1 Defence Science Technology Laboratory, Porton Down, Salisbury, Wiltshire SP4 0JQ, UK
2 Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK
3 Biological Defense Research Directorate, Naval Medical Research Center, Silver Spring, MD 20910, USA
Correspondence
Caroline Redmond
credmond{at}dstl.gov.uk
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ABSTRACT |
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INTRODUCTION |
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Much of the work published on the fine structure and composition of the exosporium to date has focused upon B. cereus (Gerhardt & Ribi, 1964; Matz et al., 1970
; Beaman et al., 1971
; Charlton et al., 1999
). This layer is chemically complex, consisting of protein, amino and neutral polysaccharides, lipids and ash (Matz et al., 1970
). B. cereus exosporium antigens first appear at the engulfment stage (stage III) of sporulation (DesRosier & Lara, 1984
). Bacillus thuringiensis (Garcia-Patrone & Tandecarz, 1995
), B. cereus (Charlton et al., 1999
) and B. anthracis (Sylvestre et al., 2002
) spores contain specific glycoproteins in their surface layers. The B. anthracis collagen-like spore surface glycoprotein (BclA) reported by Sylvestre et al. (2002)
is a structural component of the filaments of the hairy nap and is highly immunogenic. Several of the proteins from the exosporium of B. cereus (Todd et al., 2003
) and B. anthracis (Steichen et al., 2003
) have been described, but the overall protein composition, structure and function of the exosporium have yet to be elucidated fully. The exosporium may play a role in the interaction of the spore with the infected host. For example, there may be an association between the exosporium of B. anthracis spores and the macrophage (Dixon et al., 2000
; Guidi-Rontani et al., 2001
), and exosporially located components, like the alanine racemase and inosine hydrolase reported here, may influence spore germination within the macrophage.
This study has identified several novel proteins tightly associated with the exosporium of B. anthracis that are likely to represent integral exosporium proteins, as they are retained after salt and detergent washes.
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METHODS |
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Spore preparation.
Spores of B. anthracis were prepared on solid New Sporulation medium (3·0 g Difco Tryptone l-1; 6·0 g Oxoid bacteriological peptone l-1; 3·0 g Oxoid yeast extract l-1; 1·5 g Oxoid Lab Lemco l-1; 1 ml 0·1 % MnCl2.4H2O; 25 g Difco Bacto agar l-1) at 37 °C, until the cultures contained more than 95 % free spores. Spores were harvested by centrifugation at 10 000 g for 10 min, and washed approximately 10 times in ice-cold sterile distilled water to remove contaminating vegetative cells and debris. Microscopic examination established that spore preparations contained less than 5 % vegetative cells and debris. The washed spore preparations were stored at -20 °C for 1 week to 30 months.
Exosporium removal and purification.
Spores were defrosted and centrifuged at 10 000 g for 10 min at 4 °C. Prior to sonication, pellets were resuspended to between approximately 5x109 and 2·5x1010 spores ml-1 in 50 mM Tris/HCl, 0·5 mM EDTA buffer (pH 7·5). All subsequent manipulations were at 4 °C. To fragment and release exosporium material, spores were sonicated (amplitude 12 µm) using a Sanyo Soniprep 150 ultrasonic disintegrator, for seven 1 min bursts, each separated by 2 min cooling on ice. Exosporium fragments were separated from spores by pelleting the latter at 9000 g for 5 min. The spore pellet(s) were washed once in 50 mM Tris/HCl, 0·5 mM EDTA (pH 7·5) and the exosporium-containing supernatants were pooled, then centrifuged again (10 000 g, 15 min) to pellet the remaining spores.
For all isolates of B. anthracis, any residual endospores in the exosporium-containing supernatant were removed by filtration through 0·45 and/or 0·2 µm low-protein-binding filters (Acrodisc, Gelman Sciences). The filtrate, containing exosporium, was subjected to a sterility check by inoculating 10 vols nutrient or L-broth with 10 % of the exosporium preparation and incubating at 37 °C with shaking (200 r.p.m.) for 7 days. After centrifugation to concentrate any cells, the incubated material was plated on L-agar, in parallel with the appropriate controls. The plates were incubated at 37 °C for 7 days and absence of growth of any colonies permitted clearance for use at a lower level of containment. Once confirmed as sterile, the exosporium-containing filtrate was concentrated approximately 15- to 50-fold using an Amicon ultrafiltration cell fitted with a polyethersulfone membrane (Millipore, NWWL 10 000) or by pelleting in an ultracentrifuge at 184 000 g for 1 h at 4 °C.
Salt and detergent washing of exosporium.
The series of washes used on the exosporium was modified and simplified from a method for purifying B. subtilis spore coat fractions described by Nicholson & Setlow (1990). The technique was optimized using B. anthracis UM23Cl2 exosporium and subsequently applied to exosporium from the ACDP 3 strains of B. anthracis. The exosporium was washed with 1 M NaCl, followed by one or two washes in TEP buffer (50 mM Tris/HCl pH 7·2, 10 mM EDTA, 2 mM PMSF) containing 0·1 % (w/v) SDS. To remove the SDS, a final wash was in TEP buffer alone. Each wash involved resuspension of the purified exosporium followed by centrifugation in a Hettich microcentrifuge at 31 870 g for 40 min.
Protein determination.
Protein concentrations were determined using the BCA protein assay kit (Pierce) according to the manufacturer's instructions.
Alkali extraction of spore proteins.
Alkali extraction was carried out on washed B. anthracis UM23Cl2 spores, as detailed by Sylvestre et al. (2002). Spores were incubated in alkaline reducing buffer without SDS (50 mM Tris/HCl, pH 10, 8 M urea and 2 %
-mercaptoethanol) for 15 min or 1 h, and the spores removed by centrifugation.
Electron microscopy.
The samples were diluted in sterile distilled water, placed on Formvar-coated grids and examined after negative staining in 1 % (w/v) phosphotungstic acid in phosphate buffer (pH 7·2). After 1530 s the excess phosphotungstic acid was withdrawn using filter paper. Samples were examined immediately using a Philips CM12 transmission electron microscope at an accelerating voltage of 60 kV.
Gel electrophoresis.
Samples were mixed with an equal volume of sample buffer (125 mM Tris/HCl, pH 6·8; 20 %, v/v, glycerol; 4 %, w/v, SDS; 5 %, v/v, -mercaptoethanol; 0·0005 %, w/v, bromophenol blue) and boiled for 5 min, then held on ice for 210 min and centrifuged briefly (30 s at 13 000 g) to remove any insoluble material. The proteins were separated by SDS-PAGE in Tris-glycine buffer (24 mM Tris, 192 mM glycine and 0·1 %, w/v, SDS; Laemmli, 1970
) at 125 V using pre-cast gels (Invitrogen), or on NuPAGE Bistris pre-cast gels in MES buffer (Invitrogen). To separate low-molecular-mass proteins, samples were boiled in Tricine gel sample buffer (900 mM Tris/HCl, pH 8·45; 24 %, v/v, glycerol; 8 %, w/v, SDS; 0·015 %, w/v, Coomassie blue G; 0·0005 %, w/v, phenol red; 5 %, v/v,
-mercaptoethanol) and separated on precast 16 % Tris-Tricine gels (Invitrogen) at 125 V using the Tricine running buffer (100 mM Tris; 100 mM Tricine; 0·1 %, w/v, SDS). Gels were stained with Colloidal Blue stain (Invitrogen) according to the manufacturer's instructions. For molecular mass determination, SigmaMarker molecular mass standards were used on both Tris-glycine and Tricine gels.
N-terminal sequencing.
Proteins separated by SDS-PAGE were electrophoretically transferred onto PVDF membranes, using 10 mM CAPS transfer buffer (pH 11·0) with 10 % (v/v) methanol. The membranes were stained with 0·1 % (w/v) Coomassie 250 in 40 % (v/v) methanol/1 % (v/v) acetic acid, destained with 50 % (v/v) methanol and air-dried. N-terminal sequences were determined by automated Edman degradation in an Applied Biosystems gas-phase sequencer.
In-gel digestion and mass spectrometric analysis of protein samples.
Bands identified for analysis from the SDS-PAGE gels were excised, reduced/carboxymethylated and subjected to in-gel tryptic digestion. The resulting peptides were extracted from the gel and purified by HPLC using a C18 cartridge. The peptide-containing fractions were collected and analysed by delayed extraction-matrix assisted laser desorption ionization-time of flight mass spectrometry (DE-MALDI-TOF-MS) and some peptides were subjected to sequencing using nanospray-MS/MS. All mass spectrometric analyses were performed at M-Scan Ltd, Wokingham, UK.
Sequence data.
Sequences from protein bands were used to search preliminary sequence data for B. anthracis obtained from the Institute for Genomic Research (TIGR) website at http://www.tigr.org, using tBLASTN (Altschul et al., 1997), and to search ORFs identified in preliminary data for B. cereus ATCC 14579 from http://www.integratedgenomics.com. The wild-type Ames strain examined during this study was the parental strain for the double-cured Ames strain used for genome sequencing (Read et al., 2003
).
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RESULTS AND DISCUSSION |
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Three of the nine other proteins identified in unwashed exosporium (alanine racemase, CotY and ExsY) were also identified in washed exosporium (Fig. 3). GroEL has already been reported as an exosporium-adsorbed protein in B. cereus, removable by salt washing (Charlton et al., 1999
; Todd et al., 2003
). The Zn-dependent metalloprotease immune inhibitor A (InhA), a major component of unwashed exosporium preparations of B. cereus, was not detectable in B. anthracis exosporium.
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Identification of proteins in the washed exosporium
Salt- and detergent-washed exosporium preparations from all three strains examined showed a generally reproducible pattern of proteins on SDS-PAGE on a 16 % acrylamide, Tris-Tricine-buffered gel (Fig. 3), except for the variable EA1 S-layer proteins (as in lane 3, bands 1, 2 and 3).
A number of bands were extracted and mass spectrometry (MS/MS sequencing of tryptic peptides) provided internal sequences for all bands tested except the largest (>200 kDa; Fig. 3). Several protein components were identified their amino acid sequences, predicted from the B. anthracis genome sequence, are shown in Table 2
.
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The band at 17 kDa (Fig. 3, band 6) contained sequences that correspond to a protein encoded by B. anthracis and very recently described and designated BxpB by Steichen et al. (2003)
. The predicted molecular mass of 17 kDa corresponds to that observed experimentally. The equivalent protein detected in B. cereus exosporium shares 97 % aa identity and has been designated ExsF (Todd et al., 2003
). A related paralogue is also encoded in the B. anthracis genome sequence, but the sequence PVIND is the only one found that could be from this paralogue, and it is also present in BxpB/ExsF.
The band at 14 kDa (Fig. 3, band 7) contains sequences that have been identified as derived from at least two, and possibly three, separate proteins. One is a homologue of (30 % identity to) B. subtilis spore coat protein CotB (Table 2
), of predicted size 19·4 kDa. CotB (Donovan et al., 1987
; Driks, 1999
) is exposed on the spore surface of B. subtilis, and has even been used as an anchor for non-native antigens (Isticato et al., 2001
). Its location in B. anthracis will be determined using immunological techniques. The other sequences from the 14 kDa band (FEAFAP and SANLT) are present in the sequences of two proteins, ExsY and CotY, as named in Todd et al. (2003)
. These are paralogues (84 % identity) and are more distant homologues of the B. subtilis CotZ and CotY proteins, which are present in the insoluble outer layers of the B. subtilis spore coat (Driks, 1999
). ExsY is required for exosporium assembly (M. Johnson, S. J. Todd & A. Moir, unpublished). The cotY gene forms part of a complex operon in B. subtilis which consists of five genes, cotVWXYZ. It is hypothesized that the complex transcriptional pattern of this cluster is necessary to maintain an optimal amount of each protein for correct assembly of the spore coat in B. subtilis (Zhang et al., 1993
). There are no ORFs homologous to CotV, W or X proteins in the B. anthracis genome.
Lai et al. (2003) also reported the presence of this CotB homologue and proteins similar to ExsY or CotY in spore coat extracts prepared by boiling B. anthracis spores in SDS-PAGE loading buffer for 10 min.
The band at 10 kDa (Fig. 3, band 8) contains peptides from a predicted protein, which we have named ExsK. The ExsK protein has not previously been described as a coat or exosporium component. There is a significantly divergent homologue encoded in the B. cereus ATCC 14579 genome sequence with 64 % aa identity. Direct comparison of ExsK with B. subtilis proteins by FASTA gives the highest, though not significant score (e=0·12) as the B. subtilis outer coat protein CotX. ExsK has no valid homologues in B. subtilis, but has some similarity in composition near the N-terminus (a very charged region at residues 2937, preceding a cysteine-rich region) to the equivalent region of CotX.
Peptide FEAFAP was also present in the 10 kDa band (Fig. 3, band 8), suggesting that either or both ExsY and CotY sequences are present. The band at about 6·5 kDa (Fig. 3
, band 9) contained a peptide sequence (SIDDDD) specific to CotY. Presumably these proteins in the 10 and 6·5 kDa positions are partial degradation/processing products, as the predicted size of the intact proteins would be 1617 kDa.
Peptide sequences were obtained from the second-largest band (Fig. 3, band 11) (MFSSD and FEAFAP), demonstrating the presence of BxpB (ExsF) and either or both of ExsY and CotY in this band. These proteins are therefore present in the gel in high-molecular-mass complexes, as well as in a monomeric form (BxpB/ExsF) or in a proteolytically cleaved form (ExsY/CotY).
Alkali extraction of proteins from spores
Spores of B. anthracis UM23Cl2 were incubated with alkaline reducing buffer without SDS, as used by Sylvestre et al. (2002) to extract a fraction containing BclA, a major exosporium glycoprotein. Large bands in an SDS-PAGE protein profile of the extracted proteins (Fig. 4
) were subjected to MS/MS and N-terminal sequencing. The diffuse multiple band (>250 kDa) in the 15 min extract (Fig. 4
, lane 2) contained peptides from ExsY (SVDDDS) and ExsF (MFSSD) and yielded the N-terminal sequence for glycoprotein BclA (AFDPNLVGPT). Specific staining confirmed the presence of glycoprotein in this region of the gel (data not shown). The presence of BclA at this position in unwashed and washed exosporium of B. anthracis exosporium was also demonstrated by immunoblotting with specific antibodies (data not shown). On incubation for 60 min in alkaline extraction buffer, a smaller band at 205 kDa increased in intensity (Fig. 4
, lane 3), indicating some dissociation; peptides PVIND and PVELI from ExsF were both identified in this band. The difficulties in dissociating proteins ExsY, CotY and ExsF to their monomeric sizes suggest that these proteins in the spore are involved in relatively stable multimeric complexes, along with the glycoprotein BclA, in the exosporium.
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Of the exosporium proteins identified by Steichen et al. (2003), BclA and BxpB (ExsF) are also reported here, but their BxpC protein has not been sequenced in this study. Of the proteins identified by Todd et al. (2003)
in washed B. cereus ATCC 10876 exosporium that are also encoded in the B. anthracis genome, those designated ExsB, D, E, G and CotE have as yet not been identified directly in B. anthracis exosporium, although ExsF is reported here. As sequence data have only been obtained for relatively few of the proteins separated from washed B. anthracis exosporium, it may be that some or all of these proteins remain to be identified.
This is believed to be the only published analysis of the exosporium of the fully virulent, double-plasmid-containing Ames strain of B. anthracis. There are no obvious differences in gross exosporium profile between the wild-type and cured strains, and all of the proteins identified here are encoded chromosomally. Therefore the cured strains represent good models for studying the exosporium from spores of B. anthracis.
In addition to the demonstrated salt- and detergent-resistant components, unwashed exosporium contains other proteins, some described here, that may contribute to the in vivo activities of the exosporium. Some of these proteins may not be exosporium-bound, but may be located between the exosporium and the rest of the spore, and associate during isolation. By no means all of the exosporium proteins, whether loosely associated/adsorbed, or tightly bound, and salt- and detergent-resistant, have yet been identified. The role of the exosporium of B. anthracis, and the contribution to its structure of the CotB, ExsF, ExsY, CotY and ExsK proteins defined here, are issues that remain to be addressed in future experiments.
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ACKNOWLEDGEMENTS |
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Received 4 August 2003;
revised 14 November 2003;
accepted 25 November 2003.