.; Munson, R. S., Jr.; Lagergard, T.; Radolf, J. D.; Hansen, E. J. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 4056-4061. (11) Cortes-Bratti, X.; Chaves-Olarte, E.; Lagergard, T.; Thelestam, M. J. Clin. Invest. 1999, 103, 107-115.

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