The Enolase of Borrelia burgdorferi Is a Plasminogen Receptor
Released in Outer Membrane Vesicles
A. Toledo,aJ. L. Coleman,a,bC. J. Kuhlow,aJ. T. Crowley,aand J. L. Benacha
Department of Molecular Genetics and Microbiology, Center for Infectious Diseases, Stony Brook University, Stony Brook, New York, USA,aand State of New York
Department of Health, Stony Brook University, Stony Brook, New York, USAb
The agent of Lyme disease, Borrelia burgdorferi, has a number of outer membrane proteins that are differentially regulated dur-
enhances the ability of the bacteria to disseminate in the host. Outer membrane vesicles of B. burgdorferi contain enolase, a
of the spirochetes is assisted by borrowed proteolytic activity that
tors (17). Plasmin, a serine protease, is bound to the organism as
plasminogen (PLG), a proenzyme present in body fluids that is
assist borreliae in degrading extracellular matrices and basement
membranes, with the ultimate result of facilitating dissemination
(21–23, 44, 59).
Borreliae have a close relationship with the host’s PLG activa-
tion system. As mentioned above, these spirochetes can fix plas-
immune response to produce PLG activators (19, 20, 32, 42, 46)
borreliae are important stimulators for the production of matrix
metalloproteinases as part of their proinflammatory repertoire
(34, 35, 43). B. burgdorferi has an A-T rich genome with a corre-
sponding abundance of lysines (31), which are the most common
amino acids in PLG receptors. Thus, it is no surprise that B. burg-
lysine-rich OspA is a PLG receptor (33), but, other than in the
initial stages of tick feeding, this interaction is not likely to be
important in the dissemination within the mammalian host, as
expression of OspA is downregulated (68). In contrast, OspC, a
lipoprotein expressed after ticks begin to feed and in the early
stages of infection of the mammal, is also a PLG receptor and one
PLG receptors of B. burgdorferi include the Erp lipoproteins (12).
for dissemination as well (36, 59). Recent investigations have
shown that both B. burgdorferi (10, 40) and RFB (41, 65, 67) have
molecules that function as receptors for multiple ligands. Com-
orrelia burgdorferi, the agent of Lyme disease, invades distant
tissues from its site of entry in the skin (4, 14). Dissemination
plement regulator-acquiring surface proteins of B. hermsii and B.
burgdorferi (CspA) can bind extracellular matrices, factor H, and
distinct nonoverlapping domains (65). Factor H binding protein
plement and fixing of an active plasmin onto the surface for dis-
semination in borreliae.
by PLG activators to form plasmin. The active enzyme consists of
five kringle domains, each with three disulfide bonds that contain
the lysine binding sites and the catalytic domain. PLG binding is
an important part of the pathogenesis of infections by Gram-
detail. Two glycolytic enzymes from Gram-positive bacteria have
been implicated in the binding of PLG (5, 6). GAPDH
cells, where they perform their traditional enzymatic functions in
catalyze 2-phosphoglycerate to phosphoenolpyruvate. However,
there is evidence of the presence of enolase on the surface of
Received 19 August 2011 Returned for modification 1 October 2011
Accepted 28 October 2011
Published ahead of print 14 November 2011
Editor: J. L. Flynn
Address correspondence to J. L. Benach, email@example.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
0019-9567/12/$12.00 Infection and Immunityp. 359–368 iai.asm.org
Gram-positive (5, 62) and Gram-negative (69) bacteria, fungi,
and protozoa (3, 58). The surface location of enolase in several
types of prokaryotic and eukaryotic cells is intriguing, since this
enzyme does not have known cell surface protein motifs such as a
signal peptidase cleavage site, cell wall anchors or sequences, or
tococci binds PLG through both terminal and internal lysine res-
protozoa (Trichomonas) and Candida. The internal and terminal
lysine residues of the enolase of B. burgdorferi are also conserved,
suggesting that this enzyme could be an important PLG receptor
in this organism. Furthermore, the enolases of other bacteria are
immunogenic, suggesting that this could also be true for B. burg-
Outer membrane vesicles (OMV) are released naturally by
bacterial cell, and recent proteomic studies have now shown a
significant representation of cytosolic and inner membrane mol-
ecules (51). OMV have been considered a part of the stress re-
sponse (56), and their functions and roles in infection have been
recently reviewed (30). Among the most important functions are
the release of toxins and virulence factors, interaction with other
bacteria and host cells, and modulation of the host response (30).
The Borrelia OMV have been studied both in cultured organisms
and in vivo. Borrelia OMV induce B cell responses under experi-
lipoprotein and glycolipid contents (64) and the presence of both
of OMV from green fluorescent protein (GFP)-labeled spiro-
chetes during blood feeding in ticks was noted. An in vivo study
showed that, depending of the conditions to which the organisms
are exposed in feeding ticks, release of OMV can be induced and
burgdorferi have shown that OMV are released near sites of cell
(49, 50). In this study, we document the presence of enolase in
lysine-dependent manner, is immunogenic, and does not appear
to be exposed on the surface of the intact organism. The role of
enolase in the OMV could be that of fixing plasmin in the perib-
MATERIALS AND METHODS
Bacteria, cultures, and sera from laboratory animals and Lyme disease
at 33°C. A New Zealand White rabbit (Charles River, Wilmington, MA)
was intradermally inoculated with 50 ?g of recombinant enolase in com-
recombinant protein in incomplete Freund’s adjuvant to develop a mea-
ries, Bar Harbor, ME) infected intradermally with 2 ? 104spirochetes
were collected at various intervals after inoculation. Serum from a rabbit
infected with B. burgdorferi were collected following the attachment of
infected Ixodes scapularis females. Sera from patients with disseminated
Lyme disease (with joint or nervous system involvement) and with previ-
for determination of their reactivity to recombinant enolase.
Vesicle isolation purification and mass spectrometry. B. burgdorferi
bacteria were harvested by centrifugation for 12 min at 4,000 ? g and
incubated at 37°C in fresh complete BSK-II media for 2 h followed by
centrifugation. The supernatants were collected and filtered twice using
supernatants were pelleted by ultracentrifugation for 1 h at 100,000 ? g
and resuspended in 40% OptiPrep (Axis Shield, Norton, MA). A discon-
centrifuged for 16 h at 100,000 ? g. The OMV floated to the interface
between the 20% layer and the 25% layer, where a white band was visual-
purified (based on similar protein contents) OMV were pooled from
OptiPrep solution by resuspension in 50 ml of 20 mM HEPES (pH 7.5)
and were centrifuged for 1 h at 100,000 ? g. The pelleted, purified OMV
were resuspended in 20 mM HEPES (pH 7.5). The OMV were analyzed
matography and tandem mass spectrometry (MS/MS) or multidimen-
sional protein identification technology (MudPIT).
Recombinant protein. N-terminal polyhistidine-tagged enolase was
generated in pET-28a(?) vector (EMD Chemicals Inc., Gibbstown, NJ)
by amplification of the enolase gene (bb0337 ) by the use of primers
EnolNterF (5=-CATATGGGTTTTCACATTTATGA-3=) and EnolNterR
(5=-CTCGAGAATTTTTTGTTTAATAGAATA-3=) (The Midland Certi-
fied Reagent Company, Midland, TX) followed by digestion with restric-
England BioLabs). The resultant plasmid’s insert was sequenced on both
The recombinant enolase was expressed in Escherichia coli Rossetta
(DE3) (Novagen, Madison, WI) upon induction with IPTG (isopropyl-
?-D-thiogalactopyranoside). The culture was subjected to sonication and
the debris pelleted by centrifugation. The recombinant protein, in the
soluble fraction, was purified following a His-Trap procedure. Protein
purification was assessed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) followed by Coomassie brilliant blue stain-
ing. Protein concentrations were measured by a bicinchoninic acid pro-
tein assay. (Thermo Fisher Scientific, Rockford, IL.)
Western blot assays. A total of 3 ?g of the recombinant enolase and
feri grown at 34°C (pH 6.4 or pH 7.6) and at 23°C (pH 7.6) or the OMV
of phosphate-buffered saline (PBS) and received 10 ?l of 3? SDS-PAGE
components of each sample were separated by electrophoresis using
12.5% SDS-PAGE, and the proteins were transferred to nitrocellulose
membranes (GE Healthcare, Piscataway, NJ). The antibodies used in the
anti-DnaK (mouse IgG1) antibody (16), murine monoclonal anti-OspA
(mouse IgG1) antibody (16), murine monoclonal antiflagellin (mouse
were resolved with infrared-labeled goat anti-rabbit IgG IRDye700CW
and goat anti-mouse IgG IRDye800CW (Rockland Immunochemicals,
Gilbertsville, PA) and visualized by scanning with an Odyssey infrared
imaging system (LiCor Biosciences, Lincoln, NE).
ELISA. Microtest 96-well enzyme-linked immunoassay (ELISA)
plates (BD Biosciences, Bedford, MA) were coated overnight at 4°C with
human PLG (Sigma-Aldrich, St. Louis, MO) (10 ?g/ml), recombinant
enolase (10 ?g/ml), Borrelia whole lysate (10 ?g/ml), or bovine serum
albumin (BSA) (10 ?g/ml) in coating buffer (50 mM NaCO3, 50 mM
NaHCO3). After three PBS washes, the ELISA plates were blocked with
1% BSA for 1 h at 37°C and washed again. Recombinant enolase, PLG
sera (1:100 in PBS) were added and incubated for 1 h at 37°C and washed
with PBS. Several ELISA experiments were carried out as follows. (i) For
Toledo et al.
iai.asm.org Infection and Immunity
detection of enolase bound to PLG, rabbit antienolase serum was incu-
bated for 1 h and washed three times with PBS followed by incubation
with a goat anti-rabbit IgG-alkaline phosphatase conjugate (Sigma) at
37°C for 1 h. (ii) For detection of the PLG bound to enolase, rabbit anti-
human PLG (Boehringer, Rheim, Germany) was incubated for 1 h and
washed three times with PBS followed by incubation with a goat anti-
rabbit IgG-alkaline phosphatase conjugate (Sigma) at 37°C for 1 h. (iii)
For detection of rabbit, mouse, or human antibodies to the recombinant
enolase or to the whole B. burgdorferi cell lysate, goat anti-rabbit IgG-
alkaline phosphatase (Sigma), goat anti-mouse IgG-alkaline phosphatase
(Sigma), and goat anti-human IgG-alkaline phosphatase (KPL, Gaithers-
burg, MA) were each incubated for 1 h at 37°C. Finally, the wells were
washed three times with PBS before the alkaline phosphatase substrate
(Sigma) was added. Plates were incubated at 37°C, and optical densities
(OD) were read using a SpectraMax M2 ELISA plate reader (Molecular
Devices, Sunnyvale, CA).
binding to PLG, increasing concentrations (0 to 400 mM) of NaCl
(Sigma) were added to the plates together with the PLG. For assays ana-
lyzing the role of heparin binding domains in the enolase-PLG interac-
tion, increasing concentrations (0 to 500 IU) of heparin (Sigma) were
used. To determine the role of lysines in the enolase-PLG interaction, the
lysine analog ?-aminocaproic acid (Sigma) (1 mM) was added with PLG
to the enolase-coated plates.
Carlsbad, CA), and incubated in Hank’s balanced salt solution (HBSS)
(Invitrogen) with rabbit antienolase serum (1/100) for 1 h at 33°C to
determine whether the enolase binds the surface of intact borreliae. After
being washed in cold PBS (Invitrogen), the spirochetes were added to the
wells of a Teflon-coated indirect immunofluorescence assay (IFA) slide
anol. A 1:1,000 solution of goat anti-rabbit IgG conjugated to fluorescein
isothiocyanate (FITC) (Abcam, Cambridge, MA) was added to each well
followed by incubation for 1 h in a wet chamber. Slides were washed with
PBS, dried before the slow-fade mounting medium (Invitrogen) was
placed on the slides, and viewed using a Nikon Eclipse E400 microscope.
copy (TEM). B. burgdorferi was incubated with rabbit antienolase serum
in HBSS for 1 h at 33°C. The spirochetes were fixed to polyvinyl formal-
and blocked for 30 min at room temperature in 1% BSA (Sigma). Grids
gold-labeled antibodies (Jackson Immunochemicals, West Grove, PA) at
a dilution of 1:250 for 1 h at room temperature. Subsequently, grids were
the peritoneal cavity of rats (1) were used for detection of enolase. These
University of Connecticut, and were fixed to the grids in the manner
described above for the cultured organism.
PK treatments. Proteinase K (PK) treatment of whole B. burgdorferi
bacteria was done in a manner similar to methods published previously
(13, 25, 29). B. burgdorferi cells were harvested from complete BSK-II
were resuspended in HBSS, divided into equal aliquots of approximately
108cells, centrifuged, and then resuspended in 1 ml (each) of Dulbecco’s
PBS (Invitrogen) containing 5 mM MgCl2(PBS-Mg) alone or PBS-Mg
with PK (Boehringer) at a concentration of 50 or 250 ?g/ml. This was
followed by incubation with gentle agitation for 1 h at room temperature
in HBSS containing a protease inhibitor cocktail (EDTA-free; Roche Di-
and each received 12.5 ?l of 5? SDS-PAGE sample buffer with
2-mercaptoethanol and was then boiled for 5 min. The entire content of
each sample was subjected to 12.5% SDS-PAGE, and the protein was
transferred to nitrocellulose. Enolase was detected using hyperimmune
protein A (OspA) and periplasmic flagellar protein FlaB were detected by
antibodies were infrared-labeled goat anti-rabbit IgG IRDye700CW and
scanning with an Odyssey infrared imaging system (LiCor Biosciences,
For digestion of recombinant enolase, 4 ?g of purified protein was
incubated for 1 h at room temperature (23°C) with or without PK (250
?g/ml) in 40 ?l of PBS-Mg. Proteolysis was stopped by addition of 10 ?l
of 5? SDS-PAGE sample buffer and boiling for 5 min. Following 12.5%
Coomassie brilliant blue staining.
For proteolytic digestion of OMV with PK, 10 ?g of purified OMVs
was treated with a range of PK concentrations for 1 h at (23°C) and de-
veloped as described above for intact spirochetes and recombinant eno-
lase. Controls included the use of an antibody to OspA and rabbit anti-
RNA extraction and quantitative real-time PCR. B. burgdorferi cul-
tures were grown to a density of 108spirochetes/ml under two different
sets of conditions (33°C at pH 6.4 and 23°C at pH 7.6), and RNA was
extracted using TRI reagent (MRC Inc., Cincinnati, OH) following the
manufacturer’s instructions. The RNA was treated once at 37°C for 60
min with DNase I (Roche) to remove any contaminating DNA. A final
purification step using an RNeasy Midi kit (Qiagen, Valencia, CA) was
carried out to eliminate DNase I as well as any trace of DNA contamina-
tion. Total RNA was quantified using an ND-1000 spectrophotometer
(NanoDrop Products, Wilmington, DE). The RNA samples devoid of
contaminating DNA were reverse transcribed to cDNA with TaqMan re-
verse transcription reagents (Applied Biosystems, Carlsbad, CA). Real-
primers specific to flaB (bb0147/342RT [5=-AGAGCAACTTACAGACG
AAAT-3=] and bb0147/472RT [5=-AGTGATGCTGGTGTGTTAAT-3])
and enolase (bb0337; bb0337/680RT [5=-TAAAGAAGGCAGGATATGA
AC-3=] and bb0337/814RT [5=-GCCCAATATTCAACCAT-3=]) at a final
concentration of 2 ?M and were used in an experimental procedure with
a 7500 Fast real-time PCR system (Applied Biosystems). Preincubation
cycle parameters were as follows: 1 cycle at 95°C for 5 min followed by 40
cycles of 95°C for 15 s, 55°C for 6 s, and 72°C for 6 s.
Statistics. Data were analyzed using Welch’s test with the GraphPad
Enolase and other PLG receptors are present in OMV. The pro-
tein content of the OMV of B. burgdorferi was analyzed by mass
jor proteins detected were OspB and OspA, which accounted for
ingly, the third most abundant protein detected was GroEL
(bb0560), a cytosolic heat shock protein homolog that has also
been associated with binding of membrane proteins in Borrelia
(60) and other bacteria (11, 37). Two proteins from the glycolytic
cycle, enolase (bb0337) and glyceraldehyde-3-phosphate dehy-
drogenase (bb0057), were also found (2). Both of these enzymes
OMV mass spectrometry results were confirmed by Western blot
analysis (Fig. 1), where reactivity of an OM protein, OspA, was
detected but no reactivity to DnaK, a known cytosolic molecule,
was detected. CspA, also a PLG receptor (40), was also present in
the OMV. It is of interest that the OMV have multiple molecules
that can bind PLG in a known lysine-dependent manner.
The generation of His tag enolase was performed as described
Enolase, a Plasminogen Receptor in Vesicles
January 2012 Volume 80 Number 1iai.asm.org 361
in Materials and Methods, and the different pools obtained were
analyzed by SDS-PAGE to confirm the presence and purity of the
protein before they were used to immunize a rabbit. Briefly, the
enolase gene (bb0337) was amplified and the amplicon, as well as
the plasmid, purified and treated with restriction enzymes. Both
products were purified again to eliminate the small cut fragment
with primers that target both the plasmid and the insertion to
used for the protein purification.
The preimmunization serum of a rabbit used to develop anti-
bodies showed no reactivity to the recombinant enolase (Fig. 2A,
lane 1) and developed antibodies after immunization (Fig. 2A,
lanes 2 to 5). Likewise, preimmunization rabbit serum exhibited
no reactivity to whole Borrelia cell lysate (Fig. 2B) but recognized
native enolase after immunization (Fig. 2C).
B. burgdorferi enolase binds PLG in a dose-dependent man-
(60) and in OMV suggests that it is localized to the OM of the
spirochete and that it could therefore play a role in PLG binding.
plates and incubated with recombinant enolase. The binding was
measured by ELISA. Addition of ?-aminocaproic acid, a known
lysine analog, significantly (P ? 0.001) inhibited the binding of
interaction on the presence of lysine residues (Fig. 3A). In addi-
tion, lysines play an important role in PLG binding, interacting
with the kringle domains present in the proenzyme. Enolase is a
a characteristic shared by many other Borrelia proteins. Indeed,
protein alignment of the enolase of B. burgdorferi (AAC66719)
receptor, showed homology with both internal and terminal PLG
binding domains, suggesting a conserved function for interacting
with host proteases (alignments not shown). Assays using differ-
a dose-dependent manner (Fig. 3B). In contrast, neither heparin
nor NaCl inhibited the binding of enolase to PLG, indicating that
the interaction is not dependent on ionic forces (Fig. 3C and D).
munofluorescence assay (IFA), transmission electronic micros-
TABLE 1 Proteins identified in outer membrane vesicles in two independent experiments (A and B) using mass spectrometry in order of abundance
Putative identification GeneMM (kDa)a
% in expt:
Outer surface protein B (OspB)
Outer surface protein A (OspA)
60-kDa chaperonin (GroL)
Periplasmic serine protease DO (HtrA)
Carboxyl-terminal protease (Ctp)
Surface lipoprotein P27 (p27)
Putative uncharacterized protein
Basic membrane protein A (BmpA)
Outer membrane protein P13 (p13)
Outer surface 22-kDa lipoprotein
Oligopeptide ABC transporter (OppA-3)
P66 protein (p66)
aMM, molecular mass.
FIG 1 Detection of enolase in an outer membrane vesicle preparation by
presence of the protein in a purified outer membrane vesicle preparation.
Monoclonal antibodies to OspA and DnaK were used as controls.
lase. (A) Immunoblot showing recognition of the recombinant enolase (3
nant enolase; lanes 2 to 5, results for rabbit antienolase serum obtained at
intervals in the immunization period. (B) Immunoblot. Preimmunization
rabbit serum (1/100) does not react to whole Borrelia lysate. (C) Rabbit anti-
ular mass markers are shown at the left of each panel.
Toledo et al.
iai.asm.orgInfection and Immunity
copy (TEM), and proteinase K treatment followed by Western
blot analysis were performed to assess the location of the enolase
in the Borrelia membrane. The TEM images collected from cul-
tured and mouse-derived spirochetes after using the rabbit anti-
enolase serum and labeling with 6-nm-diameter gold particles
showed minor binding, but the preimmunization rabbit serum
showed the same level of binding, indicating a nonspecific result
(data not shown). IFA confirmed the TEM data. Identical results
were obtained with spirochetes grown in dialysis chambers, indi-
cating that host adaptation did not result in changes in enolase
expression or localization within the organism (data not shown).
PK treatment using whole bacteria (Fig. 4A) did not degrade the
however, the enolase band remained constant and no changes
were observed even at high concentrations (250 ?g/ml) of pro-
mic space and therefore should not be affected by PK treatment.
formed to show that enolase is actually degraded by the protease
B. burgdorferi is not expressed on the surface of the OM.
PK treatment of OMV followed by Western blot analysis to
determine whether enolase in the OMV is accessible to degrada-
tion by proteolysis was performed. PK degraded the enolase as
serine protease, which was also detected in the OMV (Table 1),
was not degraded by PK proteolysis.
FIG 3 (A) Enolase binds plasminogen in a lysine-dependent manner. Binding of human plasminogen to immobilized enolase in the presence and absence of
with human plasminogen (10 ?g/ml) and adding different concentrations (0 to 2 ?M) of enolase. Bound enolase was detected using rabbit antienolase serum.
Binding of enolase (10 ?g/ml) to immobilized plasminogen (10 ?g/ml) in the presence of increasing concentrations (0 to 50 IU) of heparin was not inhibited.
Data represent the means of the results of three separate experiments.
FIG 4 Digestion of whole B. burgdorferi and recombinant enolase by pro-
teinase K. (A) B. burgdorferi (108) were treated for 1 h at room temperature
SDS-PAGE and Western blot to determine whether portions of enolase were
surface exposed. Individual gel lanes received approximately 3 ? 107control
or PK-treated spirochetes. (B) Recombinant enolase (4 ?g) was incubated for
1 h at room temperature in PBS–5 mM MgCl2with or without PK (250 ?g/
ml), separated by SDS-PAGE, and stained with Coomassie brilliant blue. The
results shown are from a representative experiment.
Enolase, a Plasminogen Receptor in Vesicles
January 2012 Volume 80 Number 1 iai.asm.org 363
The enolase of B. burgdorferi is immunogenic. In several or-
ganisms, such as Trichomonas vaginalis, Candida albicans, and
Streptococcus suis, enolase has been shown to be both surface ex-
posed and immunogenic (58, 66, 75). To determine whether the
Borrelia enolase could be immunogenic, sera from infected mice
and from patients with Lyme disease were tested for reactivity to
B, respectively). In all cases, mouse sera that reacted against the
whole Borrelia lysate also reacted with the recombinant enolase.
This reactivity confirmed the immunogenicity of the enolase in
experimental needle inoculations. Reactivity to enolase in rabbits
also developed as the result of infections via tick bite (Fig. 6C),
showing that the enolase triggered an antibody response during
the course of a tick-borne infection.
levels of reactivity to the recombinant enolase (OD at 405 nm
[OD405] ? 0.4), and the others had OD levels that were twice as
high as that of the pooled-serum negative control (OD405? 0.14)
(Fig. 6E). These results indicate that enolase is recognized in the
antibody repertoire in Lyme disease but at different levels of reac-
Enolase expression. B. burgdorferi is maintained in an enzo-
undergoes a dramatic switch in gene expression. The spirochete
upregulates a large number of genes, most notably ospC, in re-
sponse to the host milieu (73). The switch in gene expression can
strated that, after normalization to flaB, the genes were expressed
posttranscriptional changes that could affect the overall quantity
OspC as the positive control and FlaB as the loading control. Us-
ing the temperature and pH combinations listed in Materials and
Methods, differences in the enolase protein contents were de-
tected by both methods (Fig. 7), suggesting that enolase is post-
transcriptionally regulated under the different sets of conditions.
The levels of enolase seen at 34°C and pH 7.6 were similar to
those seen at 34°C and pH 6.4, indicated that posttranscriptional
regulation is dependent on temperature (data not shown).
Both pathogenic and nonpathogenic species of Gram-negative
bacteria shed OMV (9, 45, 52, 55). Studies of OMV from diverse
allow bacteria to interact with each other within species, with
other bacteria, and with eukaryotic cells. Both characterization of
OMV functions and biochemical analysis have demonstrated a
role in the transport of active virulence factors to host cells. OMV
mediate bacterial binding and invasion, cause cytotoxicity, and
modulate the host immune response (30, 51). Therefore, OMV
participate in host-pathogen interactions and are potent bacterial
virulence factors. In Borrelia, the mechanisms of release of OMV
(27, 64, 70, 74). Here we list the most abundant protein compo-
etry in two independent experiments. The results demonstrated
that OspA and OspB are the two major components of the OMV
and that OspB is at least four times as abundant as OspA. Other
surface proteins detected among the 15 most abundant proteins
were P27 (bba60), CspA (bba68), BmpA (bb383), P13 (bb0034),
outer surface 22-kDa protein (bb0365), and P66 (bb0603). Inter-
estingly, other putative cytoplasmic proteins such as GroEL
(bb0649), GAPDH (glyceraldehyde-3-phosphate-dehydrogenase)
Of particular note in the protein composition of the OMV is
the presence of several known PLG receptors (OspA, OspB, and
CspA) for Borrelia and several known PLG receptors (including
other bacteria (5, 6, 53, 54, 56, 57, 61, 63). Borreliae have an ex-
faces in the appropriate orientation so that it can function as a
bound protease that assists the spirochetes in dissemination (23,
36, 59) and in degradation of matrix protein (22). Fixing plasmin
PLG activation system. B. burgdorferi can induce the production
inflammatory cells can activate the PLG bound to the Borrelia,
with the possible result of enhancing dissemination of the spiro-
chetes. Additional interactions include those involving PLG acti-
vation inhibitors (39) and metalloproteases (34, 35, 43). Few bac-
teria have such a complete association with a mammalian system.
Therefore, it is of interest that the OMV of B. burgdorferi contain
multiple PLG receptors that, given the proper orientation of the
proteolytic activity, can further enhance these interactions.
much research interest. These glycolytic enzymes do not have cell
wall anchor sequences or obvious signal peptidase cleavage sites
for export to the outer cell wall or for specific release into the
environment. Like its counterparts in Gram-positive bacteria, the
enolase of B. burgdorferi lacks the motifs that would be consistent
brane. Furthermore, the sequence of enolase of B. burgdorferi has
been found to have homology to the internal and terminal PLG
binding sequences of other bacterial enolases (8, 24).
FIG 5 Digestion of purified Borrelia vesicles by proteinase K. Vesicle samples
(10 ?g per lane) were treated with proteinase K (PK) at the concentrations
indicated and analyzed by Western blotting.
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iai.asm.orgInfection and Immunity
Nonetheless, the outer-surface location of the enolase of
Gram-positive organisms is well established, and evidence for its
not able to demonstrate a surface location for enolase in cultured
or in vivo B. burgdorferi by fluorescence or electron microscopy
the presence of enolase in OMV by mass spectroscopy and by
Western blot analysis. In addition, we have shown that enolase is
accessible to degradation by PK proteolysis in the OMV. As men-
tioned before, the enolase lacks signal peptides or cell wall an-
chors, so it is unclear how it is transported to and associated with
the membrane of the live spirochete but that its position changes
when it is released in OMV, becoming accessible for the PK and
therefore for PLG as well.
Although the specific functions of the Borrelia OMV are not
known, the presence of several PLG receptors, including enolase
(whose binding to PLG has been characterized in this study),
point to a role in fixing plasmin to the peribacterial environment.
Plasmin-coated B. burgdorferi as well as relapsing fever Borrelia
FIG6 Enolase is an immunogenic enzyme. (A and B) Serologic reactivity of experimentally infected mice (1:100) to whole Borrelia lysate (A) and recombinant
Bars C, negative control representing pooled sera from mice, rabbits, and patients. ???, P ? 0.001; ?, P ? 0.05.
Enolase, a Plasminogen Receptor in Vesicles
January 2012 Volume 80 Number 1iai.asm.org 365
isms. Borrelia with plasmin on the surface can also degrade extra-
cellular matrices to promote dissemination (23, 36, 59). Active
proteolysis in the peribacterial environment induced by released
OMV could promote further degradation of matrix proteins and
possibly the production of more easily acquired peptides, leading
to a function in bacterial nutrition. Such external proteolytic ac-
tivity would require the presence of some or all of the PLG recep-
orientation for the plasmin. Note that there is no evidence for
protease release by B. burgdorferi, thus enhancing the importance
of the idea of borrowed proteolytic activity.
shifts in gene expression that change the protein profiles of the
sion of enolase (bb0337) in Borrelia does not change as a result of
blood induction or temperature shift (73), as real-time PCR did
not reveal any differences in enolase expression when we com-
bined high temperature (34°C) and low pH (6.4). However, the
overall quantity of the enzyme that we detected was significantly
higher at 34°C than at 23°C, showing that there is posttranscrip-
tional regulation of the enolase. The mechanism that governs this
regulation is not known.
Regardless of its function(s) in the OMV, enolase is a B cell
immunogen recognized by sera from experimentally infected
mice (inoculated with cultured organisms via needle injection),
tick-infected rabbits, and Lyme disease patients. In this regard,
how this glycolytic enzyme is transported to the OMV and how it
becomes an immunogen are important issues that link bacterial
physiology and pathogenesis.
from the National Institutes of Health to J.L.B.
Richard T. Marconi generously provided the OspC type A gene in a
pET46 LIC that was used to generate the recombinant OspC that was
subsequently purified and used to develop antibodies for this study. Me-
lissa Caimano and Justin Radolf generously provided the host-derived
ported by U54-AI-0715558 (Lipkin).
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