A surface enolase participates in Borrelia burgdorferi-plasminogen interaction and contributes to pathogen survival within feeding ticks

Abstract

Borrelia burgdorferi, a tick-borne bacterial pathogen, causes a disseminated infection involving multiple organs known as Lyme disease. Surface proteins can directly participate in microbial virulence by facilitating pathogen dissemination via interaction with host factors. We show here that a fraction of the B. burgdorferi chromosomal gene product BB0337, annotated as enolase or phosphopyruvate dehydratase, is associated with spirochete outer membrane and is surface exposed. B. burgdorferi enolase, either in a recombinant form or as a membrane-bound native antigen, displays enzymatic activities intrinsic to the glycolytic pathway. However, the protein also interacts with host plasminogen, potentially leading to its activation and resulting in B. burgdorferi-induced fibrinolysis. As expected, enolase displayed consistent expression in vivo, however, with a variable temporal and spatial expression during spirochete infection in mice and ticks. Despite an extracellular exposure of the antigen and a potential role in host-pathogen interaction, active immunization of mice with recombinant enolase failed to evoke protective immunity against subsequent B. burgdorferi infection. In contrast, enolase immunization of murine hosts significantly reduced the acquisition of spirochetes by feeding ticks, suggesting that the protein could have a stage-specific role in B. burgdorferi survival in the feeding vector. Strategies to interfere with the function of surface enolase could contribute to the development of novel preventive measures to interrupt the spirochete infection cycle and reduce the incidences of Lyme disease.

Fig 1

Plasminogen interacts with B. burgdorferi and involvement of enolase. The data represent means plus the SEM from three independent experiments. (A) Plasminogen-B. burgdorferi interaction. B. burgdorferi were fixed onto the microtiter plates with glutaraldehyde and incubated with human plasminogen (hPg) in a concentration-dependent manner. Binding was detected using anti-plasminogen and secondary antibodies. The differences between hPg (0.05 to 2) and control (no hPg) are significant (*, P < 0.05). (B) Fibrinolytic activity of plasminogen-bound B. burgdorferi. Spirochetes were incubated in the absence (lane 1) or presence of plasminogen (lane 2), or together with plasminogen and tPA (lane 3), spotted into the Matrigel, and incubated for the appearance of halo areas indicative of fibrinolytic activity. (C) Enolase antibodies competitively reduce B. burgdorferi-plasminogen interaction. B. burgdorferi were coated onto microtiter wells as detailed in Fig. 1A and incubated with anti-enolase or anti-BBA52 antibodies prior to incubation with hPg. The difference between anti-enolase antibodies and control (no Ab) is significant (*, P < 0.05). (D) Plasminogen binds to recombinant enolase. Enolase (1 μg) was immobilized on microtiter well plates and incubated with hPg in a concentration-dependent manner. Bound proteins were detected using anti-plasminogen and secondary antibodies. The differences between hPg (1 to 3 μg) and no hPg (0) wells are significant (*, P < 0.05). (E) A lysine analog inhibits enolase-Pg interaction. Immobilized enolase (1 μg) was incubated with hPg (1 μg) in the presence of different concentrations of inhibitor (εACA). The differences between wells incubated with εACA or without (−) are significant. * P < 0.05.

Fig 2

Enolase is surface exposed on B. burgdorferi cells. (A) Anti-enolase antibody binds to the surfaces of intact spirochetes. B. burgdorferi cells were incubated using glutaraldehyde, which immobilizes the cells onto the microtiter wells without compromising membrane permeability. Cells were incubated with anti-enolase or anti-FlaB antibodies, and bound antibodies were detected using secondary antibodies. The data represent the means with the SEM from three independent experiments. The differences between anti-enolase antibodies and controls are significant (*, P < 0.05). (B) Enolase is associated with borrelial membrane fraction. Triton X-114-extractable membrane (detergent-phase) or soluble (aqueous-phase) proteins were immunoblotted by antibodies against enolase or known membrane protein (OspA). (C) Enolase is sensitive to proteinase K-mediated degradation of B. burgdorferi surface proteins. Viable spirochetes were incubated in the absence (−) or presence (+) of proteinase K for removal of protease sensitive surface proteins and processed for immunoblot analysis using anti-enolase antibodies. B. burgdorferi OspA and FlaB antibodies were utilized as controls for surface-exposed and subsurface proteins, respectively. (D) Densitometric analysis of proteinase K-mediated degradation of enolase. Relative densities of B. burgdorferi enolase in the absence or presence of proteinase K, as determined by immunoblot analysis with anti-enolase antibodies shown in Fig. 2C, were determined by a densitometric scan. Differences between the levels of enolase in the absence and presence of proteinase K treatment are significant (*, P < 0.05).

Fig 3

Enzymatic activities of recombinant and membrane bound B. burgdorferi enolase. (A) Activity of the recombinant enolase. Enzyme activity was measured by a coupled enzymatic assay by measurement of catalysis of 2-phosphoglycerate to phosphoenolpyruvate for a period of 35 min using 1.6 μg of recombinant enolase or a control B. burgdorferi membrane protein BBA52. (B) Substrate saturation by recombinant enolase. Different concentrations of the substrate 2-phosphoglycerate (1 to 6 mM) were incubated with fixed amount of enolase (2 μg). (C) Enolase activity on the surface of intact B. burgdorferi. The conversion of 2-phosphoglycerate to phosphoenolpyruvate was used to measure the enolase activity in B. burgdorferi cells and E. coli. (D) Enolase activity assay conditions did not affect B. burgdorferi viability. Aliquots of B. burgdorferi were isolated before or after enzymatic assays as described in panel C and subjected to spirochete viability analysis. A representative fluorescence labeling of live and dead spirochetes before or after enzyme treatment, as assessed under a laser confocal microscope, is presented. Live B. burgdorferi was stained with Syto 9 (green, arrows), and the dead spirochetes were stained with propidium iodide (red).

Fig 4

Enolase expression in vivo. The relative expression levels of enolase in the murine hosts and in representative life stages of ticks were analyzed, and the results are presented as copies of enolase transcript per 1,000 copies of flaB transcripts. Total RNA was isolated from multiple tissues of B. burgdorferi-infected mice (six mice/group) at days 7, 14, 21, and 28 after challenge, unfed nymphs following larval molting (Unf), and fed nymphs (Fed) after 3 days of feeding. BB0337 transcripts were measured using qRT-PCR. Transcripts encoding enolase were abundant in all murine tissues tested and were detected in these stages of the ticks. Bars denote the mean ± the standard error of four representative qPCR measurements from two independent infection experiments. The enolase transcript level in joints at day 28 is significantly different from that of other time points or tissue locations (P < 0.05).

Fig 5

Enolase antibody lacks bactericidal properties and does not interfere with B. burgdorferi infectivity. (A) Detection of antibodies against enolase in immunized mice. Groups of mice (three animals/group) were immunized with recombinant enolase or PBS (control) mixed with adjuvant. B. burgdorferi lysates (Bb) and recombinant protein (Eno) immunoblotted with serum were collected from individual enolase-immunized mice (indicated as 1, 2, and 3). Note that the recombinant His tag protein migrates as a slightly higher molecular protein compared to the native protein. (B) Borreliacidal activities of enolase antibodies in vitro as assessed by the growth of spirochetes following antiserum treatment. B. burgdorferi was treated with enolase antiserum (Eno Ab) or normal mouse serum (NMS) as a control. After 24 h, 1 μl of medium was inoculated into 2 ml of fresh medium, and the growth of the spirochetes was assessed using dark-field microscopy at the indicated time periods. (C and D) Effect of enolase immunization on B. burgdorferi infection in mice. Mice were immunized with enolase as detailed in Fig. 5A, and 10 days after the final immunization the mice were infected with B. burgdorferi (10⁵ spirochetes/mouse). The spirochete burdens in both groups of mice were assessed by qRT-PCR analysis by measuring the copies of the B. burgdorferi flaB that normalized with murine β-actin transcript levels. B. burgdorferi levels were examined in blood collected 3, 5, and 7 days after challenge (C) or from skin, joint, and heart locations (D) at 12 days after infection.

Fig 6

Active immunization of mice with enolase reduces B. burgdorferi acquisition by Ixodes nymphs. Mice were immunized with enolase or PBS and infected with B. burgdorferi. After 12 days, nymphs (15 ticks/mouse) were allowed to engorge on the mice and were collected after 5, 48, and 72 h of feeding. Bars denote the mean value ± the SEM of four representative quantitative PCR measurements from two independent animal infection experiments. Differences between mice immunized with enolase and controls at all time points were significant (*, P < 0.05).

Fig 7

Partial alignment of amino acid sequences from different microorganisms showing positions of potential Pg-binding domain and catalytic motif in the primary sequence of enolase. The shaded boxes represent the Pg-binding domain and catalytic motif, whereas the positions of two extreme C-terminal lysine residues that also mediate Pg binding are denoted by arrows. The GenBank accession numbers for the enolase from respective organisms are as follows: Borrelia burgdorferi B31, AAC66719.1; Leptospira interrogans serovar Lai strain 56601, AAN49150.1; Streptococcus pneumoniae, AJ303085.1; Bacillus anthracis strain Ames Ancestor, AAT34498.1; Paracoccidioides brasiliensis 01, EF558735.1; and Candida albicans, L10290.1.