Identification and cloning of the gene encoding BmpC: an outer-membrane lipoprotein associated with Brachyspira pilosicoli membrane vesicles

Darren J. Trott1, David P. Alt3, Richard L. Zuerner3, Dieter M. Bulach2, Michael J. Wannemuehler6, Judi Stasko4, Kirsty M. Townsend1 and Thaddeus B. Stanton5

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The intestinal spirochaete Brachyspira pilosicoli causes colitis in a wide variety of host species. Little is known about the structure or protein constituents of the B. pilosicoli outer membrane (OM). To identify surface-exposed proteins in this species, membrane vesicles were isolated from B. pilosicoli strain 95-1000 cells by osmotic lysis in dH2O followed by isopycnic centrifugation in sucrose density gradients. The membrane vesicles were separated into a high-density fraction (HDMV; {rho}=1·18 g cm–3) and a low-density fraction (LDMV; {rho}=1·12 g cm–3). Both fractions were free of flagella and soluble protein contamination. LDMV contained predominantly OM markers (lipo-oligosaccharide and a 29 kDa B. pilosicoli OM protein) and was used as a source of antigens to produce mAbs. Five B. pilosicoli-specific mAbs reacting with proteins with molecular masses of 23, 24, 35, 61 and 79 kDa were characterized. The 23 kDa protein was only partially soluble in Triton X-114, whereas the 24 and 35 kDa proteins were enriched in the detergent phase, implying that they were integral membrane proteins or lipoproteins. All three proteins were localized to the B. pilosicoli OM by immunogold labelling using specific mAbs. The gene encoding the abundant, surface-exposed 23 kDa protein was identified by screening a B. pilosicoli 95-1000 genome library with the mAb and was expressed in Escherichia coli. Sequence analysis showed that it encoded a unique lipoprotein, designated BmpC. Recombinant BmpC partitioned predominantly in the OM fraction of E. coli strain SOLR. The mAb to BmpC was used to screen a collection of 13 genetically heterogeneous strains of B. pilosicoli isolated from five different host species. Interestingly, only strain 95-1000 was reactive with the mAb, indicating that either the surface-exposed epitope on BmpC is variable between strains or that the protein is restricted in its distribution within B. pilosicoli.


Abbreviations: DIG-AMP, digoxigenin-labelled ampicillin; HDMV, high-density membrane vesicles; IM, inner membrane; LDMV, low-density membrane vesicles; LOS, lipo-oligosaccharide; OM(P), outer membrane (protein); PBP, penicillin-binding protein; SP, soluble protein; SSR, short sequence repetitive element; TM, total membrane

The GenBank accession numbers for the sequences reported in this paper are AY363613, AY363614 and AY376355.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Brachyspira pilosicoli is the agent of porcine intestinal spirochaetosis (also known as porcine colonic spirochaetosis), a production-limiting disease of swine characterized by non-bloody, mucus-containing diarrhoea, poor feed conversion and depressed growth rates (Hampson & Trott, 1999; Ochiai et al., 1997; Stanton, 2002; Thomson et al., 1998; Trott et al., 1996d). B. pilosicoli infection also results in delayed onset of egg production and pasty, wet faeces in poultry (Stephens & Hampson, 2002; Trampel et al., 1994). This spirochaete has been isolated from a wide range of other monogastric hosts, including dogs (Duhamel et al., 1998), monkeys (Duhamel, 2001), game birds (Trott et al., 1996c) water birds (Oxberry et al., 1997) and humans (Lee & Hampson, 1994). In humans, a high incidence of B. pilosicoli infection (~30 %) has been reported in individuals in developing communities (Trott et al., 1997) and in AIDS patients and homosexual males in more affluent societies (Trivett-Moore et al., 1998). Rates of colonization amongst other groups of individuals are extremely low. Day-old SPF chicks (Muniappa et al., 1996; Trott et al., 1995), adult chickens (Stephens & Hampson, 2002), newly weaned pigs (Duhamel, 1996; Thomson et al., 1997; Trott et al., 1996a) and laboratory mice (Sacco et al., 1997) have been used to demonstrate the pathogenic capability of B. pilosicoli strains isolated from both humans and animals; however, the mechanisms whereby B. pilosicoli infection results in colitis and mild diarrhoea are currently unknown.

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 ({rho}=1·10 g cm–3) compared to the IM ({rho}=1·16 g cm–3) 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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and culture conditions.
Brachyspira pilosicoli strain 95-1000 was obtained from Professor David Hampson, School of Veterinary and Biomedical Science, Murdoch University, Western Australia. Strain 95-1000 was originally cultured from the colonic scrapings of a grower pig with porcine intestinal spirochaetosis and is pathogenic, producing polar attachment in experimentally infected swine (Trott et al., 1996a). Spirochaetes were cultivated in stirred, pre-reduced medium under an atmosphere of 99 % N2/1 % O2 as described previously (Stanton & Lebo, 1988; Trott et al., 1996b).

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 ml–1 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 g–1 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 4–20 % 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 ml–1) 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 (15–25 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 {lambda} 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 {lambda} 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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Table 1. Oligonucleotide primers used for amplification and sequencing of inserts p151 and p421 containing B. pilosicoli bmpC

 
Expression of BmpC in E. coli.
E. coli strain SOLR (Stratagene) containing either recombinant phagemid p151 or a non-reactive phagemid, p371, were grown overnight with shaking at 37 °C in 50 ml LB broth containing ampicillin (50 µg ml–1) and 1 mM IPTG. Cells were pelleted and used either as whole-cell preparations or were separated into periplasmic, soluble, cytoplasmic membrane and OM fractions (Oliver & Beckwith, 1982) for analysis by SDS-PAGE and Western blotting. Bands that were immunoreactive with mAb 2E10 were identified by incubation with HRPO-conjugated goat anti-mouse antibodies and visualized using 4-chloro-1-naphthol colour reagent. For sequence analysis of the N terminus of the expressed protein, OM fractions of E. coli containing p151 were resolved on a Tris/Tricine 4 % stacking/15 % separating SDS-PAGE gel and blotted onto a PVDF membrane. The expressed protein band identified in E. coli SOLR containing p151 was excised from the Coomassie-blue-stained PVDF membrane and submitted to the Protein Facility of Iowa State University (Ames, IA, USA) for N-terminal amino acid sequencing by Edman degradation and HPLC analysis (Matsudaira, 1987).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Osmotic lysis of B. pilosicoli 95-1000
Membrane fractions from B. pilosicoli cells were initially prepared by the method of Plaza et al. (1997). Analysis of the fractions obtained by this technique using known B. pilosicoli cellular markers demonstrated that the membrane fractions were free of flagella and the cytoplasmic NOX protein. However, there was no difference between the fractions in the composition of key IM and OM markers and their SDS-PAGE profiles were very similar.

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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Fig. 1. Transmission electron micrographs of osmotic-induced lysis of the B. pilosicoli 95-1000 OM following mixing in dH2O. (a) Ultrathin section of B. pilosicoli 95-1000 cells prior to osmotic lysis. Bar, 0·15 µm. (b) Ultrathin section of B. pilosicoli cells following 2 h of gentle mixing in dH2O. Bar, 0·3 µm. (c) Negatively stained B. pilosicoli protoplasmic cylinder following 2 h of gentle mixing in dH2O. The periplasmic flagella have unwound from the protoplasmic cylinder, but are still attached by their insertion discs. Bar, 0·3 µm.

 
Harvested membranes were fractionated by sucrose density gradient ultracentrifugation. Broken flagella and remnant protoplasmic cylinders could be separated from membrane vesicles based on their higher density. As determined by electron microscopy, these cellular components formed a pellet ({rho}>1·20 g cm–3) at the bottom of the tube. The HDMV and LDMV bands were visualized by staining with octadecyl rhodamine B chloride and were harvested by needle aspiration. Examination of both negatively stained and thin sections of HDMV (Fig. 2a) and LDMV (Fig. 2b) by electron microscopy showed that both fractions consisted of unilamellar membrane vesicles. A second round of sucrose density ultracentrifugation was used to further purify the HDMV and LDMV fractions. The respective densities were 1·18 and 1·12 g cm–3.



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Fig. 2. Transmission electron micrographs of negatively stained B. pilosicoli 95-1000 membrane vesicles obtained by osmotic lysis and isopycnic centrifugation. (a) HDMV, {rho}=1·18 g cm–3. Bar, 0·3 µm. (b) LDMV, {rho}=1·12 g cm–3. Bar, 0·2 µm.

 
Assay of membrane vesicle fractions for cellular and membrane markers
The protein electrophoresis profiles of the HDMV and LDMV fractions differed considerably (Fig. 3). The HDMV fraction contained prominent 23, 45 and 55 kDa proteins (Fig. 3, lane 2), which were present, but reduced in the LDMV fraction (Fig. 3, lane 3). Known cellular markers were used to further characterize the composition of the fractions.



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Fig. 3. SDS-PAGE profiles of B. pilosicoli 95-1000 membrane vesicle fractions obtained by osmotic lysis and sucrose density ultracentrifugation. Lanes 1, TM fraction; 2, HDMV; 3, LDMV; 4, SP fraction. Lanes were loaded with 10 µg protein. The relative mobilities of molecular mass markers are indicated on the left.

 
NADH oxidase (48 kDa) was detected in the SP fraction and was absent from the TM, HDMV and LDMV fractions (Fig. 4a). Similarly, a 44 kDa flagella sheath protein was only identified in the TM fraction (Fig. 4b). Non-specific binding was not observed in immunoblots probed with polyclonal antibodies to either NADH oxidase or FlaA1. These data indicate that the B. pilosicoli membrane vesicle fractions obtained by osmotic lysis and isopycnic centrifugation were free of both cytoplasmic and flagella contamination.



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Fig. 4. Analysis of B. pilosicoli 95-1000 membrane vesicle fractions obtained by osmotic lysis and sucrose density ultracentrifugation for specific cellular and membrane markers. (a) SP marker (Western blot probed with polyclonal antibody against the 48 kDa B. hyodysenteriae NADH oxidase). (b) Flagella protein marker (Western blot probed with polyclonal antibody against the 44 kDa B. hyodysenteriae flagella sheath protein). (c) OM marker 1 (Western blot probed with mAb against a 29 kDa B. pilosicoli OMP). (d) OM marker 2 (Silver stained SDS-PAGE gel showing the lipid A portion of B. pilosicoli LOS). (e) DIG-AMP PBP assay. Lanes: 1, TM fraction; 2, HDMV; 3, LDMV; 4, SP fraction. Lanes were loaded with 5 µg protein, except for (d) where each lane was loaded with 75 µg protein. The relative mobilities of molecular mass markers are indicated on the left.

 
When probed with an mAb to the surface-exposed 29 kDa B. pilosicoli protein (Fig. 4c) the LDMV showed a higher concentration of the OM-associated protein compared to the HDMV. A similar profile was obtained when the proteinase-K-treated membrane vesicle fractions were silver-stained for LOS (Fig. 4d). PBPs are known to be present in spirochaete IMs (Radolf et al., 1989) and can be detected by using a digoxigenin-ampicillin (DIG-AMP) assay (Weigel et al., 1994). At least five major PBPs were observed in B. pilosicoli 95-1000 TM preparations, having molecular masses of 21, 37, 48, 74 and 83 kDa (Fig. 4e, lane 1). Binding of DIG-AMP was completely inhibited by unlabelled ampicillin, indicating the specificity of this assay for PBPs. PBPs were detected in the HDMV fraction at reduced levels compared to the TM fraction, although there was significant enrichment of the 21 kDa protein (Fig. 4e, lane 2). The proportion of PBPs was markedly reduced in LDMV compared to the other two fractions (Fig. 4e, lane 3).

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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Table 2. Characteristics of proteins reacting with B. pilosicoli-specific mAbs

 
Each mAb was used to probe TM, HDMV, LDMV and SP fractions to determine the distribution of the proteins in these fractions. In addition, to identify which of the mAbs were likely to bind to integral membrane proteins, the mAbs were also used to probe insoluble, detergent-phase and aqueous-phase Triton X-114 extracts of B. pilosicoli 95-1000 whole cells (Fig. 5). The proteins identified by the mAbs showed differences in their pattern of distribution in the membrane vesicle and Triton X-114 fractions. The 23 and 61 kDa proteins were distributed in TM, HDMV and LDMV membrane vesicle fractions, but comparatively reduced in the Triton X-114 detergent fractions. Furthermore, there was no difference in the pattern of distribution between the detergent and insoluble phases, suggesting that both proteins were relatively insoluble in Triton X-114. Triton X-114 detergent extraction appeared to result in cleavage of the 61 kDa protein to a smaller product. In contrast, the 24 and 35 kDa proteins were enriched in both the LDMV fraction and the Triton X-114 detergent phase, but not in the aqueous and insoluble Triton X-114 phases. This suggests the possibility that both are integral OMPs or lipoproteins anchored to the OM. The mAb reacted with a 79 kDa protein that was present in both TM and SP fractions. However, this protein could not be detected in the HDMV fraction and was present in only trace amounts in the LDMV fraction, suggesting that it had either undergone proteolysis or had become detached from the membrane during osmotic lysis and isopycnic ultracentrifugation. However, it may be conceivable that the 79 kDa protein is a periplasmic protein that has contaminated the membrane fractions.



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Fig. 5. Probing of Brachyspira pilosicoli 95-1000 membrane vesicle fractions and Triton X-114 extracts with B. pilosicoli membrane-protein specific mAbs obtained by immunizing mice with LDMV. (a) 4F2; (b) 2G3; (c) 1G2; (d) 2H3; (e) 2E10. Lanes: 1, TM fraction; 2, HDMV; 3, LDMV; 4, SP fraction; 5, Triton X-114 insoluble material; 6, Triton X-114 detergent phase; 7, Triton X-114 aqueous phase. Lanes were loaded with 5 µg protein. The relative mobilities of molecular mass markers are indicated on the left.

 
Immunogold labelling of B. pilosicoli whole cells
Immunogold labelling of negatively stained whole cells with mAb 2E10 (directed against the 23 kDa protein) identified an antigen that was present in abundance on the outer surface of B. pilosicoli 95-1000 cells (Fig. 6a). Immunogold labelling of B. pilosicoli cells that had been subjected to osmotic lysis and had lost their OM showed that the protein did not appear to be associated with the protoplasmic cylinder (Fig. 6b). Localization of the protein to the B. pilosicoli 95-1000 OM was also confirmed by immunogold labelling of fixed-cell cross-sections (data not shown). Immunogold labelling with mAbs 1G2 and 2H3 also identified proteins that were surface-associated, but not as abundant as the 23 kDa protein identified by 2E10 (data not shown). Immunogold labelling of both whole cells and protoplasmic cylinders with mAbs 4F2 and 2G3 did not conclusively localize the 79 kDa or the 61 kDa proteins to either the OM or the IM.



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Fig. 6. (a) Immunogold labelling of an intact B. pilosicoli 95-1000 cell with mAb 2E10, raised against a 23 kDa B. pilosicoli membrane protein. The protein is present in abundance on the outer surface of the cell. Bar, 0·2 µm. (b) Following removal of the B. pilosicoli OM by osmotic lysis, there is no immunogold labelling of the cell wall/cytoplasmic membrane complex. Bar, 0·25 µm.

 
Identification, cloning and sequence analysis of BmpC
The 23 kDa protein was an abundant constituent of the B. pilosicoli OM. Therefore, to facilitate identification and sequencing of the gene encoding the 23 kDa protein, mAb 2E10 was used to screen a B. pilosicoli genomic library in {lambda} ZAP II. Two recombinant phagemids (p151 and p421) containing DNA-encoding proteins that were reactive with mAb 2E10 and a third phagemid containing an unreactive insert (p371) were selected and were found to be stable in E. coli SOLR cells. In whole-cell lysates of E. coli SOLR cells containing p151 or p421, mAb 2E10 reacted strongly with a protein of approximately 28 kDa and weakly with a band of 48 kDa (Fig. 7, lane 8). Furthermore, the 28 kDa band was enriched in the E. coli OM fraction but was only present in trace amounts in the periplasmic fraction compared to the soluble and cytoplasmic membrane fractions. In contrast, the 48 kDa band was reduced in the cytoplasmic membrane fraction compared with the other fractions. E. coli SOLR containing p371 was consistently unreactive with mAb 2E10, confirming that the reactivity was associated with the DNA insert and not the carrier strain (Fig. 7, lane 7).



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Fig. 7. Immunoblot of E. coli cell fractions containing plasmid p151 or p371. Fractions containing p371 (lanes 1, 3, 5, 7) and p151 (lanes 2, 4, 6, 8) were electrophoresed, transferred to PVDF and probed with mAb 2E10. Periplasmic fraction (lanes 1 and 2), soluble fraction (lanes 3 and 4), cytoplasmic membrane fraction (lanes 5 and 6) and OM fraction (lanes 7 and 8) were examined. Prestained molecular mass markers (lane 9) are indicated on the right.

 
The cloned inserts carried on p151 and p421 were determined to be 3·7 and 4·0 kb respectively. Analysis of the 4·9 kb contiguous sequence determined from these cloned inserts identified four ORFs (Fig. 8). Sequence comparisons with the SWISS-PROT database identified an aldehyde oxidoreductase (54 % sequence identity over 392 aa with Enterococcus faecalis aldehyde oxidoreductase), a putative synthase (45 % sequence identity over 271 aa with an E. coli putative synthase) and a protein provisionally designated Alp (ankyrin-like protein) that contained multiple ankyrin repeats. A 561 bp unidentified ORF showed no significant homology with any protein in the database. To further associate one of these four ORFs with the 23 kDa protein, an N-terminal amino acid sequence was determined from the recombinant 28 kDa protein expressed in E. coli SOLR. An N-terminal sequence of 12 residues (MNKKILSIFVMV) was obtained, enabling the definitive identification of the protein reacting with mAb 2E10 as the 561 bp unidentified ORF. The deduced amino acid sequence of this protein would encode a 166 aa polypeptide with a predicted size of 20 kDa and a 21 aa signal peptide (MNKKILSIFVMVMALSLLSIS).



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Fig. 8. Organization of the 4·9 kb B. pilosicoli contiguous sequence obtained from cloned inserts p151 and p421. The location and orientation of the four genes identified in the sequence, including bmpC, are indicated with a single-headed arrow.

 
A series of 7 bp short sequence repetitive elements (SSRs) with a consensus sequence of 5'-AATCAGC-3' was identified in the intergenic region upstream of the 561 bp unidentified ORF, terminating at 237 bp prior to the start codon. Sixteen copies were present in p421, whereas 17 copies were present in p151. The contiguous sequence was also amplified and sequenced by PCR from B. pilosicoli 95-1000 to determine how many copies of the SSR were present in the genomic DNA. Surprisingly, only eight copies were identified, indicating the possibility of replication errors due to slipped strand mispairing. Putative –35 (ATAAACA) and –10 (TATAAT) promoter regions and a ribosome-binding site (AGGAG) were identified upstream of the 561 bp ORF. The hydrophobic precursor signal peptide shared an identical putative spirochaete lipoprotein signal peptidase II recognition site (SISC) with BlpG, one of four paralogous OM lipoproteins recently identified in B. hyodysenteriae (Cullen et al., 2003; Haake, 2000). The +2 and +3 amino acids of the mature protein were both Asn (N) residues, an uncharged amino acid identified in the +2 and/or +3 positions of the other identified Brachyspira OM lipoproteins (Cullen et al., 2003; Lee et al., 2000; Thomas & Sellwood, 1993;). The Kyte–Doolittle hydropathy plot predicted that apart from the hydrophobic precursor region, the protein contained no transmembrane domains. These analyses together with the results of immunogold labelling of B. pilosicoli whole cells suggest that the ORF is expressed as a lipoprotein on the B. pilosicoli OM, tethered to the membrane by a lipid moiety attached to the Cys at amino acid residue 22. Acylation of the mature protein may explain the apparent difference in size based on its deduced amino acid sequence with the size estimation based on its migration in SDS-PAGE gels. In accordance with the previous nomenclature adopted for Brachyspira membrane proteins (Lee et al., 2000; Thomas & Sellwood, 1993), we propose that this unique B. pilosicoli OM lipoprotein be designated BmpC (Brachyspira membrane protein C) and that the gene encoding the protein be designated bmpC. Although the BmpC sequence was unique and did not share close homology with any other identified protein, 24 of the first 32 aa (75 %) were identical to B. hyodysenteriae SmpA. Notably SmpA and BmpC both possess signal peptides that are 21 aa in length and share an identical –1 to +4 amino acid sequence of SCNNK (Thomas & Sellwood, 1993).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identifying B. pilosicoli OMPs expressed during infection is a prerequisite for the analysis of the parasite–host interaction at a molecular level. A necessary starting point is to isolate these proteins relatively free of contamination from the other cellular components. However, isolating the OM of B. pilosicoli by adopting previously published non-detergent based techniques for spirochaetes was found to be problematic (Blanco et al., 1994; Bledsoe et al., 1994; Plaza et al., 1997; Radolf et al., 1995a; Radolf et al., 1995b; Skare et al., 1995). Haake & Matsunaga, (2002) also experienced similar difficulties in obtaining Leptospira OM vesicles in sufficient quantities for characterization without concomitant contamination by IM components and eventually obtained relatively pure OM vesicles by gently mixing leptospiral whole cells in a hypertonic alkaline buffer containing Tris, NaCl and EDTA followed by isopycnic centrifugation. The inability to apply the same OM isolation techniques to different genera of spirochaetes demonstrates key fundamental differences in OM composition and function between Treponema, Borrelia, Leptospira and Brachyspira. In particular, Brachyspira OMs appear to be unusual in that they contain a significant proportion of sterols (cholesterol and cholestanol) and have a relatively low density (Plaza et al., 1997; Stanton, 1987; Trott et al., 2001). These unique OM characteristics may result in the Brachyspira OM being more susceptible to osmotic stress than other pathogenic spirochaetes, manifested by the rapid loss of Brachyspira OM integrity in low ionic strength buffers (S. Humphrey & D. J. Trott, unpublished data).

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 cm–3 and the fractions were shown to be free of flagella and cytoplasmic protein contamination. The low density ({rho}=1·12 g cm–3), 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.


   ACKNOWLEDGEMENTS
 
We thank Karen Halloum and Loren Jones for expert technical assistance. We also thank Dr Michael V. Norgard for the initial gift of 500 µl DIG-AMP, Dr Mario Jacques for providing the FlaA1 antisera and Dr David Hampson for providing B. pilosicoli strains and mAb to the 29 kDa OMP. The excellent advice of Sam Humphrey, Dr Justin Radolf and Dr Frank Gherardini is greatly appreciated and we especially thank Dr Paul Cullen for critical review of the manuscript.


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Received 9 September 2003; revised 11 November 2003; accepted 15 December 2003.



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