1 School of Veterinary Science, The University of Queensland, St Lucia, Queensland, Australia
2 Bacterial Pathogenesis Research Group, Department of Microbiology, Monash University, Victoria, Australia
3 Bacterial Diseases of Livestock Research, National Animal Disease Center, Ames, IA, USA
4 Microscopy Services, National Animal Disease Center, Ames, IA, USA
5 Pre-Harvest Food Safety Research, National Animal Disease Center, Ames, IA, USA
6 Veterinary Medical Research Institute, Iowa State University, Ames, IA, USA
Correspondence
Darren J. Trott
d.trott{at}mailbox.uq.edu.au
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ABSTRACT |
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The GenBank accession numbers for the sequences reported in this paper are AY363613, AY363614 and AY376355.
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INTRODUCTION |
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A characteristic feature of both natural and experimental B. pilosicoli infections is the attachment of large numbers of spirochaetes by one end to the epithelium of the caecum and large intestine, forming a false brush border (Hampson & Trott, 1999). B. pilosicoli also localizes within intestinal crypts and can invade the lamina propria (Duhamel, 2001
; Muniappa et al., 1996
). The B. pilosicoli outer membrane (OM) is the interface between the bacterium and the host. By analogy with other pathogens, surface-exposed proteins of B. pilosicoli should have a significant role in colonization, intimate attachment and pathogenesis. However, little is known about the OM structure of B. pilosicoli and very few OM proteins (OMPs) have been identified for Brachyspira. For B. pilosicoli, a putative 29 kDa pyruvate oxidoreductase (Rayment et al., 1998
) and a 36 kDa glucose/galactose-transport/chemoreceptor have been identified (Zhang et al., 2000
), whereas in Brachyspira hyodysenteriae, the agent of swine dysentery, identified OMPs include SmpA (Thomas & Sellwood, 1993
), and the Vsp (Gabe et al., 1998
; McCaman et al., 1999
; McCaman et al., 2003
) and BmpB/Blp families of paralogous genes (Cullen et al., 2003
; Lee et al., 2000
).
Isolating and characterizing surface-exposed B. pilosicoli proteins and investigating their interactions with the host first requires a technique for OM enrichment. However, selective removal of the spirochaete OM is hampered by its labile nature and by the periplasmic location of the flagella which contaminate OM preparations obtained using techniques reliably applied to other non-spirochaete bacteria. Solubilization of Brachyspira spp. OMPs in detergents selectively releases the periplasmic flagella or causes cell lysis and release of cytoplasmic proteins (Chatfield et al., 1988; Gabe et al., 1995
; Joens et al., 1993
; Trott et al., 2001
). The detergent Triton X-114 has been used to selectively release spirochaete OMPs (Cunningham et al., 1988a
; Cunningham et al., 1988b
; Lee & Hampson, 1995
; Lee & Hampson, 1996
; Tenaya et al., 1998
; Thomas et al., 1992
; Zuerner et al., 1991
). However, due to the labile nature of the Brachyspira OM, it is possible that proteins anchored to the inner membrane (IM), could also be solubilized in the detergent phase (Tenaya et al., 1998
; Trott et al., 2001
). Membrane vesicle fractionation may offer an advantage over detergent solubilization as it maintains proteins in their location within the OM. Membrane vesicle fractions have been obtained from B. hyodysenteriae by French pressure cell disruption and density gradient ultracentrifugation (Plaza et al., 1997
). The B. hyodysenteriae OM has a very low density (
=1·10 g cm3) compared to the IM (
=1·16 g cm3) and is unusual in containing cholesterol as a major lipid bilayer constituent. The presence of cholesterol may explain the extreme fragility of the Brachyspira OM and the difficulty previous researchers have experienced in isolating OMPs free of contamination from other cellular components (Plaza et al., 1997
).
The goal of this study was to identify and characterize B. pilosicoli OMPs and clone their genes. Membrane vesicles generated by osmotic lysis of whole cells were purified by isopycnic gradient ultracentrifugation. An OM-rich fraction was used as antigen to obtain mouse mAbs that were then used to identify selected proteins in B. pilosicoli strain 95-1000, including a 23 kDa protein that was found in abundance on the OM surface. The gene encoding this protein was isolated, sequenced and expressed in Escherichia coli. Analysis of the sequence implies that this protein, designated BmpC, is a lipoprotein.
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METHODS |
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Preparation of B. pilosicoli total membrane (TM) and soluble protein (SP) extracts.
Unless indicated, all procedures were performed at 4 °C. TM extract consisting of OM, IM and flagella, and SP extract were prepared from a 1 l culture of B. pilosicoli 95-1000 cells in exponential growth phase (OD620=0·8, approximately 5x108 cells ml1 by direct counts). The cells were harvested by centrifugation (4000 g, 8 min), washed in buffer I (20 mM HEPES, 50 mM NaCl, pH 7·6) and resuspended in 15 ml buffer I containing 10 % sucrose, 2 mM EDTA, 0·00425 % PMSF and 20 µl each of DNase type I and RNase type A. The cells were passed twice through a French pressure cell at 15 000 p.s.i., centrifuged at 10 000 g for 20 min and the supernatant was collected and centrifuged at 100 000 g for 1 h. The supernatant (SP extract) was removed and concentrated tenfold in a Microcon 10 concentrator whilst the pellet (TM extract) was washed twice and resuspended in 500 µl buffer I. Both fractions were stored at 70 °C.
Isolation of B. pilosicoli membrane vesicles.
A 4 l culture of B. pilosicoli 95-1000 was grown at 39 °C to an OD620 of 0·8 and incubated on ice overnight. Unless otherwise stated, all procedures were performed at 4 °C. The cells were harvested by centrifugation (4000 g, 8 min), washed in buffer I and resuspended in buffer I containing 1 µM octadecyl rhodamine B chloride (Molecular Probes) and 0·00425 % PMSF. The cells were then harvested by centrifugation (4000 g, 8 min) and resuspended in sterile dH2O (70 ml g1 wet wt). The cell suspension was mixed at room temperature for 2 h on a Nuova stir plate set at speed 1 using a 4x0·8 cm magnetic stir bar. Whole cells and protoplasmic cylinders were removed by centrifugation at 8000 g for 20 min and 10 000 g for 20 min, respectively, and the supernatant was then centrifuged at 100 000 g for 1 h to sediment the membrane vesicles. Membrane vesicles were resuspended in 10 ml buffer I containing 10 % sucrose and 0·00425 % PMSF. The membrane vesicle suspensions (5 ml) were layered onto a (w/w) sucrose gradient made up in buffer I containing 5 ml 21 % sucrose, 16 ml 35 % sucrose and 11 ml 45 % sucrose and subjected to ultracentrifugation using a Beckman SW28 rotor at 100 000 g for 16 h. High and low-density membrane vesicle fractions (HDMV and LDMV, respectively) were harvested from the side of the tube using a 21G needle and syringe and sedimented by centrifugation at 100 000 g for 1 h. HDMV and LDMV fractions were resuspended in buffer I containing 10 % sucrose and further purified by isopycnic centrifugation (1 ml 33 % sucrose, 3·5 ml 38 % sucrose and 1 ml 43 % sucrose for HDMV, and 1 ml 21 % sucrose, 2·5 ml 33 % sucrose and 1 ml 38 % sucrose for LDMV) using a Beckman SW55 rotor at 100 000 g for 16 h. The membrane vesicle fractions (the top layer from the LDMV gradient and the bottom layer from the HDMV gradient) were collected by needle aspiration, centrifuged at 150 000 g for 3 h, resuspended in buffer I and stored at 70 °C for further analysis. In separate experiments, membrane vesicle fractions (1 ml) were collected from the bottom of each gradient using a Beckman gradient fractionator. The density and protein concentration of the fractions were determined using a Bausch and Lomb refractometer and by measuring A280 in a Beckman DU 640 spectrophotometer, respectively.
Triton X-114 extraction.
Triton X-114 extraction and phase-partitioning of membrane proteins was performed using B. pilosicoli 95-1000 cells grown to an OD620 of 0·8 as described by Cunningham et al. (1988b).
Transmission electron microscopy.
Membrane vesicles or cells were diluted 1 : 5 in dH2O and 10 µl aliquots were negatively stained with an equal volume of 2·5 % phosphotungstic acid (pH 7) (Trott et al., 1996d). For cross sections, membranes or cells were fixed in 2 % paraformaldehyde/0·05 % glutaraldehyde for 1 h at 4 °C, harvested by centrifugation at 14 000 g for 20 min, washed twice in cacodylate buffer (pH 7·4) and resuspended in 100 µl warm (45 °C) cacodylate buffer containing 2 % agarose. The agar pieces were dehydrated in an ethanol series, embedded in Epon and ultrathin sections were cut and transferred onto 400 mesh grids. Grids were stained with uranyl acetate and lead citrate and examined at 80 kV by using a Phillips model 410 transmission electron microscope.
SDS-PAGE and Western blotting.
Protein concentrations were determined using the modified Lowry assay (Markwell et al., 1978). Protein preparations (5 or 10 µg) were separated by SDS-PAGE in precast 420 % acrylamide gradient gels with a Mini-Protean II gel electrophoresis apparatus (Bio-Rad) using standard techniques (Laemmli, 1970
). Proteins were transferred to nitrocellulose membranes with a Transblot electrophoretic transfer cell (Bio-Rad) (Towbin et al., 1979
). Immunodetection of proteins in Western blots was performed using the Enhanced Chemiluminescence System according to the manufacturer's recommendations (Amersham Pharmacia Biotech).
Assays for cellular and membrane markers.
The composition of the various fractions of B. pilosicoli was investigated by assaying for specific proteins and cellular components with known cellular locations. NADH oxidase, a cytoplasmic marker was detected on Western immunoblots by using polyclonal gnotobiotic pig antisera raised against the partially purified 48 kDa NOX protein of B. hyodysenteriae (Stanton & Jensen, 1993). FlaA1, a flagellar sheath protein, was detected using rabbit antisera raised against the 44 kDa B. hyodysenteriae protein (Li et al., 1993
). The OM markers used were a 29 kDa B. pilosicoli OMP, detected using a mouse mAb (Lee & Hampson, 1995
), and lipo-oligosaccharide (LOS). LOS was demonstrated by incubating membrane and SP extracts (75 µg) with proteinase K (2 mg ml1) at 55 °C for 2 h. The extracts were then resolved on a 14 % acrylamide, 9 M urea separating gel containing a bilayered stacking gel and LPS was detected by silver staining (Inzana & Apicella, 1999
; Tsai & Frasch, 1982
). Detection of penicillin-binding proteins (PBPs) (IM marker) was performed as described by Weigel et al. (1994)
. The FlaA1 polyclonal antisera and the 29 kDa OMP mAb were kindly provided by Professor Mario Jacques, Université de Montréal, St Hyacinthe, Quebec, Canada, and Professor David Hampson, School of Veterinary and Biomedical Science, Murdoch University, Western Australia, respectively.
mAb production.
BALB/c mice were immunized by the intraperitoneal route at days 0 and 15 and by the intravenous route on day 24 with LDMV (1525 µg protein) mixed 70 : 30 with TiterMax adjuvant (CytRx Corp.). Spleen cells were harvested on day 40 and fused with SP 2/0 mouse myeloma cells using polyethylene glycol. The fusion mixture was distributed into 96-well plates and resulting hybridomas were grown in Dulbecco's Modified Eagle Medium supplemented with 15 % (v/v) fetal calf serum. Hybridomas were screened for antibody production by dot-blot analysis using 50 ng TM from B. pilosicoli 95-1000 as antigen. Hybridomas with high antibody titres were then screened by slot-blot analysis against B. pilosicoli TM proteins with and without proteinase K treatment to remove those that bound to B. pilosicoli LOS determinants. Whole-cell extracts of the type strains of B. hyodysenteriae, Brachyspira intermedia, Brachyspira innocens, Brachyspira murdochii and Brachyspira alvinipulli were then used to further select hybridomas. Five hybridomas producing mAbs (4F2, 2G3, 1G2, 2H3 and 2E10) that reacted with B. pilosicoli membrane protein epitopes, but not with whole-cell extracts derived from the type strains of other species of Brachyspira, were identified, cloned and concentrated using the Cellmax Hollow Fibre System (Cellco Inc.). Purified mAbs were tested for reactivity in Western blots of whole-cell protein profiles using 13 genetically diverse B. pilosicoli strains isolated from different host species. These included five isolates from pigs (95-1000, P43/6/78T, Will3D4, Win3 and V883), three isolates from humans (WesB, V1H78, Rosie 2299), one isolate from a dog (V1D1) and four isolates from avian species (QU-1, 13316, 92-S76 and R4). The antibody isotype of each mAb was determined using an ELISA-based mouse monoclonal isotype kit (Bio-Rad) according to the manufacturer's instructions.
Immunogold labelling.
Cells from 1 ml B. pilosicoli 95-1000 culture were harvested by centrifugation, washed in 10 mM Tris/150 mM NaCl (pH 7·4) and resuspended in 200 µl TBS containing 2 % bovine serum albumin. A 200 µl volume of the appropriate mAb was added and the cells were gently mixed and incubated at 37 °C for 30 min in a Coy anaerobic chamber. The cells were pelleted (4000 g, 7 min), washed twice and resuspended in 100 µl TBS containing 1 % BSA. They were then absorbed onto carbon-coated grids for 5 min and incubated in a 1 : 10 solution of goat anti-mouse colloidal gold conjugate (BC-GAR-30; EBSciences) in TBS containing 1 % BSA. The grids were washed three times in 25 mM Tris, pH 7·2, twice in dH2O and stained with 2 % phosphotungstic acid before being examined with a Phillips model 410 transmission electron microscope operating at 80 kV.
Cloning and sequencing.
A genomic library of strain 95-1000 was prepared in ZAP II (Stratagene). The library was screened for phage plaques binding the 2E10 mAb using the picoBlue immunoscreening kit (Stratagene). Two positive clones (p151 and p421) were isolated and the inserts were excised from
ZAP II recombinant phage and subcloned into the pBluescript SK phagemid vector according to the manufacturer's instructions. Plasmid preparations of these clones were then purified by CsCl centrifugation. The insert sequences were obtained by PCR amplification by using both commercially available primers (T3 and T7-1) and synthesized oligonucleotide primers based on the 3'-OH end of the upstream insert sequences (Table 1
). In addition the contiguous sequence was amplified directly from B. pilosicoli 95-1000 whole cells by PCR and sequenced for comparison with p421 and p151. Sequencing was done using standardized dye-termination sequencing reactions separated on ABI PRISM model 377 DNA sequencers at the DNA Sequencing Facility at Iowa State University (Ames, IA, USA). Sequence data were compiled and analysed using Sequencher Version 4.05 (Gene Codes Corp.). The deduced hypothetical ORF was used to search for homology against the GenBank nucleotide database and the deduced amino acid sequence was compared with the SWISS-PROT protein database. Sequence data for the cloned inserts p151 and p421 and the contiguous sequence amplified from B. pilosicoli 95-1000 were submitted to GenBank and were assigned the accession numbers AY363613, AY363614 and AY376355, respectively.
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RESULTS |
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Our strategy for the isolation of OMPs was based on electron microscopy observations that resuspension of B. pilosicoli cells in dH2O caused lysis of the OM, whilst maintaining the integrity of the protoplasmic cylinder (S. Humphrey, personal communication). We therefore developed a membrane vesicle enrichment technique based on osmotic lysis in dH2O. Following 2 h of gentle stirring, examination by phase-contrast microscopy showed that the majority of cells had a reduced cell diameter, suggesting that their OM had been removed. However, a proportion of the cells (<20 %) still retained their original cell diameter or had lost their helical shape and formed spherical bodies. As determined by electron microscopy, the cells with reduced cell diameter consisted of protoplasmic cylinders that had lost their OM (Fig. 1a, b). The flagella had unwound from the protoplasmic cylinder but were still attached to the terminal ends of the cell (Fig. 1c
).
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In overview, the LDMV fraction was free of flagella and cytoplasmic components and contained OM markers. IM components were present, but were substantially reduced when compared to the TM fraction. On the basis of these marker assays, LDMV represented enriched OM vesicles and proteins identified in this fraction were likely to be OM-associated.
Production of mAbs using LDMV as a source of antigen
To further characterize B. pilosicoli-specific OM-associated proteins, the LDMV fraction was used to immunize mice for mAb production. The mAbs were screened and comprised three groups. The first represented mAbs that reacted with proteinase-K-resistant material, most probably LPS. The second group of mAbs cross-reacted with proteins from other Brachyspira species. A third group of five mAbs reacted with B. pilosicoli-specific proteinase-K-sensitive proteins of 23, 24, 35, 61 and 79 kDa, based on electrophoretic migration. Three of these mAbs were of the IgG1 subclass, one of the IgG2b subclass and one of the IgA class. mAbs 1G2 and 2G3 (directed against the 35 and 61 kDa proteins, respectively) reacted with all 13 B. pilosicoli strains tested, whereas mAb 2E10, directed against the 23 kDa protein, only reacted with B. pilosicoli 95-1000 (Table 2).
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DISCUSSION |
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This paper describes a novel OM enrichment method that takes advantage of the observation that B. pilosicoli cells rapidly lose their OMs whilst maintaining the integrity of the protoplasmic cylinder when suspended in dH2O. B. pilosicoli 95-1000 membrane vesicles were separated into LDMV and HDMV fractions with respective densities of 1·12 and 1·18 g cm3 and the fractions were shown to be free of flagella and cytoplasmic protein contamination. The low density (=1·12 g cm3), the presence of two OM markers (LOS and a 29 kDa OMP) and the relative absence of the IM marker (PBPs) in LDMV, suggested that this fraction represented an enrichment of OM vesicles. LDMV was therefore used to develop key immunologic reagents for further investigation of B. pilosicoli OMP constituents.
We identified five B. pilosicoli-specific mAbs that reacted with proteins in the LDMV fraction. Comparison of the distribution of these proteins in the other membrane fractions and Triton X-114 extracts also demonstrated key differences. The 24 and 35 kDa proteins were enriched in the LDMV fraction compared with the TM and HDMV fractions and both these proteins were enriched in the Triton X-114 detergent phase compared to the insoluble phase, confirming that they are likely to be integral membrane proteins or lipoproteins anchored to the OM. Immunogold labelling results were consistent with the surface location of these proteins.
BmpC, identified by mAb 2E10, was one of three major membrane-associated proteins identified by SDS-PAGE in the TM, HDMV and LDMV fractions. However, in contrast to the 24 and 35 kDa proteins, BmpC did not show selective partitioning into the Triton X-114 detergent phase, suggesting that it was either present on both the OM and the IM of B. pilosicoli or that the protein was poorly soluble in this non-ionic detergent. Haake & Matsunaga (2002) have previously demonstrated the poor solubility of a Leptospira OM porin (OmpL1) in Triton X-114. In addition, immunogold labelling confirmed that BmpC was surface-exposed on the B. pilosicoli 95-1000 OM surface. Immunogold labelling was not apparent on protoplasmic cylinders stripped of their OM by osmotic lysis in dH2O.
Cell fractionation showed that recombinant BmpC was expressed predominantly in OM and CM fractions of E. coli strain SOLR without any apparent deleterious effects on the host strain. However, there was a noticeable size difference in the migration of recombinant and native BmpC during SDS-PAGE. It would seem that the majority of BmpC is not expressed on the OM of E. coli in its mature lipoprotein form, given that expression of the mature lipoprotein together with the N-terminal precursor peptide sequence (MNKKILSIFVMVMALSLLSIS) would yield a protein with a predicted molecular size approaching 28 kDa, which is close to the size of the recombinant protein as determined by SDS-PAGE. In addition, the N-terminal amino acid sequence obtained for recombinant BmpC matched the first 12 aa of the peptide leader sequence. No other proteins in E. coli SOLR containing p151 showed reactivity with anti-BmpC mAb, except an additional band of approximately 48 kDa that also suggests inefficient processing or post-translational modification of BmpC in E. coli, such as the formation of a dimer.
BmpC and B. hyodysenteriae SmpA share highly similar 21 aa signal peptide sequences. However, when expressed in E. coli, recombinant SmpA predominantly partitioned into the OM as both uncleaved prolipoprotein and fully processed, mature lipoprotein forms, with the amount of each form regulated by the stage of the growth cycle (Thomas & Sellwood, 1993). The differences demonstrated between recombinant BmpC and SmpA highlight the fundamental difficulties associated with the expression of Brachyspira lipoproteins in E. coli. Typically, to obtain stable expression of spirochaetal lipoproteins in E. coli in their lipidated form, it is necessary to modify the N-terminal sequence to facilitate appropriate processing, as reported for the pDUMP plasmid vector (Cullen et al., 2002
).
Analysis of the amino acid sequence of BmpC shows that it has some features in common with B. hyodysenteriae SmpA and the BmpB/BlpA family of paralogous lipoprotein genes (Cullen et al., 2003; Lee et al., 2000
; Thomas & Sellwood, 1993
). Most notably, the signal peptides of SmpA and BmpC showed significant homology and BlpG and BmpC possessed identical atypical signal peptidase II recognition sites of SISC. In Leptospira interogans, it is hypothesized that the sorting of leptospiral lipoproteins is governed by mechanisms similar to E. coli, in that the +2 and +3 amino acids of the mature, lipidated protein appear to be critical in determining the membrane location. A negatively charged amino acid in the +2 or +3 position targets the lipoprotein to the IM, whereas positively charged amino acids target the OM and neutral amino acids suggest the protein is expressed in both locations (Haake & Matsunaga, 2002
; Cullen et al., 2003
). The +2 and +3 positions of B. pilosicoli BmpC are both uncharged Asn molecules, whereas in B. hyodysenteriae SmpA and BmpB/BlpA they are Gly and Asn (Cullen et al., 2003
; Lee et al., 2000
). By this definition, SmpA, BmpB/BlpA and BmpC would localize to both the OM and IM. However, all three proteins appear to be expressed exclusively on the Brachyspira OM. The lipoprotein membrane targeting system identified in Leptospira does not appear to be universal amongst the spirochaetes and it seems more likely that the mechanisms governing the trafficking and localization of membrane-anchored lipoproteins are different between Leptospira and Brachyspira.
Because BmpC is abundant on the B. pilosicoli 95-1000 cell surface, we considered it worthy of further analysis, since surface-exposed proteins are likely to mediate interactions between a bacterium and its host environment. A number of interesting features associated with BmpC warrant further investigation.
First, when tested against 13 genetically heterogeneous B. pilosicoli isolates obtained from four different host species, mAb 2E10 was only reactive with strain 95-1000 (Table 2). This possibly suggests that BmpC is unique to strain 95-1000. Other explanations, however, are that surface-exposed epitopes of the protein vary in sequence, specifically the target region of the BmpC-specific mAb or BmpC is expressed differently in other strains. These characteristics are likely to be important for B. pilosicoli to survive immune surveillance in its mammalian host. Variability in surface-exposed regions of OMPs has been previously demonstrated in B. hyodysenteriae and has been suggested as a potential mechanism for chronic infection and evasion of the immune response (Cullen et al., 2003
; Gabe et al., 1998
; McCaman et al., 1999
).
Second, bmpC is located downstream from a series of 7 bp SSRs. Variation in the number of repeats was demonstrated between both phagemids containing bmpC and the contiguous sequence amplified from B. pilosicoli 95-1000. These variations demonstrate the occurrence of slipped strand mispairing. Slipped strand mispairing induced by intragenic SSRs or those located between the 35 and 10 promoter regions, is often used by pathogenic bacteria such as Neisseria meningitidis and Haemophilus influenzae as a mechanism for generating variability in surface-exposed proteins (van Belkum et al., 1998). SSRs of 7 bp in length are highly unusual in prokaryotes, but the fact that they are located upstream of the promoter region in B. pilosicoli may indicate that they have no effect on the expression of bmpC. It also may be possible that the same SSR is located in other regions of the B. pilosicoli chromosome, having an as yet unknown regulatory function.
Finally, B. pilosicoli bmpC is located immediately upstream of a gene (provisionally designated alp) encoding a protein containing multiple ankyrin repeats. Ankyrins are spectrin-binding structural proteins in red blood cells that bridge the exoskeleton to the cytoplasmic plasma membrane surface. In bacteria, many ankyrin-like proteins have been identified and they are normally located near genes involved in nutrient uptake or tolerance to adverse environmental conditions. For example, AnkA, an ankyrin-like protein of Ehrlichia phagocytophila may play a role in altering host-cell gene expression (Caturegli et al., 2000), whereas AnkB, identified in Pseudomonas aeruginosa, is involved in the protective response to oxidative stress induced by hydrogen peroxide (Howell et al., 2000
). Notably, ankyrin-binding proteins have also been implicated in host-cell interactions by pathogenic organisms, including Treponema pallidum (Weinstock et al., 1998
). The Coxiella burnetii genome contains 13 proteins with ankyrin repeats. In the absence of other genes encoding typical structures for adhesion in the C. burnetii genome, ankyrin-like proteins may serve a function in attachment to the host-cell extracellular matrix prior to internalization (Seshadri et al., 2003
).
The current study illustrates the effectiveness of isolating and identifying B. pilosicoli OMPs through membrane vesicle fractionation and production of B. pilosicoli-specific mAbs in the absence of a genomic database. Identification, cloning and sequencing of the genes encoding the four remaining membrane-associated proteins will facilitate further understanding of the Brachyspira OM and its unique host interactions.
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ACKNOWLEDGEMENTS |
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Received 9 September 2003;
revised 11 November 2003;
accepted 15 December 2003.
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