<i>B. burgdorferi</i> enolase is surface exposed.

<p>(A) Demonstration of outer surface exposure of enolase by <i>in situ</i> protease degradation. Whole <i>B. burgdorferi</i> were incubated with proteinase K for 30, 60 or 120 minutes or 120 minutes in buffer with no protease. Proteases were then inactivated, bacteria were lysed, proteins were separated by SDS-PAGE, and the integrity of enolase, OspC (outer surface protein), and FlaB (periplasmic protein) were analyzed by immunoblot. Two separate protease degradation experiments were performed, with blots repeated 4 times. A representative blot is shown. (B) Densitometric analysis of <i>in situ</i> protease degradation immunoblots. Images from blots were scanned and peak areas for 120-minute proteinase K treatment and no protease control were assessed using ImageJ software (<a href="http://imageJ.nih.gov/ij" target="_blank">http://imageJ.nih.gov/ij</a>). Data represent the means and standard errors from 4 separate blots. *, <i>P</i> = 0.03, Student's <i>t</i> test assuming unequal variances. (C) Binding of exogenous <i>B. burgdorferi</i> enolase to the surface of whole bacteria. Binding of <i>B. burgdorferi</i> enolase (0–50 µg/ml) to immobilized <i>B. burgdorferi</i> (2×10⁷/ml) was analyzed by ELISA, with bound enolase detected by rabbit polyclonal antiserum directed against human enolase. Data represent the means and standard errors from three separate experiments with six replicates per enolase concentration.</p

Role of lysines in enolase/plasminogen binding activity.

<p>Binding of plasminogen to immobilized enolase (10 µg/ml) was analyzed by ELISA. Plasminogen (25 µg/ml) was added to enolase-coated wells in the presence or absence of 0–30 mM ε-aminocaproic acid (lysine analog). Bound plasminogen was detected using a specific antiserum. BSA was used as a negative control. Data represent the means and standard errors from three separate experiments with 12 replicates per condition. ***, <i>P</i><0.001, Student's <i>t</i> test assuming unequal variances.</p

Conservation of enolase.

<p>(A) Alignment of enolases from mouse, human, yeast and <i>B. burgdorferi</i>. Dark shaded residues indicate identity; light gray residues indicate similarity. Arrows indicate the 3 conserved residues that make up the enzymatic active site; stars indicate the three conserved residues required for binding the cofactor manganese. (B) A rabbit polyclonal antiserum directed against human enolase recognizes enolase from <i>B. burgdorferi</i>. Top panel: Western blot; Bottom panel: Coomassie-stained SDS-PAGE gel. Molecular weight markers are indicated on the left. Lane 1: fresh BSKII medium; Lane 2: spent BSKII medium from late-log phase cultures of <i>B. burgdorferi</i>; Lane 3: <i>B. burgdorferi</i> cell lysate; Lane 4: recombinant enolase from <i>B. burgdorferi</i>.</p

Enolase-bound plasminogen is converted into plasmin.

<p>Enolase-coated wells of microtiter plates were incubated with plasminogen, urokinase (uPA), and/or a plasmin-specific chromogenic substrate. Proteolytic activity was measured by absorbance at 405 nM. Data represent the means and standard errors from three different experiments with six replicates per condition. ***, <i>P</i><0.001 compared to the activation of plasminogen bound to the control protein, BSA, Student's <i>t</i> test assuming unequal variances.</p

Immunoelectron microscopic analysis of <i>B. burgdorferi</i> enolase.

<p>A) Anti-enolase was localized intermittently across the outer surface of <i>B. burgdorferi (arrows)</i>. Gold particles were also observed on membrane blebs in proximity to the spirochete (asterisks). The boxed area in the inset indicates the region demonstrated in image (A). B) Omission of the primary antibody resulted in a complete loss of immunoreactivity. C) Anti-OspC immunolabeling demonstrated moderately heavy labeling on the outer surface of the spirochete (Arrows). Magnification bar  = 0.2 µm.</p

BB0347, from the Lyme Disease Spirochete <i>Borrelia burgdorferi</i>, Is Surface Exposed and Interacts with the CS1 Heparin-Binding Domain of Human Fibronectin

<div><p>The causative agent of Lyme disease, <i>Borrelia burgdorferi</i>, codes for several known fibronectin-binding proteins. Fibronectin a common the target of diverse bacterial pathogens, and has been shown to be essential in allowing for the development of certain disease states. Another borrelial protein, BB0347, has sequence similarity with these other known fibronectin-binding proteins, and may be important in Lyme disease pathogenesis. Herein, we perform an initial characterization of BB0347 via the use of molecular and biochemical techniques. We found that BB0347 is expressed, produced, and presented on the outer surface of intact <i>B. burgdorferi</i>. We also demonstrate that BB0347 has the potential to be important in Lyme disease progression, and have begun to characterize the nature of the interaction between human fibronectin and this bacterial protein. Further work is needed to define the role of this protein in the borrelial infection process.</p></div

Primers used in this study.

*<p>All primers were designed during the course of this study, except Fla3 & 4, which have been described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075643#pone.0075643-Chenail1" target="_blank">[44]</a>.</p

Recombinant BB0347 binds Fn in a dose-dependent manner.

<p>Fn-binding was determined by ELISA using BSA (negative control), RevA (positive control), and BB0347 as coating proteins. Fn was added to wells (0, 10, and 20 µg/mL) allowed to bind, and any unbound Fn was washed away. Values were blanked against the negative control to eliminate background from non-specific binding. BB0347 bound Fn in a dose-dependent manner as determined by an increased binding of αFn antibodies in wells treated with BB0347. Results are representative of three independent experiments–of at least 4 replicates for each concentration–with similar results, and error bars are indicative of ±SEM. Significance was determined by comparing the values of each concentration of Fn to the 0 Fn control values for both proteins after blanking with the BSA negative control.</p

BB0347 is expressed and produced in culture.

<p><b>A)</b> Expression of BB0347 was verified by RT- PCR with <i>flaB</i> mRNA as a control. Lane 1: <i>flaB</i> from genomic DNA, lane 2: <i>flaB</i> from cDNA, lane 3: no RT control, lane 4: <i>bb0347</i> from genomic DNA, lane 5: <i>bb0347</i> from cDNA, lane 6: no RT control, L: ladder. <b>B)</b> Western blotting shows that BB0347 protein is produced in the spirochete at all sampled time points. <b>C)</b> QRT- PCR of <i>bb0347</i> at two different temperatures of incubation with <i>flaB</i> as a standard. <b>D)</b> Western blot using αBB0347 and αFlaB against whole-cell lysates from spirochetes grown at either 34 or 23°C to similar cellular densities. All figures are representative of at least two independent experiments with similar results, and error bars indicate ±SEM.</p

BB0347 is surface exposed in <i>B. burgdorferi</i> MI-16.

<p><b>A)</b> Intact spirochetes were treated with different proteases for 30 min or 2 hrs. Western blots were run against whole cell lysates after these treatments and blotted with αBB0347, αOspC, or αFlaB. After two hours, no difference was observed between the control and protease-treated spirochetes in the αFlaB-blotted membranes, but OspC and BB0347 were almost completely degraded (Key: C: No protease control, K: proteinase K, P: pronase, T: trypsin). <b>B)</b> Intact bacteria were coated onto glass slides and fixed with 10% Formalin. Antibodies against the same proteins listed in (A) were used to stain and a secondary antibody conjugated with Dylight 488 was used for detection. DAPI was used as a secondary stain to localize spirochetes (see Materials and Methods). An additional control in which the <i>B. burgdorferi</i> membrane was compromised by desiccation, was included to verify that αFlaB antibodies were effective on fixed spirochetes. Key: Panel 1) αOspC, 2) αBB0347, 3) αFlaB 4) αFlaB with membrane disruption. BB0347 was detected in intact spirochetes, further verifying the surface exposure. White bars indicate a length of 10 µM, and the magnification is 1000×. Results are indicative of three independent experiments with similar outcomes.</p