differential proteomic analysis of the bacillus anthracis ... · secreted proteins, which expands...

JOURNAL OF BACTERIOLOGY, May 2006, p. 3551–3571 Vol. 188, No. 100021-9193/06/$08.00�0 doi:10.1128/JB.188.10.3551–3571.2006Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Differential Proteomic Analysis of the Bacillus anthracis Secretome:Distinct Plasmid and Chromosome CO2-Dependent Cross Talk

Mechanisms Modulate Extracellular Proteolytic Activities†Theodor Chitlaru,1 Orit Gat,1 Yael Gozlan,2 Naomi Ariel,1 and Avigdor Shafferman1*

Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research,1 andLife Science Research Israel Ltd.,2 Ness-Ziona 74100, Israel

Received 11 January 2006/Accepted 19 February 2006

The secretomes of a virulent Bacillus anthracis strain and of avirulent strains (cured of the virulenceplasmids pXO1 and pXO2), cultured in rich and minimal media, were studied by a comparative proteomicapproach. More than 400 protein spots, representing the products of 64 genes, were identified, and aunique pattern of protein relative abundance with respect to the presence of the virulence plasmids wasrevealed. In minimal medium under high CO2 tension, conditions considered to simulate those encoun-tered in the host, the presence of the plasmids leads to enhanced expression of 12 chromosome-carriedgenes (10 of which could not be detected in the absence of the plasmids) in addition to expression of 5pXO1-encoded proteins. Furthermore, under these conditions, the presence of the pXO1 and pXO2plasmids leads to the repression of 14 chromosomal genes. On the other hand, in minimal aerobic mediumnot supplemented with CO2, the virulent and avirulent B. anthracis strains manifest very similar proteinsignatures, and most strikingly, two proteins (the metalloproteases InhA1 and NprB, orthologs of geneproducts attributed to the Bacillus cereus group PlcR regulon) represent over 90% of the total secretome.Interestingly, of the 64 identified gene products, at least 31 harbor features characteristic of virulencedeterminants (such as toxins, proteases, nucleotidases, sulfatases, transporters, and detoxification fac-tors), 22 of which are differentially regulated in a plasmid-dependent manner. The nature and theexpression patterns of proteins in the various secretomes suggest that distinct CO2-responsive chromo-some- and plasmid-encoded regulatory factors modulate the secretion of potential novel virulence factors,most of which are associated with extracellular proteolytic activities.

Bacillus anthracis is a gram-positive spore-forming bacte-rium that is the etiological agent of anthrax, a lethal diseasesporadically affecting humans and animals, in particular herbi-vores. In its most severe manifestation, B. anthracis infection isinitiated by inhalation of spores, which are taken up by alveolarmacrophages and germinate into fast-dividing vegetative cellswhich secrete toxins and virulence factors during growth (81,99). If untreated by prompt antibiotic administration, the bac-teria invade the bloodstream, resulting in massive bacteremiaand consequently generalized systemic failure and death. B.anthracis is considered to represent a potential biothreat agent,owing to the severity of the anthrax disease, the ease of respi-ratory contamination, and the perpetual environmental stabil-ity of the infective spores. The recent deliberate disseminationof B. anthracis (15) accelerated the efforts to identify new B.anthracis virulence-related determinants for the design ofnovel diagnostic, preventive, and/or therapeutic strategies.

Fully virulent B. anthracis strains harbor two native plas-mids, pXO1 and pXO2, which encode critical pathogenicityfactors. The absence of either one of the two plasmids resultsin a pronounced attenuation of B. anthracis virulence. ThepXO2 plasmid encodes proteins involved in the biosynthesis of

the poly-D-glutamic acid capsule, which may inhibit phagocy-tosis of bacteria during infection; pXO1 encodes the threetoxin components protective antigen (PA), lethal factor (LF)(a zinc-dependent metalloprotease which proteolytically inac-tivates protein kinase kinases 1 and 2), and edema factor (EF)(a calmodulin-dependent adenylate cyclase), which form twobinary toxins, lethal toxin and edema toxin. PA, the commoncomponent of both toxins, is not toxic by itself, yet it plays thecentral role of binding a specific receptor on the host cells andtranslocating LF and EF into the cytosol of infected cells,where they exert their detrimental activities. Anthrax is ac-knowledged as a toxinogenic disease, owing to the lethality ofpure toxin preparations (77); on the other hand, additional B.anthracis secreted proteins are most probably involved in theonset and course of the disease and in survival of the bacteriain the host.

The regulatory circuits governing the virulence of B. anthra-cis are still to be fully deciphered, yet certain observationssuggest that the virulence of the bacteria entails cross talkmechanisms which link expression of plasmid-encoded andchromosomally encoded genes. The regulatory AtxA protein,encoded by pXO1, is essential for expression of the toxin andcapsule synthesis genes in vivo (a situation which can be mim-icked by growing the bacteria in minimal medium under highbicarbonate-CO2 conditions) (75). Two additional regulatoryproteins, AcpA and AcpB, encoded by pXO2, were suggestedto act downstream of AtxA and to affect capsule synthesis (35,36). AtxA was found also to influence expression of chromo-

* Corresponding author. Mailing address: Israel Institute for Bio-logical Research, P.O. Box 19, Ness-Ziona 74100, Israel. Phone: 972 89381595. Fax: 972 8 9401404. E-mail: [email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

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somal genes, either directly or via AcpA and AcpB. In addi-tion, the protein AbrB, which is a chromosomally encodedtransition state regulator, was suggested to negatively controlthe activity of the toxin gene promoters (123, 131) via AtxA.

Secreted proteins include factors involved in pathogenicity,in particular in gram-positive bacteria (79). Such proteins mayserve as possible targets for diagnostic purposes and/or thera-peutic intervention. Bacteria of the Bacillus cereus phyloge-netic group (B. cereus and Bacillus thuringiensis), to which B.anthracis belongs, secrete a diversity of factors that are essen-tial for virulence, including toxins, hemolysins, proteases, andlecithinases. Notably, in these bacteria the secretion of certainvirulence factors is regulated by a pleiotropic regulator, PlcR(2, 82, 87, 110), which is inactive in B. anthracis (2). It has beensuggested that the evolutionary inactivation of the PlcR regu-lon in B. anthracis was due to incompatibility with the AtxA-controlled regulon and reflects the fact that the PlcR targetgenes are not essential for anthrax pathogenicity (96). Severalstudies have postulated that secreted proteases, other thanthose belonging to the silenced PlcR regulon, are responsiblefor some clinical manifestations of anthrax (1, 9, 117, 140).Such proteases could damage host tissues, interfere with im-mune effectors of the host, and/or provide nutrients for bacte-rial survival (103). Some chromosomally encoded B. anthracisextracellular proteases were suggested to be controlled byCot43, a novel regulatory gene encoded by pXO1 (9).

The availability of the B. anthracis genomic DNA sequence(109, 121) paved the way for high-throughput genomic, tran-scriptomic, and proteomic analyses of B. anthracis (6, 7, 8, 17,25, 41, 50, 64, 78, 85, 121, 144) in an effort to elucidate patho-genicity mechanisms by identification of novel virulence factorsor in search for specific therapeutic and/or diagnostic targets.Indeed, bioinformatic surveys of the B. anthracis genome (7, 8,121) suggested that proteins other than PA, LF, and EF, mayparticipate in anthrax pathogenesis. Furthermore, many B. an-thracis open reading frames (ORFs) encode potentially se-creted or membrane-bound proteins exhibiting homology toknown virulence factors from other bacteria (7, 8).

A preliminary proteomic study carried out in our laboratoryexamined membrane proteins prepared from a nonvirulent B.anthracis strain and led to the recognition of a number ofimmunodominant exposed proteins (8, 25). Here, we docu-ment an extended proteomic study, focusing on B. anthracissecreted proteins, which expands the data set of expressed B.anthracis proteins from both virulent and nonvirulent strains(6, 25, 41, 50, 64, 78, 85, 144). Based on identification of morethan 400 two-dimensional electrophoresis (2-DE)-separatedprotein spots, we report the expression of 64 proteins whichrepresent the most abundant B. anthracis secreted proteins,many of which resemble factors involved in the virulence ofother pathogens. Comparison of the relative abundances ofproteins in pXO1- and pXO2-containing and plasmid-curedstrains reveals about 30 ORFs which are either preferentiallyexpressed or repressed in the virulent B. anthracis strain underconditions which are considered to simulate those encounteredwithin the mammalian host. The pattern of expression of thesespecific proteins demonstrates that B. anthracis possesses dis-tinct regulatory pathways which involve plasmid- or chromo-some-encoded CO2-inducible responsive factors.

MATERIALS AND METHODS

Bacterial cultures and sample preparation for 2-DE. The B. anthracis strainsused in this study are: the fully virulent Vollum strain (pXO1� pXO2�) and theattenuated strains �Vollum (pXO1� pXO2�) and �14185 (pXO1� pXO2�).Strain �14185 is a pXO1-deleted derivative of the nonproteolytic vaccine strainV770-NP1-R (148) (ATCC 14185).

Cells were cultured in either FAG medium (27), brain heart infusion (BHI)(Difco/Becton Dickinson, MD), or NBY medium (containing 0.8% [w/vol] nu-trient broth [Difco], 0.3% yeast extract [Difco], and 0.5% glucose) for up to 24 hat 37°C with vigorous agitation. The rich FAG and BHI media were selectedfollowing a preliminary pilot study in which different media routinely used for B.anthracis cultures were examined for their ability to support maximal cell yieldand to promote expression of a rich complementary repertoire of extracellularproteins (data not shown). For identification of CO2-induced proteins, cells weregrown at 37°C in NBY supplemented with 0.9% NaHCO3 in hermetically sealedfilled flasks without agitation (referred to hereafter as NBY-CO2). Under theseconditions, which we define as semiaerobic, no aeration of the culture occursduring growth, except for the small amount of air present at the onset of theculture. After 12 h of growth, the bacterial cultures typically reach optical den-sities at 660 nm of approximately 15, 12, and 6 in FAG, BHI, and NBY, respec-tively. In NBY-CO2, an optical density at 660 nm of 1 to 2 is observed after 12 h.In all cases, no visible change in optical density was observed between 12 and 36 hof culture. The low-nutrient-content (compared to FAG or BHI) NBY-CO2

medium promotes very efficient toxin production and capsule synthesis (whichcan be visualized by negative staining using India ink [Becton Dickinson, MD]).

Secreted proteins were collected from the culture-conditioned media essen-tially as described by Antelman and coworkers (4). In brief, cultures were cen-trifuged for removal of cells and subjected to filtration using 0.22-�m filters. Onehundred milliliters of FAG or BHI conditioned medium or 200 ml of NBYmedium was incubated in the cold overnight in the presence of 10% trichloro-acetic acid and then precipitated by centrifugation for 30 min in a Sorvall S34instrument (12,000 rpm). Pellets of trichloroacetic acid-precipitated proteinswere washed four times in a large volume of 96% ethanol and then resuspendedby scraping and extensive pipetting in 5 ml of an isoelectric focusing (IEF)sample solution composed of 8 M urea, 4% (wt/vol) 3-[(3-cholamidopropyl)di-methylammonio]-1-propanesulfonate (CHAPS), 40 mM Tris, 2% dithiothreitol(DTT), and 0.2 (wt/vol) Bio-Lyte 3/10 (Bio-Rad). The clear solution containedapproximately 0.3 mg/ml protein.

2-DE separation of proteins and spot quantitation. The secreted protein mixture(100 �g/run, representing approximately 7 ml of FAG or BHI culture medium or 15ml of NBY medium) was resolved first by IEF on pH 3 to 10 (nonlinear) ready-made, 17-cm, immobilized pH gradient strips (Immobiline DryStrips; Pharmacia),applied to a Protean IEF cell (Bio-Rad). IEF was carried out at 10,000 V to a totalof 50,000 V-h, initiated by a slow step at 250 V for 30 min. Strips were then processedfor the second-dimension separation by a 10-min incubation in 6 M urea, 2% sodiumdodecyl sulfate (SDS), 0.375 M Tris-HCl (pH 8.8), 20% glycerol, 2% (wt/vol) DTT,followed by a 10-min incubation in a similar solution in which the DTT was replacedby 2% iodoacetamide. Strips were applied to 12.5% SDS-polyacrylamide gels, andelectrophoresis was carried out on an Ettan DALT II System (Pharmacia). Gelswere stained with Coomassie blue G-250 (Bio-safe Coomassie; Bio-Rad) and spotsdetected and analyzed by scanning on a GS-800 calibrated densitometer assisted bythe PDQuest 2-D software (Bio-Rad). In each case, at least two independentlyobtained secretomes representing the same biological sample were evaluated by2-DE, and for each sample, at least three gels were used. Apparent induction orrepression of a particular protein was considered to have occurred if the relativeintensities of its respective pairwise spots in the compared gels exhibited at least afivefold difference. Reference protein spots with essentially identical intensities wereused for quantitative comparisons. It is conceivable that the use of a more sensitivestaining procedure could detect additional protein spots, yet the Coomassie bluestaining (approximate detection level of 50 ng protein/spot) enabled visualization ofthe most abundant protein species as well as the major changes associated with theculture conditions.

In-gel trypsin digestion and MALDI-TOF identification of proteins. Proteinspots were cut from 2-DE gels, destained for 1 h in 30% CH3CN, 50 mMNH4HCO3, and subjected to in-gel overnight digestion with 10 �l of 6.25-�g/mltrypsin (Promega). Gel fragments were subjected to two rounds of vacuum dryingand resuspension in double-distilled water, and then peptide elutions were carriedout first in 1% trifluoroacetic acid (TFA) (20 min, room temperature) and then in50% CH3CN (20 min, room temperature). Finally, the eluted tryptic peptides weredried under vacuum and resuspended in 10 ml of 25% CH3CN, 0.1% TFA. Twomicroliters was mixed with an equal volume of 10-mg/ml �-cyano aqueous solutioncontaining 50% ethanol, 25% CH3CN, and 0.1% TFA; applied to a matrix-assisted

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laser desorption ionization–time-of-flight (MALDI-TOF) target; and allowed tocrystallize in a vacuum oven at 60°C. Mass spectra were acquired on a MicromassTofSpec 2E instrument in positive ion reflectron mode, using a source voltage of20,000, a pulse voltage of 2,600 to 3,000, and a laser intensity of 20%. Externalcalibration using standard peptides was applied. Tryptic fragments originating fromtrypsin were used as internal standards for correction of mass spectra. MALDI-TOFspectra were processed using the MassLynx mass spectrometry software (Micro-mass) and compared to hypothetical tryptic digestion fragments of all ORFs in theB. anthracis Ames ancestor strain genome DNA sequence by two alternative mo-dalities: (i) online, using the ProFound search engine at http://bioinformatics.genomicsolutions.com/knexus.html (confidence of identification was based on a Zscore of above 2), or (ii) using an in-house-generated identification program de-scribed previously (8, 25). Throughout this paper, chromosomal ORFs are identifiedaccording to the NCBI locus tag identifier of the B. anthracis Ames ancestor chro-mosome and plasmid-located ORFs are identified by their ORF identifier accordingto Okinaka and coworkers (109). Accession numbers of the Ames ancestor genomeare NC_007530 for the chromosome, NC007322 for pXO1, and NC007323 forpXO2 (www.ncbi.nlm.nih.gov/genomes/MICROBES/anthracis.html). Identificationof proteins was based on peptide coverage of more than 30% and peptide massdeviation between observed and calculated values of less than 100 ppm. In cases inwhich the two-dimensional electrophoretic migration of proteins indicated a molec-ular weight lower than predicted, such as for proteins which undergo proteolyticprocessing, their MALDI-TOF tryptic-digest spectra served for determining frag-

ments present in the mature forms, by establishing the positions of the detectedpeptides on the primary amino acid sequences of the proteins. For the sake ofsimplicity, protein spots representing the products of the same ORF were assignedthe same spot number throughout this study.

Sequence analysis of B. anthracis ORFs. Throughout this study, functionalannotation of the B. anthracis Ames strain chromosomal or plasmid ORFs wasaccording to Ariel and coworkers (7, 8). Putative localization of the product ofeach ORF was assigned based on integration of results of analyses for thepresence of signal peptides, lipoprotein-anchoring signals, transmembrane seg-ments, gram-positive-specific anchoring motifs, and peptidoglycan-anchoring do-mains, as previously described (8). The amino acid sequences of all proteinsidentified were subjected to BLAST analysis against all available bacterial ge-nomes of the NCBI data bank for identification of ortholog and paralog genes.Potential export signal peptides were identified using the SignalP (version 3.1)server (http://www.cbs.dtu.dk); in cases when signal sequences could not bepredicted by the computerized analysis, the N terminus of the respective proteinsequence was inspected individually for the presence of typical secretion signal Nand H regions as well as consensus cleavage sites (135, 136, 139). In one case(protein BA1197), the amino acid sequence of the protein lacks a characteristicsignal sequence. Reexamination of the genomic DNA sequence upstream of theputative translation starting point revealed a correct type II signal peptidase(SPase) cleavage signal in an alternative reading frame; this signal is 100%homologous to that present in the B. cereus ortholog. We assume that a DNA

FIG. 1. Overview of the 2-DE maps of the various B. anthracis secretomes inspected in this study. Representative gels of proteins secreted bydifferent virulent and nonvirulent B. anthracis strains under the indicated growth conditions are shown. All secretomes were collected at thestationary phase of the bacterial cultures (20 h) (see Materials and Methods), and equivalent amounts of protein were loaded on gels.

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sequence mistake resulting in a frameshift in the deduced protein sequenceoccurred approximately 40 base pairs upstream of the presently assigned trans-lation initiation point.

Putative conserved functional domains were identified by sequence analysisusing the CDD database on the NCBI protein analysis server (91). The predicted

ligand specificities of all solute-binding domains (SBPs) of ABC transportersdetected in this study are according to the TransportDB classification (http://www.membranetransport.org/other).

Zymography. In-gel proteolytic activity assays were carried out essentially asdescribed by Caballero and coworkers (20). Twenty-five-microliter bacterial con-

FIG. 2. 2-DE separation of proteins secreted by B. anthracis strain Vollum cultured in rich FAG and BHI media. The numbers in the left lowercorners of the panels correspond to those in Fig. 1. For a detailed identification of the various spots marked on the gels, see Table 1. Spots withinboxes are isoforms of same protein. MW, molecular weight (in thousands); NL, nonlinear.



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FIG. 3. 2-DE separation of proteins secreted by B. anthracis strain Vollum cultured in NBY medium. Representative 2-DE gels of the NBY-O2and NBY-CO2 medium secretomes of the virulent B. anthracis Vollum strain are shown. The numbers in the left lower corners of the panelscorrespond to those depicted in Fig. 1. For a detailed identification of the various spots marked on the gels, see Table 1. All spots within boxesrepresent isoforms of same protein. White boxes indicate full-length or partial fragments of PA. Arrows indicate the major proteins InhA1 (spot2), NprB (spot 46), and HtrA (spot 11) (see text). MW, molecular weight (in thousands); NL, nonlinear.



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ditioned medium samples were electrophoresed under nonreducing conditionsusing 10% SDS-polyacrylamide gel electrophoresis with 0.1% gelatin, casein, orpurified PA (27). The gels were washed three times in 2.5% Triton X-100 andincubated for 24 h at 37°C in either casein substrate buffer (50 mM Tris [pH 7.6],1 �M ZnCl2, 100 mM NaCl) or gelatin/PA substrate buffer (50 mM Tris [pH 8],10 mM CaCl2, 1 �M ZnCl2, 150 mM NaCl). The gels were stained with Coo-massie blue G-250 and destained with water.

Preparation of anti-NprB antibodies. The protein NrpB was extracted from2-DE gels of the NBY-O2 secretome of B. anthracis �Vollum and purified usingthe Maxi GeBAflex tube dialysis system (Gene Bio-Application, Israel). Threedoses of gel-purified NrpB were administrated to six mice (5 to 10 �g/animal) at2-week intervals (first injection in complete Freund adjuvant [Sigma]; second andthird injections in incomplete Freund adjuvant). Mice were sacrificed 2 weeksafter the third administration, and their sera were separated from the hematocrit,pooled, and used as specific anti-NrpB antibodies. The specificity of the sera wasconfirmed by Western blot analysis. Mice were handled according to the Na-tional Institutes of Health’s guide for the care and use of laboratory animals andthe guidelines of the local commission for animal care.

RESULTS

Experimental design. B. anthracis secreted proteins wereobtained from different cultures of the wild-type virulent Vol-lum strain (pXO1� pXO2�) and �Vollum plasmid-cured(pXO1� pXO2�) bacteria (145). The studies included also theB. anthracis �14185 strain (27, 94), a plasmid-cured derivate ofthe vaccine ATCC 14185 strain that is defective in proteolyticactivity (42). The specific mutations conferring the nonproteo-lytic phenotype of this strain are not known, yet inspection ofits extracellular proteome served for determining whether ornot the secretome signatures are distorted by the possibleeffect of bacterial secreted proteolytic activity (e.g., see theeffect of the abundant extracellular protease NprB [see below],which is undetected in the �14185 cultures).

Proteins secreted by these B. anthracis strains were collectedfrom essentially three types of cultures (Fig. 1): (i) bacteriagrown aerobically in the rich media BHI and FAG, conditionswhich enable optimal protein expression resulting in a richrepertoire of secreted proteins; (ii) bacteria grown in thelower-nutrient-content NBY medium under aerobic conditions(NBY-O2); and (iii) bacteria grown in NBY medium supple-mented with bicarbonate under semianaerobic conditions(NBY-CO2; see Materials and Methods). The last culture con-ditions, characterized by a high CO2 content, were used tomimic those encountered by the bacteria in the host duringinfection, which are known to induce expression of the B.anthracis toxins and capsule (75). The semianaerobic condi-tions used for the NBY-CO2 culture are necessary for preserv-ing a high CO2 concentration in the culture. It is thereforeimportant to emphasize that the terms NBY-CO2 and NBY-O2

pertain to the relative CO2 concentration rather than to an-aerobic or aerobic conditions.

Composition of the B. anthracis secretome. The protein sig-natures of the three different strains under the various condi-tions (Fig. 1) were obtained by 2-DE fractionation of equiva-lent proteins amounts. Coomassie blue-stained gels (asexemplified in Fig. 2 and 3) were scanned for quantification ofthe various protein spots. More than 500 protein spots could bevisualized on the various 2-DE gels. The identities of the pro-teins represented by the spots were determined by MALDI-TOF analysis of their tryptic digestion products (Table 1; seeTable SI in the supplemental material). In some cases theidentities of different spots could be unequivocally established

by their positions on the proteomic maps; in most of the cases,the identity of a certain spot was confirmed by MALDI-TOFanalysis. Overall, approximately 400 differently migrating pro-tein spots from the various gels were inspected by MALDI-TOF analysis. Under the various conditions used in this study,the major proteins constituting the B. anthracis secretomesrepresent the products of 64 ORFs (Table 1). Twenty-twoproteins have not been previously detected in the B. anthracissecretome and are marked “novel” in Table 1. The vast ma-jority of the proteins detected in this study are chromosomallyencoded, regardless of the presence or absence of the virulenceplasmids. In fact, with the exceptions of PA, LF, and EF, onlythree pXO1-encoded proteins were identified: pXO1-15,pXO1-90, and pXO1-130. Remarkably, no pXO2-encodedproteins were detected in the secretome. Nevertheless, thecharacteristic pXO2-encoded capsule of B. anthracis was de-tected under conditions of high CO2 tension (not shown).

At least 31 proteins identified in the secretome are stronglyrelated to virulence of other pathogenic bacteria (Table 2).This observation suggests that B. anthracis secretes, in additionto its “classic” lethal toxin and edema toxin, a large repertoireof proteins which may be essential for its virulence.

Secretion signal peptides and cell wall retention sequencesin proteins identified in the secretome. (i) Signal peptides.Fifty proteins in the secretome exhibit export signal peptides(Table 1). The signal sequences displayed by the B. anthracissecreted proteins are highly similar to those described forBacillus subtilis, in which the secretion process and secretomehave been extensively studied (4, 5, 70, 135, 136, 139). All 50signals conform to the consensus sequences of type I or type IISPases, and the proteins should therefore be secreted via theSec pathway. The lengths of most of the predicted signals varybetween 20 and 41 amino acids. The protein HtrA (spot 11)exhibits an unusually long secretion signal (58 amino acids). Asexpected, most of the signal peptides of the putative lipopro-teins (processed by type II SPases) are shorter (19 to 21 aminoacids) and invariably include a typical cysteine residue at the�1 position. Fourteen proteins identified in this study do notexhibit a recognizable signal sequence. This may be due to thefact that some secretion signals do not conform to the consen-sus sequence, and the presence of such proteins in bacterialsecretomes may indicate the existence of secretion pathwaysother than those known to involve a canonical export signal.The possibility that these proteins are cytosolic in nature andtheir appearance in the secretome results from cell lysis cannotbe ruled out. Yet, we note that orthologs of eight proteinswhich do not possess a recognizable signal sequence (DnaK,GroEL, enolase, SodA-2, AhpC, TiG, chitosanase, and exo-chitinase Chi36) have been detected in an extracellular form inprevious studies of many other bacteria (18, 50, 80, 107).

The 2-DE electrophoretic migrations of several proteins in-dicated putative removal of extensive segments from the full-length forms, more than would be expected from processing ofregular signal sequences. These proteins are as follows: somePA subforms (Fig. 3); the 3 sulfatases BA5470 (spot 10),BA2947 (spot 29), and BA1436 (spot 53); an InhA1 subform(spot 2) (Fig. 3); the protein QoxA (spot 22); the proteaseNprB (spot 46); and the S-layer homology (SLH) proteinBA0898 (spot 50). In all cases, examination of the respectiveMALDI-TOF spectra established the approximate boundaries



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of these truncations (see below). In the case of the threesulfatases mentioned above (Fig. 4), an explanation for theoccurrence of truncated forms may originate from the fact thatthese proteins exhibit a signal-like sequence located approxi-

mately 180 amino acids from their N termini. This phenome-non of an internally located signal peptide was reported byAntelman and coworkers (6) for two of the proteins (BA5470and BA2947). We note that each of the three sulfatases exhib-

TABLE 2. Secreted proteins with documented involvement in bacterial pathogenesis detected in this study a

Spotno.b Accession no. Protein Documented involvement in virulencec (reference[s])

1 BA4539 DnaK Chlamydia trachomatis, immunogenic (124); Listeria monocytogenes (56, 125);Vibrio cholerae (22); Salmonella enterica, required for invasion and survival inM (19, 132)

2 BA1295 InhA1 Bacillus thuringiensis, required for M escape (119); Bacillus cereus, exosporiumprotein (23)

4 BA0267 GroEL Chlamydia trachomatis, vaccine candidate (124); Listeria monocytogenes (43);Salmonella enterica and others (19)

5 BA5364 Eno Streptococci, involved in tissue invasion, immunosuppressive (32, 141); Candidaalbicans, immunogenic, protective, vaccine candidate (39); Listeriamonocytogenes (125)

6 pXO1-110 PA Bacillus anthracis toxin18 BA3189 MntA d Streptococci and other (13, 132, 133); B. anthracis (48)73 pXO1-130 AdcA d Brucella abortus, required for intracellular survival (73); Streptococcus

pneumoniae (33); Haemophilus ducreyi (45, 83)8 BA1191, BA0656 Opp family d Bacillus thuringiensis: (37, 51, 128); Listeria monocytogenes, required for

intracellular survival (16); Streptococcus pyogenes (130)20 BA1197 Opp family d

28 BA0908 Opp family d

69 BA3645 Opp family d

9 BA4322 Nucleotidase Pseudomonas aureginosa, cytotoxic in M (157); Vibrio cholerae, cytotoxic inM (118); Haemophilus influenzae, immunodominant, vaccine candidate (158)43 BA3162 Nucleotidase

10 BA5470 Sulfatase Pseudomonas aureginosa, Helicobacter pylori, Burkholderia cepacia (57);Eschericia coli (62); Clostridium difficile (126); Mycobacterium tuberculosis(104); Campylobacter jejuni (155)

29 BA2947 Sulfatase53 BA1436 Sulfatase11 BA3660 HtrA Pasteurella piscicida (61); Chlamydia pneumoniae (101); Enterotoxigenic E. coli

(138); Helicobacter pylori (55); Haemophilus influenzae (86); Yersiniaenterocolitica (60, 84); Salmonella typhimurium (44, 68); Streptococcuspneumoniae (66); Klebsiella pneumoniae (28); Streptococcus pyogenes (55, 69)

21 BA1290 Camelysin Bacillus cereus (52)27 BAS5205 Collagen adhesin Staphylococcus aureus (106, 150, 151); Pneumoviridea virulence (88)34 BA2944 Polysaccharide deacetylase Streptococcus pneumoniae (14, 143)40 BA0331 Polysaccharide deacetylase36 BA0165 Prolyl olygopeptidase Streptococcus pneumoniae (113); Salmonella enterica (102); Porphyromonas

gingivalis (10); Trypanosoma cruzi (53)38 BA1952 NlpC/P60 family protein Listeria monocytogenes (92); Staphylococcus aureus, Clostridium acetobutylicum,

Mycobacterium tuberculosis (3); Corynebacterium diphtheriae (149)41 BA2793 Chitin-binding protein Staphylococcus areus (54); Listeria monocytogenes (34)46 BA0599 NprB Listeria monocytogenes (26); Bacillus cereus (110)49 BA3737 Alanine amidase Clostridium, Enterococcus faecalis, Pseudomonas aureginosa, Neisseria

meningitidis, Streptococcus agalactiae, Staphylococcus, Yersinia pestis (122);Salmonella enterica (40); Listeria monocytogenes (21); Bacillus anthracis,immunodominant membranal proteins (8, 25)

50 BA0898 Alanine amidase

52 pXO1-107 LF Bacillus anthracis toxin57 BA4346 YfkN Clostridium perfringes (11); Yersinia enterocolitica (156)59 BA5427 LytE Listeria monocytogenes (92); Staphylococcus aureus, Clostridium acetobutylicum,

Mycobacterium tuberculosis (3); Corynebacterium diphtheriae (149); ERMdomain is involved in adhesion (90, 127)

60 pXO1-122 EF B. anthracis toxin64 BA5696 Superoxide dismutase, Mn Listeria monocytogenes, highly immunogenic and vaccine candidate (59);

Mycobacterium tuberculosis (18); Vibrio vulnificus (72); Brucella abortus (112)67 BA0345 AhpC Salmonella enterica, immunogenic (134); Mycobacterium tuberculosis (129);

Helicobacter pylori, immunogenic (111, 154); Legionella pneumophilla, requiredfor survival in M (120); Mycobacterium leprae, required for survival in M(115); Bacillus anthracis, immunodominant membranal protein (25)

a The table represents a partial list of known and potential virulence factors among B. anthracis secreted proteins.b Proteins are listed according to their respective spot numbers, except for the paralogs identified in this study (three sulfatases [spots 10, 29, and 53], two

nucleotidases [spots 9 and 43], two alanine amidases [spots 49 and 50], and two polysaccharide deacetylases [spots 34 and 40]), which are grouped. Also grouped areproteins representing solute-binding subunits of ABC transporters. The B. anthracis classic toxin components are in boldface.

c All of the selected pathogenic bacterial systems and references provided exemplify the involvement of these proteins in virulence. In some cases additional briefinformation pertaining to the role and significance of the proteins is also provided. M, macrophage.

d ABC transporter (solute-binding subunit).



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its extensive transmembrane domains (five hydrophobic loops)located within the N-terminal portion of the molecule (Fig. 4;see below), the removal of which could convert these proteinsinto the soluble form, compatible with their occurrence in thesecretome. A mechanism which ensures the dual location ofthese sulfatases, as both membrane-bound and secreted forms,may be beneficial to the bacteria in invasiveness and patho-genic colonization (57, 62, 104, 126, 155).

(ii) Membrane retention sequences. The occurrence of typ-ically cell-associated proteins in the extracellular milieu is anintriguing phenomenon, which has been documented in B.subtilis and other bacteria (4, 107, 137, 142). The exact mech-anism of the release of membrane-anchored or covalently cell-wall-attached proteins is still not resolved. In the present study,22 proteins detected in the B. anthracis secretome possessretention sequences (Table 1): 4 proteins exhibit hydrophobictransmembrane segments (the three sulfatases describedabove [BA5470/spot 10, BA2947/spot 29, and BA1436/spot 53]and the protein QoxA/spot 20), 2 proteins exhibit an LPXTGsortase recognition sequence (93) for covalent anchoring(BA3367/spot 3 and BA4346/spot 57), 9 lipoproteins repre-senting the ligand-binding subunits of ABC transporters ex-hibit a lipobox for lipoprotein covalent attachment (BA1191and BA0656/spot 8, BA3189/spot 18, BA1197/spot 20,BA0908/spot 28, BA0855/spot 30, BA5220/spot 65, BA3645/spot 69, and pXO1-130/spot 73), and 6 proteins possess SLH

domains (Sap/spot 19, EA1/spot 47, BA3338/spot 48, BA3737/spot 49, BA0898/spot 50, and pXO1-90/spot 51).

Ligand-binding subunits of ABC transporters have been re-ported to represent a major class of secreted proteins in othergram-positive bacteria (4, 137). We note that in previous pro-teomic analyses of B. anthracis membranes (25), only two of thenine ABC transporter solute-binding subunits detected in thepresent study were identified: BA5220 (spot 65) and BA3189(MntA, spot 18). MntA has recently been shown to represent animportant virulence determinant of B. anthracis (48).

The S-layer proteins Sap and EA1 are known to exist also assubunits in the extracellular medium, and Sap has been shownto be released in considerable amounts (95, 97). Interestingly,all five chromosomally encoded SLH domain-harboring pro-teins observed in the secretome were detected in the B. an-thracis membrane fraction in a previous proteomic study (25).

Functional classification of the secreted proteins. Twelve pro-teins identified in this study can be defined as proteins with anunknown function; their frequency (18%) is somewhat lower thantheir representation in the genome (30%) (7, 8, 121), a phenom-enon often encountered in proteomic studies (see reference 25for a discussion addressing this issue). Furthermore, we note that7 of the 12 proteins of unknown function exhibit recognizabledomains: BA0685 (spot 7) possesses a 3D domain (putativepeptidase domain), BA0796 (spot 56) exhibits both a 3D do-main and an SH3 domain (involved in protein-protein inter-

BA2947

pH

MW

62

47.5

BA2947

BA5470

BA1436

BA1436

DRNYIVKYLGAYNYTIYDGIQSAKASTER-ALA

DRQTVVKNLGLYTYHLFDITLQSKSSAERVF-ASG

DRVMLVKNLGLYVHQVYDLGLQAKSSSQK-AFA

4 5

BA5470

Hydrophobicity

FIG. 4. Evidence for removal of transmembrane domains from three secreted B. anthracis sulfatases identified in this study. The positions ofthe electrophoretic migrations of the three sulfatases BA2947, BA1436, and BA5470 are indicated on the 2-DE gel (enlarged portion of gel 1 fromFig. 1) depicted in the left panel, and their Kyte-Doolittle hydropathy profiles (76) are provided in the right panel. The secretion signal-likesequence is indicated; the predicted SPase I cleavage site is indicated by a dashed line. Shaded boxes under the hydropathy profiles indicate thepositions of the tryptic peptides identified in the MALDI-TOF spectra.



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actions), BA0799 (spot 39) exhibits a truncated Hly domain(involved in toxin secretion in gram-negative bacteria),BA3338 (spot 48) and pXO1-90 (spot 51) exhibit SLH anchor-age domains, the lipoprotein BA3588 (spot 17) is a putativesurface-anchored protein, and BA3367 (spot 3) exhibits a sor-tase recognition LPXTG motif. This ORF of unknown func-tion has been recently recognized as a � phage receptor (31).

Almost 80% of the polypeptides identified are functionallyannotated (see Table 1 for a short description of their func-tion), allowing their classification into six functional categories(Table 3): enzymes, ABC transporters, chaperons, proteinsactive in the process of detoxification, structural proteins, andtoxins (the last category refers to the classical anthrax toxinsPA, LF, and EF).

The numbers of secreted proteins attributed to the variousfunctional categories (Table 3) reveal that B. anthracis releasesinto the medium an unusually high number of proteins pos-sessing activities involved in polypeptide processing: 16 en-zymes have proteolytic and amino acid-degrading activity, and7 transporters exhibit specificity for oligopeptides or aminoacids. This characteristic is even more pronounced when oneconsiders the abundance rather than the number of polypep-tides possessing protein hydrolysis functions (between 20 and95% of the total protein content visualized in the 2-DE gels)(Fig. 5; see below).

With the exception of two polypeptides (InhA1 and NprB,which are orthologs of PlcR target genes [see below]), none ofthe proteins identified in the present study are potential mem-bers of the PlcR regulon, which governs expression of virulencefactors in B. thuringiensis and B. cereus (encoded by orthologsof target genes of the PlcR factor [67] or by genes exhibiting

PlcR-inducible cis-acting recognition sequences upstream oftheir coding sequences [121]). The absence of the PlcR puta-tive targets is in line with the known inactivation of the PlcRregulator in B. anthracis (2, 96).

Differential expression of secreted proteins. The main gen-eral findings following inspection of the various secreted-pro-tein signatures (presented schematically in Fig. 5) are as fol-lows: (i) as expected, a much larger protein repertoire issecreted in rich medium than in minimal medium; (ii) in min-imal aerobic medium, more than 90% of the total secretedpolypeptides are represented by two proteins; (iii) there arevery minor differences between the secretomes of the virulentVollum and avirulent plasmid-cured �Vollum strains gener-ated in rich medium or minimal medium under aerobic con-ditions; (iv) the presence of the virulence plasmids exerts a verysignificant impact on the composition of the secretome whenthe cells are cultured in minimal medium under high CO2

tension, with respect to both chromosome- and plasmid-en-coded proteins; and (v) the expression of the major extracel-lular proteases of B. anthracis is influenced by CO2 and/or bythe presence of the virulence plasmids. These observations aredetailed below (see also Table 1 and Fig. 5).

(i) Rich-medium secretomes. Approximately 500 proteinspots in a wide dynamic range of concentrations can be visu-alized by Coomassie blue staining of the proteomic maps of therich-medium secretomes (Fig. 1 and 2). Small amounts of PAand EF could be detected in the secretome of the Vollumstrain, suggesting that their repression in the absence of CO2

(75) is leaky. Except for these pXO1-related features, the Vol-lum and �Vollum rich-medium secretomes are strikingly sim-ilar and differ only slightly in terms of the abundances of some

TABLE 3. Functional classification of proteins identified in the B. anthracis secretomea

Category Function No. ofproteins Spot nos.

Enzymes Sulfatase 3 10, 29, 53Chitinase 4 41, 62, 63, 66Polysaccharide degradation 2 34, 40Nucleotidase 4 9, 43, 54, 57Phospholypid degradation 2 37, 72Protein/peptidoglycan/amino acid

degradation/modification16 2, 11, 21, 27, 31, 33, 36, 38, 42,

46, 49, 50, 52, 59, 70, 71Energy metabolism 4 5, 22, 70, 71

ABC transporters Transport of amino acids or peptides 7 8, 20, 28, 30, 65, 69Transport of metal ions 2 18, 73ATP-binding subunit of ABC transporter 1 23

Chaperonins, protein folding 4 1, 4, 11, 68

Stress (detoxification) 4 1, 4, 64, 67

Structural S-layer protein 2 19, 47

Toxins PA, LF, EF 3 6, 52, 60

Unknown function Exhibit recognizable domains 7 3, 7, 17, 39, 48, 51, 56No recognizable domains 5 32, 45, 55, 58, 61

a See Table 1 for a detailed description of the functions of individual proteins. Some proteins have a dual role and therefore are included in two functional categories:GroEL (spot 4) and DnaK (spot 1) are categorized as chaperones, yet these are heat shock proteins induced under stress conditions; HtrA (spot 11) is a protease anda chaperone involved in the response to secretion stress; LeuDH and Ald-2 (spots 70 and 71, respectively) belong both to the energy metabolism category and the aminoacid degradation group; and the toxin component LF is a metal protease and therefore is included both in the toxins and the protein degradation enzymes categories.Spot 8 represents two proteins (see Table 1); therefore, the total number of proteins in the category “transport of amino acids or peptides” is 7.



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of the proteins (Fig. 5). Thus, at least in the case of secretedproteins, possible regulatory cross talk mechanisms connectingplasmid- and chromosome-encoded genes are not manifestedunder these highly permissive conditions.

(ii) NBY-O2 medium secretomes. Under NBY-O2 mediumgrowth conditions, the number of secreted proteins is dramat-ically reduced compared to that of the rich-medium secre-tomes (Fig. 3 and 5). The secretomes of both the Vollum and�Vollum strains display unique and rather unexpected com-positions, consisting of only two major proteins which togetherrepresent more than 90% of the total proteins released by thebacteria: immune inhibitor A (InhA1, BA1295) and neutralprotease (NprB, BA0599). We note that a few additional mi-nor chromosome-encoded proteins (Fig. 5) are present in theNBY-O2 secretomes of both strains (spots 23, 56, 58, 63, 64,and 66). The abundance of these additional protein spots inNBY-O2 is similar to that observed in the rich medium, furtheremphasizing the significant induction exhibited by InhA1 andNprB. Both InhA1 and NprB are metalloprotease homologs ofvirulence factors in the phylogenetically related pathogens B.cereus and B. thuringiensis (24, 49, 50, 110). In both Vollum and�Vollum, the InhA1 protease appears in two forms that differin molecular weight (Fig. 3, spot 2). The isoform of lower mass(60 kDa) lacks the C-terminal region of the molecule. Theelectrophoretic migration of NprB (Fig. 3, spot 46) indicates anapproximate molecular mass of 45 kDa, indicative of the re-moval of about 180 amino acids from its N terminus. A similarprocessing step was reported to occur in a variety of bacterialvirulence-related secreted metalloproteases, such as thermoly-sin family proteases from Legionella pneumophila, Pseudomo-nas aeruginosa, Vibrio cholerae, and Vibrio vulnificus (103).

A pXO1-encoded protein of unknown function, pXO1-15(spot 45), can be detected in significant amounts in theNBY-O2 secretome of the Vollum strain. Except for pXO1-15,as in the case with rich medium, essentially no differences areobserved between the Vollum and the �Vollum strains.

(iii) NBY-CO2 medium secretomes. The pattern described forthe NBY-O2 secretomes is strikingly different when the cells arecultured in the same minimal medium supplemented with bicar-bonate (NBY-CO2) (Fig. 3 and 5). Thirty proteins, which areabsent from the NBY-O2 culture, can now be detected. Further-more, unlike the situation in rich medium and NBY-O2, in NBY-CO2, the protein signature of the Vollum strain differs signifi-cantly from that of the plasmid-cured �Vollum strain, and thisdifference is characterized by the presence of both plasmid- andchromosome-encoded proteins. Seventeen proteins are present inthe NBY-CO2 secretome of the Vollum strain in quantities sig-nificantly higher than in the NBY-CO2 secretome of the �Vollumstrain, while 13 proteins are more abundant in the NBY-CO2

secretome of the �Vollum strain (Fig. 1, 3, and 5; Table 1). Thesepatterns are described in more detail below.

(a) pXO1-encoded proteins abundant in the NBY-CO2 secre-tome of the Vollum strain. As could be expected, PA representsthe major protein in the NBY-CO2 secretome of the Vollumstrain (Fig. 3 and 5). Several forms of PA were detected, repre-senting either isoforms with the same mass but different isoelec-tric points or subfragments resulting from trimming or processingof the full-length molecule. The major form, migrating in the2-DE gel at a position compatible with a full-length molecule,represents more than 30% of the total protein amount in thesecretome (Fig. 3, framed spot 6) and exhibits a wide range of pI.These observations are in agreement with a recent study whichsuggested that modifications, such as deamidations, may result ina multitude of PA forms that differ in their isoelectric points(159). Several PA subfragments, representing 10% of total pro-tein visualized on the 2-DE gel, could be detected (Fig. 3, whiteframes marked 12,26 [around 40 kDa] and 13-16 [around 30kDa]). Inspection of the MALDI-TOF spectra established thatthe 40-kDa PA forms originate from the C-terminal portion ofthe molecule, while the 30-kDa PA forms span the N-terminalregion of the molecule. The presence of PA subfragments in B.anthracis culture supernatants has been observed in previousstudies (78, 146). The other toxin components, LF and EF, weredetected in much smaller amounts, in agreement with previousstudies (81).

Two additional pXO1-encoded genes were detected in theNBY-CO2 secretome of the Vollum strain: pXO1-90 (spot 51)and pXO1-130 (spot 73). Both pXO1-90 and pXO1-130 weredocumented in a previous report from our laboratory to beimmunogens expressed in vivo, and they represent potentialanthrax vaccine candidates (7). These two proteins were alsoidentified in the supernatant of B. anthracis cultures under highCO2 tension by Lamonica and coworkers (78). These proteinsmay be involved in bacterial pathogenesis: detection of theprotein pXO1-90 (an SLH-exhibiting protein of unknownfunction) is in good agreement with the observation that it istranscriptionally activated by the virulence regulator AtxA(17). The protein pXO1-130 represents a truncated version ofAdcA, the solute-binding subunit of an orphan ABC trans-porter exhibiting extensive domain similarity to proteins in-volved in the import of zinc (30, 58). AdcA homologs wereinvoked in virulence (Table 2) of other pathogenic bacteriasuch as Brucella (73), Pasteurella (46), and Haemophilus (83).Notably, the B. anthracis toxin subunit LF is a metalloproteasecofactored by zinc (74). The up-regulation of a Zn importmachinery under conditions favoring toxin synthesis maytherefore be beneficial for the bacteria.

(b) Chromosomally encoded proteins abundant in the NBY-CO2 secretome of the Vollum strain. Twelve chromosomallyencoded proteins were detected under these conditions (Table1 and Fig. 5, spots 1, 11, 17, 21, 22, 34, 38, 39, 41, 50, 59, and61). Three of these (spots 17, 39, and 61) are proteins of

FIG. 5. Relative abundances of B. anthracis proteins in various secretomes. The values represent the averaged abundance (�10%) obtained forindividual spots identified in at least three 2-DE duplicate gels representing each protein signature, which were scanned and analyzed using thePDQuest software. The absence of spots indicates an abundance lower than 0.1% of the total protein mass. Equivalent amounts (100 �g) of proteinwere loaded on each gel. Note that some protein spots (e.g., spots 23 and 56) are present in similar abundances in all secretomes and could beused for quantitative comparisons. Gray bars represent proteins possessing biological activities associated with utilization of proteins such asproteases and transporters involved in amino acid and peptide import.



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unknown function. The functional classification of the othernine proteins is discussed below.

The protein DnaK (spot 1) is a chaperone, usually involvedin heat, salt, and oxidative stress responses; consistently, DnaKcould not be detected in the NBY-CO2 secretome of �Vollumat all time points inspected (not shown). This result is puzzling,especially in view of the fact that it can be readily detected inthe NBY-CO2 secretome of the related �14185 strain (see Fig.S1 in the supplemental material). Yet, we note that in Vibriocholerae (22) and in Salmonella enterica (132), DnaK wasshown to be required for production and secretion of virulencefactors; therefore, one may speculate that it is up-regulated inthe Vollum strain as an accessory to toxin production.

The protein QoxA (spot 22), which can exist both membranebound and as a truncated product in the medium (see above), isan essential terminal electron acceptor in the electron transportchain in bacterial respiration metabolism (116). The expression ofQoxA observed in the NBY-CO2 culture of the Vollum strainmay suggest that this protein is required for anaerobic growth, asrecently documented for group B Streptococcus. Interestingly,quinol oxidase activity was recently implicated in the capacity ofthese bacteria to grow in the host (153).

Two putative polysaccharide-degrading enzymes were de-tected: a chitin-binding protein (BA2793/spot 41) and the poly-saccharide deacetylase YjeA (BA2944/spot 34). In the aerobicminimal medium culture (NBY-O2) of B. anthracis, anotherchitin-binding protein is expressed (BA2827/spot 66). It shouldbe noted that BA2793 but not BA2827 exhibits two copies ofthe FN3-type extracellular matrix adhesion domains. Such do-mains were identified in virulence-related proteins of patho-gens such as Staphylococcus aureus (54), Clostridium thermo-cellum (71), and Streptococcus pyogenes (108).

Five protein-degrading enzymes exhibit elevated levels (Fig. 3and 5): the serine protease HtrA (BA3660/spot 11), two cell wallhydrolases belonging to the NlpC/P60-family (BA1952/spot 38and LytE-BA5427/spot 59), the SLH domain alanine amidase(BA0898/spot 50), and the camelysin (BA1290/spot 21).

HtrA (spot 11) is the most abundant protein in the NBY-CO2 secretome of the Vollum strain except for PA (Fig. 3 and5). HtrA has been extensively studied in the context of secre-tion, in which HtrA fulfills two roles: it acts as a chaperoneresponsible for the correct folding of secreted proteins and as aprotease responsible for the degradation of misfolded proteins (5,29, 65, 136). The high abundance of HtrA in the NBY-CO2

secretome of the Vollum strain may stem from the fact thatunder these conditions, it is active in supporting the secretionof extremely large amounts of PA. Such a phenomenon wasdescribed for another extracellular chaperone, PrsA, whichwas shown to be necessary for production of PA in a heterol-ogous B. subtilis system (147).

The protein BA1952 (spot 38) is a putative cell wall hydro-lase exhibiting an NlpC/P60 domain and also two SH3 (Srchomology) virulence-related domains. The protease LytE (spot59) is a major cell wall hydrolase in B. subtilis (152). It harborstwo important functional domains: an NlpC/P60 domain, sim-ilar to BA1952, and a truncated N-terminal ERM domain. TheERM domain is basically a eukaryotic protein-protein interac-tion motif encountered in actin-binding proteins (90) and wassuggested to mediate Shigella entry into epithelial cells (127).

The alanine amidase BA0898 (spot 50) is an SLH protein

that was previously detected as a membrane-associated proteinof B. anthracis and recognized as an immunodominant vaccinecandidate (8, 25). While the full-length polypeptide is presentin the rich-medium secretome (Fig. 2) and in the membraneproteome (25), the form detected in the NBY-CO2 secretomeof the Vollum strain is a subfragment spanning the C-terminalregion of the molecule, devoid of the cell surface anchorageSLH domain. It is interesting to note that an S-layer proteinpossessing alanine amidase activity is present in considerableamounts in the secretome of the pathogen Clostridium difficileunder conditions which favor very high toxin production (105).

(c) Proteins abundant in the NBY-CO2 secretome of the�Vollum strain. The higher calculated relative abundance ofsome of the proteins in the �Vollum secretome compared tothat of the Vollum strain (for example, spots 2, 3, 4, 5, and 8 inFig. 5) may be a reflection of the overall lower level of proteinspecies secreted by the �Vollum strain in NBY-CO2 mediumcompared to the wild-type Vollum strain. Yet, five proteinsappear to be preferentially secreted by the plasmid-cured�Vollum strain in the NBY-CO2 culture, indicating that thepresence of the virulence plasmids not only up-regulates butalso represses some chromosomally encoded genes, a phenom-enon previously observed by transcriptome analysis (17). Theproteins which appear in the present study to be down-regu-lated by the presence of the plasmids (Fig. 5 and Table 1) arethe S-layer proteins Sap and EA1 (spots 19 and 47, respec-tively), a prolyloligopeptidase (BA0165, spot 36), a 5�-nucle-otidase (BA3162, spot 43), and an SLH domain-containingprotein of unknown function (BA3338, spot 48). The mostabundant protein is Sap. This observation is in accordance witha pioneering report of regulatory cross talk which demon-strated the link between Sap expression and the pXO1-en-coded PagR and the AtxA virulence regulators (98). In a B.anthracis heterologous production system we have observed anegative linear correlation between high production and re-lease into the medium of recombinant PA and the amount ofSap secreted (47; O. Gat and A. Shafferman, unpublisheddata), suggesting that the down-regulation of Sap under con-ditions of high toxin production is beneficial to the bacteria.

The increase in abundance of the prolylpeptidase BA0165,the nucleotidase BA3162, and the SLH protein BA3338 in the�Vollum secretome could suggest that they are not essentialfor manifestation of B. anthracis virulence, although proteinsbelonging to the first two families are documented virulencefactors in other pathogens (10, 102, 157, 158).

Tight CO2-dependent regulation of the protein NprB. Oneof the most striking B. anthracis protein signatures observed inthis study is related to the proteases InhA1 (spot 2) and NprB(spot 46), which are present in exceptionally large amounts inthe NBY-O2 cultures of both the Vollum and �Vollum strains(Fig. 3, 5, and 6A). InhA1 represents between 10 and 20% ofthe total protein in this secretome, while NprB consistentlyrepresents between 60 and 80% of the NBY-O2 secretome.Several observations suggest that the scarcity of other proteinspots in the NBY-O2 secretomes is not associated with theprotease activity of NprB: (i) apart from the NBY-O2 condi-tions detailed above, a significant induction of NprB andInhA1 can be observed in BHI medium in late stationary phase(30 h), and yet the protein signature of this culture still in-cludes a large number of spots (Fig. 6C); (ii) an NBY-O2



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secretome collected at an earlier time point (10 h) was almostindistinguishable from the one collected after 20 h in culture(Fig. 6A); and (iii) an NBY-O2 profile with few secreted pro-teins was also exhibited by B. anthracis �14185, a strain whichappears to be defective in NprB secretion (Fig. 5 and 6D).

InhA1 was detected in all B. anthracis secretomes inspected,

albeit in much smaller amounts. In contrast, the neutral pro-tease NprB could not be detected in any other secretome(Table 1; Fig. 5 and 6) except for a late-stationary-phase rich-medium culture (see below). This absence of NprB, and inparticular the shutoff of its expression in NBY-CO2, was quitestriking in view of its unusually high expression in the aerobic

FIG. 6. Evidence of tight regulation of the expression of the extracellular proteases NprB and InhA1. A, C, and D: 2-DE gels of secretomesfor the indicated strains, growth conditions, and harvesting times. Numbers in the lower left corners of some gels indicate the respective proteinmaps in Fig. 1. Spots representing NprB and InhA1 are boxed (the protein Sod, indicative of a state of stress [12, 63] is indicated in panel C. B:Western blot analysis using anti-NprB antibodies. Ten micrograms of protein of the indicated secretomes (and samples diluted 1:500 in the caseof the NBY-O2 samples) was fractionated by SDS-polyacrylamide gel electrophoresis (upper panel), and transferred to Western blots, probed withanti-NprB antibodies (1:1,000), and visualized by chemiluminescence (ECL). E: Zymograph gels carried out with the indicated NBY secretomesof the Vollum (V) or �Vollum (�) strain, using casein-, gelatin-, or PA-impregnated gels.



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NBY medium. Western blot analysis using specific anti-NprBantibodies (Fig. 6B) was used to evaluate the extent of its up-and down-regulation. We find that 500-fold dilution of theNBY-O2 secretome still allowed NprB detection. On the otherhand, Western blot analysis performed with anti-NrpB anti-serum on one-dimensional gels (Fig. 6B) and on 2-DE gels(not shown) from rich-medium secretome or from NBY-CO2

culture did not generate an NprB-specific signal. Normalizingfor the amount of protein (100 �g/2-DE gel), one could esti-mate that at least 1,000 times more NprB is present in theNBY-O2 secretome than under the other conditions tested.

Given the fact that both NprB and InhA1 are proteases, weinspected the proteolytic activity present in the supernatants ofthe NBY-O2 and NBY-CO2 cultures, by use of zymographygels containing casein and gelatin as generic protease sub-strates (Fig. 6E). We observe that a significantly lower extra-cellular protease activity is present in the NBY-CO2 culture.Notably, similar results were obtained by carrying out the zy-mography tests with PA-impregnated gels, suggesting that thisproteolytic activity may interfere with toxin accumulation inthe extracellular milieu (Fig. 6E).

Finally, we emphasize that the dramatic decrease in theamount of NprB (and to a lesser extent InhA1) occurs at highCO2 tension with equal efficiency in the Vollum, �Vollum, and�14185 strains (Fig. 1, 5, and 6). This strongly suggest that theCO2-responsive mechanism which down-regulates NprB expres-sion (InhA1 only in the case of �14185) is chromosomally con-trolled.

DISCUSSION

In this report we present a proteomic study of B. anthraciswhich addresses the influence of the presence of the virulenceplasmids on the composition of the bacterial secretome in variousmedia and growth conditions. The study expands the currentdatabase of B. anthracis secreted proteins by complementing therecent proteomic analyses reported by Antelman and coworkers(6), Gohar and coworkers (50), and Lamonica and coworkers (78)(Table 1), both with respect to the number of protein speciesidentified and, mainly, regarding the expression patterns of thevarious proteins.

Of the 400 spots inspected, more than 60 proteins wereidentified (Table 1), corresponding to the most abundant spe-cies, many of which are virulence factors in other pathogens(Table 2) and therefore represent potential targets for individ-ual investigation of their role in B. anthracis virulence. It isinteresting that the B. anthracis secretome, as evidenced in thisand other studies (6, 50, 78), does not resemble that of theclosely related B. cereus or B. thuringiensis (50). Based on thegenomic similarity of these species and on the known evolu-tionary silencing of the PlcR regulon in B. anthracis (2, 96; seebelow) one would expect that the B. anthracis secretome of astrain devoid of plasmids strain should be similar to that of a B.cereus plcR null mutant. Yet these secretomes are different(based on the analysis carried out by Gohar and coworkers[49], which involved the rich-medium secretome of plcR nulland wild-type B. cereus cells). This observation emphasizes thephenotypic difference between B. cereus and B. anthracis de-spite their genotypic similarity and strongly suggests that reg-ulatory circuits other than the PlcR regulon are active in de-

termining secretion in B. anthracis. Furthermore, this indicatesthat B. anthracis evolved its own set of secreted factors whichcontribute to its characteristic behavior and that putative vir-ulence factors particularly involved in anthrax pathogenesis arepresent in the B. anthracis secretome.

The secreted proteins could be classified among severalfunctional categories (Table 3), establishing the prevalence offunctions required for protein utilization (e.g., degradation andimport), which are overrepresented in the B. anthracis extra-cellular proteome (6, 121). Fifty out of the 64 proteins reportedhere possess secretion signal peptides (Table 1) indicative oftheir export via the Sec pathway. While most of the bacteriapossess only one SecA protein, two homologs of the SecA genewere identified in the B. anthracis genome (BA0882 andBA5421 in the “Ames ancestor” genome) (147), suggestingthat this bacterium has an alternative secretion pathway whichmay be responsible for secretion of some of the signal-lessproteins found in the secretome (114). The alternative Secpathway is often involved in the secretion of virulence factors,and it may mediate release of proteins which do not necessarilyexhibit a canonical export signal. Notably, in Mycobacteriumtuberculosis, two of the signal-less proteins exported by analternative Sec pathway are SodA and catalase-peroxidase(18), which resemble the B. anthracis SodA-2 and AhpC iden-tified in this study. Similarly, in Listeria monocytogenes, or-thologs of the signal-less proteins DnaK, GroEL, and enolase,all of which were identified in the B. anthracis secretome(Tables 1 and 2), are exported via the alternative SecA2 path-way (80). In addition to soluble proteins fully compatible withtheir localization in the extracellular milieu, the secretomecomprises 22 proteins of a membrane nature, either containingextensive transmembrane hydrophobic domains or exhibitingmotifs which mediate surface anchoring. This situation wasreported also for other bacteria, such as B. cereus, B. licheni-formis, B. subtilis, and S. aureus, and it may indicate that duallocalization of some proteins, both cell associated and secret-ed/shed, may be important to their physiological role (4, 50,107, 137, 142).

In rich medium, the Vollum and the �Vollum strains of B.anthracis secrete a large number of proteins, and only minordifferences between the secretomes of these strains are ob-served (Fig. 1, 2, and 5). However, inspection of a late-station-ary-phase BHI secretome (a stage at which the cells may ex-perience a state of stress) reveals a sharp increase in the levelsof the two proteases InhA1 and NprB (Fig. 6C). We note thatAntelman and coworkers (6) also observed an increase in ex-pression of NprB in the late stationary phase of the culture ofan other plasmid-cured avirulent B. anthracis strain (UM23C1-2). More dramatically, in the NBY aerobic medium (whichcontains much lower levels of nutrients than the rich BHImedium), unprecedentedly large amounts of these two pro-teins are observed (Fig. 1 and 3). In NBY supplemented withCO2, both of these proteases are down-regulated (Fig. 2, 5, and6B). In particular, the amount of NprB protease, which in theaerobic medium is unusually high (60 to 80% of total proteinmass in the secretome), is reduced to a nondetectable level (byat least 3 orders of magnitude) and virtually, disappears fromthe secretome. The dramatic silencing of the NprB expressionin NBY-CO2 indicates that under conditions in which the B.anthracis toxins are expressed, it is beneficial for the bacteria to



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reduce its proteolytic activity. This concept is strongly sup-ported by the zymography profiles of the secretomes (Fig. 6D):we observe that a much higher proteolytic activity acting bothon casein and gelatin is secreted in NBY-O2 than in the NBY-CO2 cultures. Interestingly, this proteolytic activity is highlyefficient in a zymography test carried out on gels impregnatedwith PA, strongly indicating that this high proteolytic activitymay be deleterious to the bacterium under conditions when itssurvival depends on the secretion of the toxins, such as thoseencountered in the host during infection. The fact that thesame phenomenon of a sharp shutoff of NprB expression oc-curs equally efficiently in the Vollum and �Vollum strainsdemonstrates that CO2-signaling regulatory circuits indepen-dent of the pXO1-carried atxA gene or the presence of pXO2plasmid are active in B. anthracis. This possibility was sug-gested by Mock and Mignot (100), based on inspection of theCO2 response of B. anthracis strains devoid of either virulenceplasmid, yet the present study may provide direct evidence thatchromosomally encoded CO2-responsive regulons also are in-volved in determining the virulence of the bacteria.

One of the most fundamental aspects of B. anthracis viru-lence in contrast to its phylogenetically close relative B. cereusis the evolutionary silencing of the PlcR regulon in B. anthracis.This genetic inactivation is irreversible due to a nonsense mu-tation in the plcR gene (2), and it was postulated that themanifestation of B. anthracis virulence is strictly dependent onthe complete abolishment of the expression of PlcR targetgenes which may be incompatible with the characteristic patho-genicity of B. anthracis (97). Our results bring strong newevidence in support of this concept; we note that among allsecreted proteins identified in this study, the only ones whichare homologous to B. cereus potential PlcR targets (67) are thestrikingly differentially expressed NprB and InhA1 proteases(although they do not exhibit PlcR-responsive DNA se-quences, they are orthologs of PlcR targets). While B. anthracisvirulence is not disturbed by PlcR target proteins due to theirstable dormant status, both NprB and InhA1, which may berequired for survival under minimal medium conditions, aresuppressed by a CO2-dependent mechanism.

In the CO2-supplemented medium the patterns of expres-sion of many proteins differ in the Vollum and the �Vollumstrains, demonstrating that regulatory cross talk mechanisms,manifested in both an inductive manner and a repressive man-ner, link the expression of plasmid- and chromosome-encodedproteins. Actually, 10 chromosomally encoded proteins (BA0307,BA0703, BA0799, BA0898, BA1290, BA2793, BA2944, BA3588,BA4539, and BA5427) which were identified in the NBY-CO2

secretome of the Vollum strain could not be detected at allunder the same conditions in the secretome of the �Vollumstrain, suggesting that these gene products are positively reg-ulated by plasmid-encoded factors. On the other hand, expres-sion of 14 proteins (BA0165, BA0267, BA0656, BA0885,BA0887, BA0908, BA1191, BA1295, BA1449, BA2947,BA3162, BA3338, BA3367, and BA5364) encoded by chromo-somal genes appears to be down-regulated by the presence ofthe virulence plasmids. Two additional proteins, BA1952 andBA3660, are present in much larger amounts in the NBY-CO2

secretome of the Vollum strain, yet they can also be detectedin �Vollum. These two proteins are actually proteases, whichappear to be the most abundant proteins of the NBY-CO2

secretome of the virulent Vollum strain (except for PA) (Fig.5) and are absent from the NBY-O2 culture. BA1952 exhibitsan NlpC/P60 domain which was observed in peptidases with awidespread role in the dynamics of the bacterial cell wall (3).Most interestingly, BA1952 contains also two SH3 (Src homol-ogy) domains. These domains represent bacterial versions ofeukaryotic motifs encountered in many signal transductionproteins (38). Furthermore, bacterial proteins possessing SH3domains were shown to be involved in the virulence of severalpathogens, such as the diphtheria toxin repressor DtxR ofCorynebacterium diphtheriae (89, 149), the invasion proteinInlB of Listeria monocytogenes (92), and the iron-dependentrepressor IdeR of Mycobacterium tuberculosis (89) (Table 3).The second abundant protein, BA3660, is HtrA, a chaperoneand protease and a well-established virulence factor and po-tential vaccine candidate in many bacteria, which also has arole in the secretion process (Table 3). Whether HtrA is onlyan accessory to toxin secretion or is by itself involved in viru-lence manifestations, such as proteolysis of host tissues, is stillto be determined, and studies which address this issue arecurrently being carried out in our laboratory.

In conclusion, the study presented here strongly suggeststhat cross talk regulatory circuits affecting expression of chro-mosome- and plasmid-encoded genes act in a CO2-dependentfashion. Overall, in comparison to bacteria cured of the viru-lence plasmids, the secretome composition of the virulent iso-genic B. anthracis strain propagated in NBY-CO2 (conditionswhich simulate those encountered in vivo) shows that the pres-ence of the plasmids preferentially affects the expression of atleast 26 chromosomal genes besides pXO1-carried genes. It isnoteworthy that at least 17 of these 26 differentially expressedproteins were invoked in virulence of other pathogens. Thepattern of expression characterizing the major secreted pro-teases identified in this study, e.g., HtrA and NprB, delineatestwo alternative but not mutually exclusive regulatory circuits:HtrA represents a class of proteins which are subject to posi-tive regulation by factors encoded on the virulence plasmidsacting in a CO2-dependent fashion, and NprB belongs to a cate-gory of proteins which are negatively regulated also in a CO2-dependent modality but by chromosomally encoded factors.

ACKNOWLEDGMENTS

We are very grateful to Sara Cohen, Baruch Velan, Anat Zvi, andArie Ordentlich for critical, constructive, and helpful comments on themanuscript.

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differential proteomic analysis of the bacillus anthracis ... · secreted proteins, which expands...

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