Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) A retraction has been published Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shin, J. J. Articles by Cabello, F. C. Search for Related Content PubMed PubMed Citation Articles by Shin, J. J. Articles by Cabello, F. C.

 Previous Article  |  Next Article 

Infection and Immunity, April 2004, p. 2280-2287, Vol. 72, No. 4
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.4.2280-2287.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Localization of BmpA on the Exposed Outer Membrane of Borrelia burgdorferi by Monospecific Anti-Recombinant BmpA Rabbit Antibodies

Junghee J. Shin,¹ Anton V. Bryksin,¹ Henry P. Godfrey,² and Felipe C. Cabello^1*

Departments of Microbiology and Immunology,¹ Pathology, New York Medical College, Valhalla, New York 10595²

Received 7 October 2003/ Returned for modification 7 November 2003/ Accepted 12 January 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
BmpA (P39) is an immunodominant chromosomally encoded Borrelia burgdorferi protein. The potential strong cross-reactivity of anti-BmpA antibodies with the other members of this paralogous protein family and the previous use of antibodies whose reactivity to the other Bmp proteins was uncharacterized have resulted in continued controversy over its localization in B. burgdorferi. In an effort to provide a definitive demonstration of the localization of BmpA, rabbit antibodies raised to recombinant BmpA (rBmpA) were rendered monospecific by absorption with rBmpB. This reagent did not react with rBmpB, rBmpC, or rBmpD in dot immunobinding, detected only a single 39-kDa band and a single 39-kDa, pI 5.0 spot on one- and two-dimensional immunoblots of B. burgdorferi lysates, respectively, and immunoprecipitated a single 39-kDa protein from these lysates. It detected BmpA in the Triton X-114-soluble and -insoluble fractions of B. burgdorferi, suggesting association with both inner and outer bacterial cell membranes. Treatment of intact B. burgdorferi with proteinase K partially digested BmpA, consistent with a limited surface exposure on the outer bacterial membrane, a suggestion confirmed by immunofluorescence of unfixed B. burgdorferi cultured in vitro and in vivo. Anti-rBmpA antibody was bacteriostatic for B. burgdorferi B31 in culture, again suggesting localization of BmpA on the exposed spirochetal outer surface. Surface localization of BmpA, growth inhibition by anti-rBmpA antibodies, and the previously reported conservation of bmpA in different B. burgdorferi sensu lato strains may indicate that BmpA plays an essential role in B. burgdorferi biology.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lyme disease, the most prevalent tick-borne disorder in the United States, is caused by infection with the spirochete Borrelia burgdorferi sensu lato (23). Structurally, B. burgdorferi consists of a protoplasmic cylinder surrounded by a cytoplasmic membrane, flagella, and a labile outer membrane that is only loosely associated with the other underlying structures (3). The genome of the B. burgdorferi B31 type strain consists of a linear chromosome of 910 kb containing 853 open reading frames (ORFs) and at least 12 linear and 9 circular plasmids (a total of 610 kbp) containing 535 ORFs (18). Approximately 5% of the chromosomal genes and 15% of the plasmid genes (150 ORFs) encode putative lipoproteins (18).

The roles of most of these lipoproteins in the biology and pathogenesis of Lyme disease are unknown. The postulated surface exposure, lipid component, abundance, and immunogenicity of B. burgdorferi lipoproteins strongly suggest that their intensive study is likely to yield additional novel diagnostics and vaccines for Lyme disease (13), but the lack of readily available genetic tools to manipulate the genome of B. burgdorferi, the biological limitations imposed by its slow growth, and the genetic instability of its extrachromosomal elements have inhibited studies of their function. Published evidence indicates that lipoproteins such as decorin-binding protein, fibronectin-binding protein, and OspA may play a role in adhesion and colonization of host tissues, VlsE lipoproteins may potentially be involved in evading the host immune system by antigenic variation, and the plasmid-encoded Erp (OspEF related) proteins are involved in binding to complement inhibitory factor H (reviewed in reference 13). B. burgdorferi lipoproteins also provide a major inflammatory stimulus through their recognition by Toll-like receptor 2 (24).

One group of putative B. burgdorferi lipoproteins, paralogous family 36, is encoded by the bmp genes (2, 37, 43). These genes are located in tandem on the B. burgdorferi chromosome at nucleotides 3391932 to 396563 (18). They share 50 to 70% identity in their nucleotide coding sequences, are conserved in all B. burgdorferi sensu lato strains examined to date (20), and encode four proteins, BmpA, BmpB, BmpC, and BmpD, that show 36 to 52% identity in their deduced amino acid sequences and display similar deduced chemical and physical properties (2, 37, 43). All Bmp proteins have a putative consensus signal peptidase II site at their N terminus, suggesting that they are lipoproteins located in the cytoplasmic or outer membranes of B. burgdorferi (2, 37, 43). The function of the Bmp proteins is unknown, but BLASTN analysis suggests that they possess significant similarity to ABC-type transporters in other bacteria. BmpA (also known as P39) is widely used as a diagnostic antigen in the serological diagnosis of Lyme disease because of its immunoreactivity with sera from patients with early and late Lyme borreliosis (26).

The immunodominance of BmpA in patients with Lyme disease (44) has fostered great interest in determining its cellular localization in B. burgdorferi. Several groups have previously explored this question using a variety of assays and polyclonal and monoclonal anti-BmpA antibodies. They concluded that BmpA, like FlaB but unlike OspA, was associated with the inner cytoplasmic membrane and was not exposed on the spirochetal outer membrane (6, 11, 45). Unfortunately, the conclusions reached in each of these studies were undercut by the incomplete characterization of the antibody specificity for Bmp proteins other than BmpA or, in the case of monoclonal anti-BmpA reagents, by a lack of characterization of the epitopes recognized and their locations on the BmpA molecule. In order to provide unequivocal evidence for the localization of BmpA in B. burgdorferi, we have developed a monospecific polyclonal anti-recombinant BmpA (rBmpA) reagent and have used it in multiple assay systems to show conclusively that BmpA is exposed on the B. burgdorferi outer membrane as well as being associated with the cytoplasmic membrane.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and cultures. Noninfectious high-passage B. burgdorferi B31 (ATCC 35210) was obtained from the American Type Culture Collection (Fairfax, Va.). Infectious low-passage B. burgdorferi N40 was provided by Linda Bockenstadt (Yale University, New Haven, Conn.). B. burgdorferi flaB mutant MC-1 was provided by Nyles W. Charon (West Virginia University, Morgantown, W.Va.). Infectious B. burgdorferi 297 was provided by Justin D. Radolf (University of Connecticut Health Center, Farmington, Conn.). All borrelial strains were stored in aliquots at -80°C. For in vitro culture, aliquots were taken from -80°C storage and grown at 34°C in BSK-H medium (Sigma Chemical Co., St. Louis, Mo.) supplemented with 6% rabbit serum (Sigma). For culture of B. burgdorferi MC-1, kanamycin (350 µg/ml) (Sigma) was added to the medium (32). In vivo culture of B. burgdorferi N40 and 297 in dialysis membrane chambers implanted in the rabbit peritoneum was performed as described previously (1). Escherichia coli M15(pREP4) (Qiagen Inc., Valencia, Calif.) (used to produce rBmpA) and E. coli BL21(RIL) (Novagen, Madison, Wis.) (used to produce rBmpB, rBmpC, and rBmpD) were grown in Luria-Bertani (LB) medium (Life Technologies, Paisley, Scotland) containing appropriate antibiotics (Sigma).

Antibodies. Rabbit anti-FlaB was a gift from Justin D. Radolf. Mouse anti-FlaB and anti-OspA monoclonal antibodies were gifts from Michael V. Norgard (University of Texas Southwestern Medical Center, Dallas, Tex.).

Cloning, expression, and purification of rBmp proteins. E. coli M15(pREP4) was transformed with pQE40-bmpA, encoding an rBmpA-dehydrofolate reductase fusion protein with a 6x His tag at the N terminus. E. coli BL21(RIL) was transformed with pET30-bmpB, encoding rBmpB with 6x His tags at both the N and C termini, with pET30-bmpC, encoding rBmpC with a 6x His tag at the N terminus, or with pET30-bmpD, encoding rBmpD with 6x His tags at both the N and C termini. Transformed E. coli containing pQE40-bmpA was grown in LB medium containing kanamycin (50 µg/ml) and ampicillin (25 µg/ml); transformed E. coli containing pET30-bmpB, pET30-bmpC, or pET30-bmpD was grown in LB medium containing kanamycin (50 µg/ml) at 32°C. All cultures were grown to 0.5 to 0.6 absorbance unit (595 nm), induced with 1 mM isopropyl thiogalactoside (Denville Scientific Inc., Metuchen, N.J.), and grown for an additional 3 h at 32°C (41). rBmpA was purified from bacterial lysates by nitrilotriacetic acid (NTA)-Ni²⁺ affinity chromatography (Qiagen, Valencia, Calif.) and Sephacryl S-300 gel filtration chromatography (Amersham Biosciences, Piscataway, N. J.); rBmpB was purified by gel filtration chromatography followed by NTA-Ni²⁺ affinity chromatography; rBmpC and rBmpD were purified by washing with 2% Triton X-100 twice followed by NTA-Ni²⁺ affinity chromatography (21). NTA-Ni²⁺ affinity and gel filtration chromatographies were performed according to the manufacturers' instructions in all cases. Purification of the four Bmp proteins was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining of proteins electrolytically transferred to nitrocellulose membranes (27).

Production of anti-rBmpA antibodies and removal of anti-BmpB reactivity. Female New Zealand White rabbits (Millbrook Breeding Labs, Amherst, Mass.), 2.5 ± 0.3 kg, were immunized intramuscularly with 70 µg of purified rBmpA emulsified in 50 µl of TiterMax Gold adjuvant (Sigma). Test bleeds were obtained from the ear artery before primary immunization and every 2 weeks thereafter; their antibody content was determined by dot immunobinding (28). Rabbits received an intramuscular injection of 25 µg of rBmpA emulsified in 50 µl of TiterMax Gold on day 100 after primary immunization and were exsanguinated under anesthesia by cardiac puncture on day 29 after the booster injection (day 130 after primary immunization). Immunoglobulin (Ig) was purified from serum by precipitation with 50% saturated ammonium sulfate, and the precipitates were extensively dialyzed against phosphate-buffered saline, pH 7.4 (PBS), and stored in aliquots at -80°C (21). Protein content was determined by absorbance at 280 nm.

Purified rBmpB (1 mg) was bound to 1 ml of Affi-gel 15 (Bio-Rad Laboratories, Hercules, Calif.) according to the manufacturer's instructions, transferred to a 5-ml polypropylene column (Qiagen), and washed with 10 bed volumes of adsorption buffer (10 mM Tris [pH 8.5], 500 mM NaCl) (21). To remove anti-rBmpB activity, anti-rBmpA antibody diluted 1:5 in adsorption buffer was cycled over the column by gravity flow for five cycles of 10 passages each for a total of 50 passages (21). The rBmpB immunoadsorbent was reactivated with 10 bed volumes of 100 mM glycine, pH 2.8, followed by 20 bed volumes of adsorption buffer after each 10-passage cycle.

SDS-PAGE, 2D-NEPHGE, and Western blotting. Mid-log-phase B. burgdorferi B31 cells (2.5 x 10⁷ to 5 x 10⁷ cells/ml) were centrifuged (4,000 x g for 30 min) and washed twice with PBS. For SDS-PAGE, lysates were prepared by resuspending B. burgdorferi pellets in 1% NP-40 and SDS-PAGE sample buffer. For two-dimensional nonequilibrium pH gradient electrophoresis (2D-NEPHGE), lysates were prepared by sonication of B. burgdorferi pellets resuspended in 10 mM Tris-HCl (pH 7.4)-5 mM MgCl₂ followed by treatment with 9.5% urea-2% NP-40-5% ß-mercaptoethanol (33). A urea-ampholine isoelectric focusing tube gel (pH 3.0 to 10.0) (Bio-Rad) was used for the first dimension of 2D-NEPHGE according to the manufacturer's instructions; 4.5 and 12% polyacrylamide gels were used for stacking and running gels, respectively, for both the second dimension in 2D-NEPHGE and for SDS-PAGE. For Western blotting, proteins were transferred electrolytically to nitrocellulose membranes (Bio-Rad) and developed and read using enhanced chemiluminescence technology (ECF Western blotting kit; Amersham Biosciences) (28) and a Storm PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.).

Immunoprecipitation. Washed B. burgdorferi B31 cells (3 x 10⁸) were lysed with buffer containing 20 mM sodium phosphate (pH 7.5), 500 mM NaCl, 0.1% SDS, 1% NP-40, 1.0% deoxycholic acid (Sigma), and 0.02% sodium azide, a somewhat different buffer from the lysis buffer otherwise used in these studies (38). Lysed bacterial cells were first cleared by incubation with an equal volume of insolubilized protein A/G (Pierce Biotechnology, Rockford, Ill.) for 2 h at 25°C, and supernatants from this incubation were then incubated overnight at 4°C with an equal volume of anti-rBmpA antibody (1:250 final dilution); this dilution had been previously determined to be optimal. Antigen-antibody complexes were precipitated by incubation for 2 h at 25°C with an equal volume of insolubilized protein A/G (21) and were analyzed by Western blotting.

Triton X-114 phase partitioning. B. burgdorferi B31 grown to a final concentration of 5 x 10⁸ cells/ml was centrifuged (2,000 x g), washed twice with PBS, suspended to 5 x 10⁹ cells/ml in 1% Triton X-114 in PBS, and incubated at 4°C on a rotating platform overnight (4). Isolation of the detergent-insoluble fraction (periplasmic core) and phase partitioning of the detergent-soluble fraction with Triton X-114 were performed as described previously, omitting the acetone precipitation step (14). The presence of immunoreactive BmpA and FlaB in the three different fractions was assessed by Western blotting with anti-rBmpA and anti-FlaB, respectively.

Proteolytic sensitivity of BmpA. Intact mid-log-phase B. burgdorferi B31 (100 µl) was incubated with soluble proteinase K at final concentrations of 0.04, 0.4, or 4 mg/ml for 45 min at 25°C in the absence or presence of 0.05% (vol/vol) Triton X-100 (9, 16). The reaction was stopped, and further proteolysis was inhibited by protease inhibitors (16). Susceptibility of BmpA, OspA, and FlaB to proteolysis was assessed by Western blotting with anti-rBmpA, anti-OspA, and anti-FlaB, respectively.

Indirect immunofluorescence of B. burgdorferi. In order to determine outer surface expression of BmpA in B. burgdorferi B31grown in vitro, intact cells were double labeled in solution with optimal dilutions of rabbit anti-rBmpA and mouse monoclonal anti-OspA or rabbit anti-rBmpA and mouse monoclonal anti-FlaB or with similar dilutions of preimmunization rabbit Ig (9). After incubation with primary antibodies, cells were washed with PBS, incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, Malvern, Pa.) and rhodamine Red -X-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) for an additional 30 min, washed with PBS, and observed under immunofluorescence microscopy. The same indirect immunofluorescence assay was performed with B. burgdorferi B31 that had been first fixed with 100% methanol to confirm expression of BmpA, OspA, and FlaB proteins in the spirochetes.

Outer surface expression of BmpA was also determined in B. burgdorferi B31 grown in vitro and B. burgdorferi N40 and 297 grown in dialysis membrane chambers implanted in rabbit peritoneal cavities (1) either adsorbed to glass slides or encapsulated in gel microdroplets (9, 10). In these cases, the unfixed organisms were stained by indirect immunofluorescence for BmpA or FlaB in the presence of 0.05% Triton X-100 or PBS (9, 10).

Expression of BmpA during growth in mice was determined by injecting five female C3H/HeJ mice (15 ± 0.3 g) (Jackson Laboratories, Bar Harbor, Maine) intraperitoneally with B. burgdorferi N40 (10⁶ cells/100 µl). Fourteen days later, B. burgdorferi N40 cells were recovered by peritoneal lavage of anesthesized mice (31), and the recovered spirochetes were fixed with methanol on glass slides and then stained for BmpA and FlaB by indirect immunofluorescence.

Inhibition of in vitro growth of B. burgdorferi by anti-rBmpA antibodies. Mid-log-phase B. burgdorferi B31 grown to approximately 1 x 10⁸ cells/ml was diluted to 2 x 10⁷ cells/ml with fresh medium (40). Cells (2 x 10⁶) were incubated with 2.5% (vol/vol) guinea pig serum (Rockland Inc., Gilbertsville, Pa.) or with serial twofold dilutions of heat-inactivated monospecific anti-rBmpA (twofold dilutions in BSK-H medium; initial dilution, 1:10; final dilution, 1:5,120) in the presence or absence of 2.5% (vol/vol) guinea pig serum as a source of complement. Preliminary experiments showed that this concentration of guinea pig serum was not borreliacidal in the absence of borrelia-specific antibodies. All samples were prepared in triplicate using 96-well round-bottom plates (Costar, Corning, N.Y.). Bacterial growth after incubation for 24, 48, and 72 h at 34°C was evaluated using Live/Dead BacLight Bacterial Viability kits (Molecular Probes, Eugene, Oreg.) according to the manufacturer's instructions. Percent inhibition of bacterial growth was calculated as follows: [1 - (number of live bacteria in the presence of antibodies/number of live bacteria present in PBS alone)] x100.

Statistical analysis. Statistical analysis was done by the appropriate use of Student's t test or by a Kruskal-Wallis test (nonparametric one-way analysis of variance) with a Dunn repeated-measures posttest. A significance level of P < 0.05 was used.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Production of monospecific rabbit anti-rBmpA. Antibodies reacting with only a single Bmp protein are critical for determining expression and localization and for quantitation of the individual Bmp proteins because of the potential cross-reactivity of their closely homologous amino acid sequences (2, 37, 43). Ammonium sulfate-purified rabbit anti-rBmpA had a titer of 10¹⁰ with 10 ng of rBmpA in dot immunobinding and a titer of 10⁵ with 10 ng of rBmpB and did not react with rBmpC or rBmpD. After repeated treatment with the rBmpB immunoadsorbed, anti-rBmpA reacted only with rBmpA (Fig. 1). On SDS-PAGE Western blots of B. burgdorferi lysates, this absorbed anti-rBmpA detected a single band of 39 kDa (Fig. 2A) but did not recognize FlaB (Fig. 2B). It detected a single 39-kDa, pI 5.0 spot (Fig. 2C) in 2D-NEPHGE Western blotting of B. burgdorferi lysates. Both of these observations are consistent with the predicted molecular weight and pI for BmpA (43). Immunoprecipitation of B. burgdorferi lysates with absorbed anti-rBmpA isolated a single 39-kDa protein (Fig. 2D). These results indicate that absorbed anti-rBmpA was monospecific.

View larger version (56K): [in this window] [in a new window]

FIG. 1. Specificity of rabbit anti-rBmpA in dot immunobinding before (lanes 1 and 2) and after (lanes 3 and 4) repeated absorption with rBmpB. Lane 1, unabsorbed anti-rBmpA (1:10,000); lane 2, unabsorbed anti-rBmpA (1:100,000); lane 3, absorbed anti-rBmpA (1:1,000); lane 4, absorbed anti-rBmpA (1:10,000).

View larger version (67K): [in this window] [in a new window]

FIG. 2. Detection of BmpA in B. burgdorferi lysates by monospecific anti-rBmpA. (A) Immunoblot of B. burgdorferi B31 lysate proteins separated by SDS-12% PAGE and developed with anti-rBmpA (lane 2), showing a single 39-kDa band (arrowhead). Lane 1, silver stain of lane 2. (B) Immunoblot of B. burgdorferi B31 lysate proteins (lanes 1 and 3) and B. burgdorferi flaB mutant MC-1 lysate proteins (lanes 2 and 4) developed with anti-rBmpA (lanes 1 and 2) and anti-FlaB (lanes 3 and 4). Anti-rBmpA detects a 39-kDa protein (arrowhead) in both strains; anti-FlaB detects a 41-kDa protein only in B. burgdorferi B31. (C) Immunoblot of B. burgdorferi B31 lysate proteins separated by 2D-NEPHGE and developed with anti-rBmpA (panel 2) showing a single 39-kDa, pI 5.0 spot. Panel 1, silver stain of panel 2. (D) B. burgdorferi B31 lysate proteins immunoprecipitated with anti-rBmpA and separated by SDS-12% PAGE detects a single 39-kDa band (arrowhead) in a reaction mixture containing anti-rBmpA and anti-rabbit Ig (lane 1) but not in a reaction mixture containing only anti-rabbit Ig and no anti-rBmpA (lane 2).

Localization of BmpA protein in B. burgdorferi by using detergent fractionation. The presence of a putative consensus signal peptidase II site in BmpA suggested that it was a lipoprotein and, as such, probably located in the cytoplasmic or outer membrane of B. burgdorferi (43). However, previous reports could not rule out a mainly periplasmic location with anchorage in the cytoplasmic or the outer membrane (6, 11, 45). As a first step to localize BmpA, B. burgdorferi cells were fractionated with 0.1% (vol/vol) (data not shown) and 1% (vol/vol) Triton X-114 (Fig. 3) and analyzed by Western blotting with anti-rBmpA and anti-FlaB. BmpA was present in all fractions (Fig. 3), and while the detergent phase of the Triton X-114 lysate containing the outer membrane proteins had somewhat more immunoreactive BmpA than the aqueous phase with its more hydrophilic proteins, BmpA was also present in the detergent-insoluble fraction containing periplasmic core proteins. In contrast, FlaB was detected primarily in the detergent-insoluble fraction, with only minimal if any immunoreactive FlaB in the aqueous phase of the Triton X-114 lysate. These data suggest that BmpA and FlaB differ considerably in their distribution within B. burgdorferi and that BmpA appears to be preferentially located on the outer membrane of B. burgdorferi cells.

View larger version (35K): [in this window] [in a new window]

FIG. 3. Fractionation of BmpA and FlaB in 2 x 108 B. burgdorferi B31 cells treated with 1% Triton X-114, separated by SDS-12% PAGE, and developed with anti-rBmpA and anti-FlaB. Whole cells, B. burgdorferi B31 treated with Triton X-114. Insoluble pellet, insoluble material from whole cells treated with Triton X-114. Aqueous phase, aqueous phase of Triton X-114-soluble fraction. Detergent phase, detergent phase of Triton X-114-soluble fraction. See Materials and Methods for details of immunoblotting.

Localization of BmpA protein in intact B. burgdorferi. To provide additional evidence for BmpA localization, intact B. burgdorferi cells were incubated with proteinase K, and the proteolysis of BmpA, OspA, and FlaB was then quantified by immunoblotting of lysed organisms. In the absence of proteinase K, the amounts of immunoreactive BmpA, OspA, and FlaB were not affected by incubation of intact B. burgdorferi with Triton X-100 (Fig. 4, lanes 1). In the presence of proteinase K, BmpA and OspA in intact B. burgdorferi were partially digested by all three concentrations of proteinase K (compare lanes 2-, 3-, and 4- with lane 1- in Fig. 4 for each protein). The amounts of immunoreactive FlaB did not decrease under these conditions. In contrast, when the B. burgdorferi outer membrane was disrupted by incubation of intact organisms in 0.05% Triton X-100, BmpA was completely digested by as little as 0.04 mg of proteinase K/ml, FlaB was completely digested by 0.4 mg of proteinase K/ml, and OspA was completely digested by 4 mg of proteinase K/ml (Fig. 4, compare lanes 2+, 3+, and 4+ with lane 1+). The partial susceptibility of BmpA to proteinase K in intact B. burgdorferi suggests that this protein, like OspA, is partially exposed on the surface of B. burgdorferi during growth in vitro, while the insensitivity of FlaB to proteinase K in intact organisms is consistent with its location under the outer cell membrane (32).

View larger version (38K): [in this window] [in a new window]

FIG. 4. Proteolytic sensitivity of BmpA, OspA, and FlaB in 2 x 107 intact B. burgdorferi B31 cells in the absence (-) or presence (+) of 0.05% Triton X-100. After incubation with proteinase K, cells were lysed, proteins were analyzed by SDS-12% PAGE, and immunoblots were developed with anti-rBmpA, anti-OspA, or anti-FlaB. Lane 1, intact B. burgdorferi incubated without proteinase K; lane 2, intact B. burgdorferi treated with 0.04 mg of proteinase K/ml (final concentration); lane 3, intact B. burgdorferi treated with 0.4 mg of proteinase K/ml (final concentration); lane 4, intact B. burgdorferi incubated with 4 mg of proteinase K/ml (final concentration). See Materials and Methods for details of immunoblotting.

Indirect immunofluorescence was used to provide additional evidence for the partial exposure of BmpA on the surface of B. burgdorferi. Intact B. burgdorferi B31 grown in vitro showed dual labeling with anti-rBmpA and anti-OspA (Fig. 5) but was not labeled by anti-FlaB (Fig. 5). After disruption of the outer membrane by methanol fixation, however, B. burgdorferi cells were labeled by all three antibodies. Confirmation of the difference in localization between BmpA and FlaB was provided by indirect immunofluorescence labeling of bacteria encapsulated in agarose microdroplets, a method that protects the labile spirochetal outer membrane from disruption (10, 13). Both gel-encapsulated B. burgdorferi B31 grown in vitro and gel-encapsulated B. burgdorferi N40 and 297 grown in vivo in dialysis chambers in rabbits showed intense, continuous fluorescence labeling with anti-rBmpA antibodies, consistent with surface labeling of B. burgdorferi, but were not labeled by anti-FlaB antibodies (data not shown). After 0.05% Triton X-100 treatment, all three strains of B. burgdorferi showed significant labeling with anti-rBmpA antibodies as well as with anti-FlaB antibodies (data not shown). BmpA detectable by indirect immunofluorescence assay with anti-rBmpA was present in B. burgdorferi N40 recovered from mice infected by intraperitoneal injection 2 weeks previously, showing that BmpA was also expressed during in vivo growth in this species (data not shown).

View larger version (18K): [in this window] [in a new window]

FIG. 5. Dual-labeling indirect immunofluorescence staining of intact unfixed or methanol-fixed B. burgdorferi B31 with preimmunization rabbit Ig (left panels), rabbit anti-rBmpA and mouse anti-OspA (center panels), or rabbit anti-rBmpA and mouse anti-FlaB (right panels). Magnification, x1,000.

Bacteriostatic and bactericidal activities of anti-rBmpA antibody. If the BmpA protein was located on the outer cell membrane of B. burgdorferi, antibody could be deposited on the bacterial cell surface and trigger biological phenomena related to this deposition, including activation of complement, bacteriostasis, and bacteriolysis (17, 30, 35, 39). B. burgdorferi cells were therefore incubated with anti-rBmpA in the absence or presence of guinea pig complement. In the absence of complement, a 1/160 dilution of anti-rBmpA caused 96% inhibition of B. burgdorferi growth at 72 h compared to cells treated with similar dilutions of preimmunization Ig (data not shown); no bacterial agglutination was seen at this dilution of anti-rBmpA (data not shown). The observed growth inhibition was bacteriostatic since the number and viability of bacterial cells were constant over this time period (Fig. 6). In the presence of complement, anti-rBmpA was bacteriostatic at 24 h (no increase in cell number and no decrease in cell viability), with a decrease in cell viability between 24 and 72 h compared to control samples containing only anti-rBmpA and no complement (Fig. 6). Agglutinated B. burgdorferi cells were visible in cultures containing anti-rBmpA and complement at all time points examined (data not shown).

View larger version (14K): [in this window] [in a new window]

FIG. 6. Bacteriostatic and bactericidal activities of anti-rBmpA on B. burgdorferi during in vitro growth. , PBS control; , 2.5% (vol/vol) guinea pig serum; •, anti-rBmpA (1:160); , anti-rBmpA (1:160) and 2.5% (vol/vol) guinea pig serum.

Top Abstract Introduction Materials and Methods Results Discussion References

 
High-titer anti-rBmpA rabbit antibody was raised and rendered monospecific by extensive absorption with rBmpB. In contrast to previously developed anti-BmpA antibodies, which were not examined for cross-reactivity with other Bmp proteins (42, 44), the present monospecific antibody reacted only with rBmpA and not with other rBmp proteins. It detected only one band (39 kDa) and a single spot (39 kDa, pI 5.0) in immunoblots of B. burgdorferi lysates by SDS-PAGE and 2D-NEPHGE, respectively, and precipitated a single 39-kDa protein from B. burgdorferi lysates, all of these values consistent with predicted values for BmpA. BmpA was at least partially exposed on the outer cell membrane of B. burgdorferi, since like OspA, it was partially digested in intact cells by proteinase K and was stained by indirect immunofluorescence in intact cells, while FlaB, as expected from its periplasmic location, was not digested or detected (32). Exposure of BmpA on the surface of B. burgdorferi was further suggested by the bacteriostatic activity of anti-rBmpA antibody on B. burgdorferi in the absence of complement and bactericidal activity in its presence. Detection of BmpA in the detergent phase of the Triton X-114-soluble fraction also points to an outer membrane localization, since outer membrane proteins typically partition into the Triton X-114 detergent-rich hydrophobic fraction (14, 36; reviewed in reference 13), and agrees with recently published work showing that BmpA is extracted into the detergent phase of B. burgdorferi lysates by Triton X-114 (12). However, our identification of appreciable amounts of immunoreactive BmpA in the Triton X-114-insoluble fraction suggests that a fraction of B. burgdorferi BmpA may be also associated with periplasmic cellular proteins and the cytoplasmic membrane (12).

The immunodominance of BmpA in patients with Lyme disease (44) has fostered great interest in determining its cellular localization in B. burgdorferi. Bunikis and Barbour reported that immunoreactive BmpA detected by monoclonal antibody H1141 was resistant to proteolysis with 200 µg of proteinase K/ml in intact B. burgdorferi cells but was readily digested after the cells were sonicated; they characterized both BmpA and FlaB as "internal" proteins associated only with the cytoplasmic membrane (6). The epitope recognized by H1141 and its location on the BmpA molecule were not determined, and its reactivity with other Bmp proteins was not examined. Sullivan and coworkers used an IgG2a monoclonal anti-BmpA antibody (NYSP39H) to localize BmpA in B. burgdorferi (45). They found that after surface labeling of intact B. burgdorferi with biotin and subsequent treatment with NP-40, immunoreactive BmpA was not biotinylated but was present in detergent-solubilized material and could be demonstrated at the cytoplasmic membrane region of the spirochete. They concluded that BmpA was present within the outer membrane but not on the outer surface of the organism, but again, neither the location of the epitope recognized by this monoclonal antibody on BmpA nor the specificity of the antibody for other Bmp proteins was examined. Cox and Radolf also concluded that BmpA and FlaB lay within the outer membrane when they were unable to stain both of these proteins in intact B. burgdorferi by using rat antisera but could readily detect them once the outer membrane had been removed by extraction with 0.05% Triton X-100 (11). Here, too, the monospecificity of the anti-BmpA serum had not been demonstrated nor was its reactivity with other Bmp proteins characterized. The cause for the inconsistencies between these earlier reports and the present work is unclear but may have resulted from the use of different methods to assess the exposure of BmpA and from the use of monoclonal antibodies that recognized only nonexposed epitopes of BmpA (11, 45). Alternatively, the amount of BmpA protein exposed on the surface of B. burgdorferi may have been so small that the reagents used by the other researchers could not detect it while our more specific and much higher-titer antibody could.

The biological activity of anti-rBmpA on B. burgdorferi observed by us is consistent with previous work demonstrating in vitro bactericidal activity of human monoclonal antibodies against native B. burgdorferi P39 protein in the absence of complement (42). While the bactericidal effect observed in this earlier study could have been secondary to an unrecognized cross-reactivity with the other Bmp proteins, to a dual specificity of the antibodies for P39 (BmpA) and the 66-kDa B. burgdorferi protein (42), or to some other technical factor, there are several reports of specific antibodies binding to particular epitopes of a B. burgdorferi surface protein and causing protein or membrane distortions resulting in cell lysis (8, 17, 39). The bactericidal/bacteriostatic effects of monospecific anti-rBmpA might even provide host protection (17, 30). Although vaccination with rBmpA did not induce protective immunity in mice, this lack of protection could have been a result of the relatively low antibody titers produced (19). The bacteriostatic action of monospecific anti-rBmpA observed in the present study suggests that a sufficiently strong immune response against this protein could be protective. Differences in bmpA expression in B. burgdorferi during the course of murine infection could also suggest the involvement of BmpA in B. burgdorferi adaptation to various hosts in the spirochete life cycle (29).

Most of the immunogenic, surface-exposed B. burgdorferi lipoproteins that have been extensively studied to date are plasmid encoded (16, 22, 30, 34, 46). Chromosomally encoded BmpA and the other Bmp proteins appear to be less prone to recombination than plasmid-encoded proteins (7), and the temperature- and pH-independent expression of bmpA mRNA (15) and BmpA (J. J. Shin and F. C. Cabello, unpublished data) suggest that its expression is not controlled by the RpoN-RpoS regulatory pathway, which appears to control expression of some plasmid-encoded lipoproteins (25, 46). However, previous experiments in our laboratory have indicated that modulation of the expression of the bmp gene cluster is achieved by growing B. burgdorferi cells in the presence of tick cells (5), suggesting that the expression of these genes may be modulated by alternative stimuli and pathways. The surface location of BmpA lipoprotein, the growth inhibition of B. burgdorferi by anti-rBmpA antibodies, the conservation of the bmpA gene in different B. burgdorferi sensu lato strains (20), and the consistent detection of bmpA expression and BmpA at different B. burgdorferi cell densities, temperatures, and pH and in different B. burgdorferi sensu lato strains (15 and Shin and Cabello, unpublished) could suggest that BmpA and perhaps other Bmp family proteins play an essential role in the metabolism of B. burgdorferi. Examination of the regulation and modulation of bmp gene expression using techniques similar to those used with BmpA in the present study and the host responses to this modulation using these techniques and isolated bmp null mutants will be critical in analyzing the mechanism(s) involved and its potential role in the adaptive strategies of B. burgdorferi.

 
We thank Nyles W. Charon for B. burgdorferi flaB mutant MC-1, Justin D. Radolf for rabbit anti-FlaB polyclonal antibody, Michael V. Norgard for mouse anti-FlaB and anti-OspA monoclonal antibodies, Justin D. Radolf and Melissa J. Caimano for help with in vivo experiments using rabbit peritoneal chambers, and Harriett Harrison for secretarial assistance in preparing the manuscript.

This work was supported by NIH grant R01 AI48856 to F.C.C.

 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Basic Science Building, New York Medical College, Valhalla, NY 10595. Phone: (914) 594-4182. Fax: (914) 594-4176. E-mail: cabello{at}nymc.edu.

Editor: D. L. Burns

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Akins, D. R., K. W. Bourell, M. J. Caimano, M. V. Norgard, and J. D. Radolf. 1998. A new animal model for studying Lyme disease spirochetes in a mammalian host-adapted state. J. Clin. Investig. 101:2240-2250.[Medline]
2
2. Aron, L., M. Alekshun, L. Perlee, I. Schwartz, H. P. Godfrey, and F. C. Cabello. 1994. Cloning and DNA sequence analysis of bmpC, a gene encoding a potential membrane lipoprotein of Borrelia burgdorferi. FEMS Microbiol. Lett. 123:75-82.[CrossRef][Medline]
3
3. Barbour, A. G., and S. F. Hayes. 1986. Biology of Borrelia species. Microbiol. Rev. 50:381-400.[Free Full Text]
4
4. Brusca, J. S., and J. D. Radolf. 1994. Isolation of integral membrane proteins by phase partitioning with Triton X-114. Methods Enzymol. 228:182-193.[Medline]
5
5. Bugrysheva, J., E. Dobrikova, H. P. Godfrey, M. Sartakova, and F. C. Cabello. 2002. Modulation of Borrelia burgdorferi stringent response and gene expression during extracellular growth with tick cells. Infect. Immun. 70:3061-3067.[Abstract/Free Full Text]
6
6. Bunikis, J., and A. G. Barbour. 1999. Access of antibody or trypsin to an integral outer membrane protein (P66) of Borrelia burgdorferi is hindered by Osp lipoproteins. Infect. Immun. 67:2874-2883.[Abstract/Free Full Text]
7
7. Casjens, S. 2000. Borrelia genomes in the year 2000. J. Mol. Microbiol. Biotechnol. 2:401-410.[CrossRef][Medline]
8
8. Coleman, J. L., R. C. Rogers, and J. L. Benach. 1992. Selection of an escape variant of Borrelia burgdorferi by use of bactericidal monoclonal antibodies to OspB. Infect. Immun. 60:3098-3104.[Abstract/Free Full Text]
9
9. Cox, D. L., D. R. Akins, K. W. Bourell, P. Lahdenne, M. V. Norgard, and J. D. Radolf. 1996. Limited surface exposure of Borrelia burgdorferi outer surface lipoproteins. Proc. Natl. Acad. Sci. USA 93:7973-7978.[Abstract/Free Full Text]
10
10. Cox, D. L., D. R. Akins, S. F. Porcella, M. V. Norgard, and J. D. Radolf. 1995. Treponema pallidum in gel microdroplets: a novel strategy for investigation of treponemal molecular architecture. Mol. Microbiol. 15:1151-1164.[CrossRef][Medline]
11
11. Cox, D. L., and J. D. Radolf. 2001. Insertion of fluorescent fatty acid probes into the outer membranes of the pathogenic spirochaetes Treponema pallidum and Borrelia burgdorferi. Microbiology 147:1161-1169.[Abstract/Free Full Text]
12
12. Crother, T. R., C. I. Champion, X. Y. Wu, D. R. Blanco, J. N. Miller, and M. A. Lovett. 2003. Antigenic composition of Borrelia burgdorferi during infection of SCID mice. Infect. Immun. 71:3419-3428.[Abstract/Free Full Text]
13
13. Cullen, P. A., D. A. Haake, and B. Adler. 2003. Outer membrane proteins of pathogenic spirochetes. FEMS Microbiol. Rev., in press.
14
14. Cunningham, T. M., E. M. Walker, J. N. Miller, and M. A. Lovett. 1988. Selective release of the Treponema pallidum outer membrane and associated polypeptides with Triton X-114. J. Bacteriol. 170:5789-5796.[Abstract/Free Full Text]
15
15. Dobrikova, E. Y., J. Bugrysheva, and F. C. Cabello. 2001. Two independent transcriptional units control the complex and simultaneous expression of the bmp paralogous chromosomal gene family in Borrelia burgdorferi. Mol. Microbiol. 39:370-378.[CrossRef][Medline]
16
16. El-Hage, N., K. Babb, J. A. Carroll, N. Lindstrom, E. R. Fischer, J. C. Miller, R. D. Gilmore, Jr., M. L. Mbow, and B. Stevenson. 2001. Surface exposure and protease insensitivity of Borrelia burgdorferi Erp (OspEF-related) lipoproteins. Microbiology 147:821-830.[Abstract/Free Full Text]
17
17. Escudero, R., M. L. Halluska, P. B. Backenson, J. L. Coleman, and J. L. Benach. 1997. Characterization of the physiological requirements for the bactericidal effects of a monoclonal antibody to OspB of Borrelia burgdorferi by confocal microscopy. Infect. Immun. 65:1908-1915.[Abstract/Free Full Text]
18
18. Fraser, C. M., S. Casjens, W. M. Huang, G. G. Sutton, R. Clayton, R. Lathigra, W. White, K. A. Ketchum, R. Dodson, E. K. Hickey, M. Gwinn, B. Dougherty, J. F. Tomb, R. D. Fleischmann, D. Richarson, J. Peterson, A. R. Kerlavage, J. Quackenbush, S. Salzberg, M. Hanson, R. van Vugt, N. Palmer, M. D. Adams, J. Gocayne, J. Weidman, T. Utterback, L. Watthey, L. McDonald, P. Artiach, C. Bowman, S. Garland, C. Fujii, M. D. Cotton, K. Horst, K. Roberts, B. Hatch, H. O. Smith, and J. C. Venter. 1997. Genomic sequence of a Lyme disease spirochete, Borrelia burgdorferi. Nature 390:580-586.[CrossRef][Medline]
19
19. Gilmore, R. D., Jr., K. J. Kappel, M. C. Dolan, T. R. Burkot, and B. J. Johnson. 1996. Outer surface protein C (OspC), but not P39, is a protective immunogen against a tick-transmitted Borrelia burgdorferi challenge: evidence for a conformational protective epitope in OspC. Infect. Immun. 64:2234-2239.[Abstract/Free Full Text]
20
20. Gorbacheva, V. Y., H. P. Godfrey, and F. C. Cabello. 2000. Analysis of the bmp gene family in Borrelia burgdorferi sensu lato. J. Bacteriol. 182:2037-2042.[Abstract/Free Full Text]
21
21. Harlow, E., and D. Lane. 1988. Antibodies. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
22
22. Hellwage, J., T. Meri, T. Heikkila, A. Alitalo, J. Panelius, P. Lahdenne, I. J. Seppala, and S. Meri. 2001. The complement regulator factor H binds to the surface protein OspE of Borrelia burgdorferi. J. Biol. Chem. 276:8427-8435.[Abstract/Free Full Text]
23
23. Hengge, U. R., A. Tannapfel, S. K. Tyring, R. Erbel, G. Arendt, and T. Ruzicka. 2003. Lyme borreliosis. Lancet Infect. Dis. 3:489-500.[CrossRef][Medline]
24
24. Hirschfeld, M., C. J. Kirschning, R. Schwandner, H. Wesche, J. H. Weis, R. M. Wooten, and J. J. Weis. 1999. Inflammatory signaling by Borrelia burgdorferi lipoproteins is mediated by Toll-like receptor 2. J. Immunol. 163:2382-2386.[Abstract/Free Full Text]
25
25. Hubner, A., X. Yang, D. M. Nolen, T. G. Popova, F. C. Cabello, and M. V. Norgard. 2001. Expression of Borrelia burgdorferi OspC and DbpA is contolled by a RpoN-RpoS regulatory pathway. Proc. Natl. Acad. Sci. USA 98:12724-12729.[Abstract/Free Full Text]
26
26. Johnson, R. C., and B. J. B. Johnson. 2002. Lyme disease: serodiagnosis of Borrelia burgdorferi serum lato infection, 494-501. In N. R. Rose, R. G. Hamilton, and B. Detrick (ed.), Manual of clinical laboratory immunology, 6th ed. ASM Press, Washington, D.C.
27
27. Kovarik, A., K. Hlubinova, A. Vrbenska, and J. Prachar. 1987. An improved colloidal silver staining method of protein blots on nitrocellulose membranes. Folia Biol. 33:253-257.
28
28. Landowski, C. P., H. P. Godfrey, S. I. Bentley-Hibbert, X. Liu, Z. Huang, R. Sepulveda, K. Huygen, M. L. Gennaro, F. H. Moy, S. A. Lesley, and M. Haak-Frendscho. 2001. Combinatorial use of antibodies to secreted mycobacterial proteins in a host immune system-independent test for tuberculosis. J. Clin. Microbiol. 39:2418-2424.[Abstract/Free Full Text]
29
29. Liang, F. T., F. K. Nelson, and E. Fikrig. 2002. Molecular adaptation of Borrelia burgdorferi in the murine host. J. Exp. Med. 196:275-280.[Abstract/Free Full Text]
30
30. Ma, J., C. Gingrich-Baker, P. M. Franchi, P. Bulger, and R. T. Coughlin. 1995. Molecular analysis of neutralizing epitopes on outer surface proteins A and B of Borrelia burgdorferi. Infect. Immun. 63:2221-2227.[Abstract/Free Full Text]
31
31. Montgomery, R. R., S. E. Malawista, K. J. Feen, and L. K. Bockenstedt. 1996. Direct demonstration of antigenic substitution of Borrelia burgdorferi ex vivo: exploration of the paradox of the early immune response to outer surface proteins A and C in Lyme disease. J. Exp. Med. 183:261-269.[Abstract/Free Full Text]
32
32. Motaleb, M. A., L. Corum, J. L. Bono, A. F. Elias, P. Rosa, D. S. Samuels, and N. W. Charon. 2000. Borrelia burgdorferi periplasmic flagella have both skeletal and motility functions. Proc. Natl. Acad. Sci. USA 97:10899-10904.[Abstract/Free Full Text]
33
33. O'Farrell, P. H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007-4021.[Abstract/Free Full Text]
34
34. Pal, U., R. R. Montgomery, D. Lusitani, P. Voet, V. Weynants, S. E. Malawista, Y. Lobet, and E. Fikrig. 2001. Inhibition of Borrelia burgdorferi-tick interactions in vivo by outer surface protein A antibody. J. Immunol. 166:7398-7403.[Abstract/Free Full Text]
35
35. Pavia, C. S., V. Kissel, S. Bittker, F. Cabello, and S. Levine. 1991. Antiborrelial activity of serum from rats injected with the Lyme disease spirochete. J. Infect. Dis. 163:656-659.[Medline]
36
36. Radolf, J. D., N. R. Chamberlain, A. Clausell, and M. V. Norgard. 1988. Identification and localization of integral membrane proteins of virulent Treponema pallidum by phase partitioning with the nonionic detergent Triton X-114. Infect. Immun. 56:490-498.[Abstract/Free Full Text]
37
37. Ramamoorthy, R., L. Povinelli, and M. Philipp. 1996. Molecular characterization, genomic arrangement, and expression of bmpD, a new member of the bmp class of gene encoding membrane proteins of Borrelia burgdorferi. Infect. Immun. 64:1259-1264.[Abstract/Free Full Text]
38
38. Roberts, D. M., M. Theisen, and R. T. Marconi. 2000. Analysis of the cellular localization of Bdr paralogs in Borrelia burgdorferi, a causative agent of lyme disease: evidence for functional diversity. J. Bacteriol. 182:4222-4226.[Abstract/Free Full Text]
39
39. Sadziene, A., M. Jonsson, S. Bergstrom, R. K. Bright, R. C. Kennedy, and A. G. Barbour. 1994. A bactericidal antibody to Borrelia burgdorferi is directed against a variable region of the OspB protein. Infect. Immun. 62:2037-2045.[Abstract/Free Full Text]
40
40. Sadziene, A., P. A. Thompson, and A. G. Barbour. 1993. In vitro inhibition of Borrelia burgdorferi growth by antibodies. J. Infect. Dis. 167:165-172.[Medline]
41
41. Sambrook, J., and D. W. Russell. 2001. Molecular cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
42
42. Scriba, M., J. S. Ebrahim, T. Schlott, and H. Eiffert. 1993. The 39-kilodalton protein of Borrelia burgdorferi: a target for bactericidal human monoclonal antibodies. Infect. Immun. 61:4523-4526.[Abstract/Free Full Text]
43
43. Simpson, W. J., W. Cieplak, M. E. Schrumpf, A. G. Barbour, and T. G. Schwan. 1994. Nucleotide sequence and analysis of the gene in Borrelia burgdorferi encoding the immunogenic P39 antigen. FEMS Microbiol. Lett. 119:381-387.[CrossRef][Medline]
44
44. Simpson, W. J., M. E. Schrumpf, and T. G. Schwan. 1990. Reactivity of human Lyme borreliosis sera with a 39-kilodalton antigen specific to Borrelia burgdorferi. J. Clin. Microbiol. 28:1329-1337.[Abstract/Free Full Text]
45
45. Sullivan, T. J., K. E. Hechemy, H. L. Harris, U. H. Rudofsky, W. A. Samsonoff, A. J. Peterson, B. D. Evans, and S. L. Balaban. 1994. Monoclonal antibody to native P39 protein from Borrelia burgdorferi. J. Clin. Microbiol. 32:423-429.[Abstract/Free Full Text]
46
46. Yang, X. F., A. Hubner, T. G. Popova, K. E. Hagman, and M. V. Norgard. 2003. Regulation of expression of the paralogous Mlp family in Borrelia burgdorferi. Infect. Immun. 71:5012-5020.[Abstract/Free Full Text]

---

Infection and Immunity, April 2004, p. 2280-2287, Vol. 72, No. 4
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.4.2280-2287.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Hughes, J. L., Nolder, C. L., Nowalk, A. J., Clifton, D. R., Howison, R. R., Schmit, V. L., Gilmore, R. D. Jr., Carroll, J. A. (2008). Borrelia burgdorferi Surface-Localized Proteins Expressed during Persistent Murine Infection Are Conserved among Diverse Borrelia spp.. Infect. Immun. 76: 2498-2511 [Abstract] [Full Text]  
• Pal, U., Wang, P., Bao, F., Yang, X., Samanta, S., Schoen, R., Wormser, G. P., Schwartz, I., Fikrig, E. (2008). Borrelia burgdorferi basic membrane proteins A and B participate in the genesis of Lyme arthritis. JEM 205: 133-141 [Abstract] [Full Text]  
• Dubytska, L., Godfrey, H. P., Cabello, F. C. (2006). Borrelia burgdorferi ftsZ Plays a Role in Cell Division.. J. Bacteriol. 188: 1969-1978 [Abstract] [Full Text]  
• Bryksin, A. V., Godfrey, H. P., Carbonaro, C. A., Wormser, G. P., Aguero-Rosenfeld, M. E., Cabello, F. C. (2005). Borrelia burgdorferi BmpA, BmpB, and BmpD Proteins Are Expressed in Human Infection and Contribute to P39 Immunoblot Reactivity in Patients with Lyme Disease. CVI 12: 935-940 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) A retraction has been published Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shin, J. J. Articles by Cabello, F. C. Search for Related Content PubMed PubMed Citation Articles by Shin, J. J. Articles by Cabello, F. C.