The Tick Salivary Protein Salp15 Inhibits the Killing of Serum-Sensitive Borrelia burgdorferi Sensu Lato Isolates

Borrelia isolates and growth conditions.Serum-sensitive strains B. garinii A87S and VSBP and intermediately resistant strains B. burgdorferi VS215 and B31 were used in this study. Both B. garinii strains are human isolates, while both B. burgdorferi strains are tick isolates. Spirochetes were cultivated at 33°C in Barbour-Stoenner-Kelley medium plus sodium bicarbonate (BSK-H medium) supplemented with 6% rabbit serum (Sigma).

Purification of recombinant I. scapularis and I. ricinus Salp15.For the purification of Iscap Salp15 (GenBank accession number AAK97817), salp15 was cloned in frame in Drosophila melanogaster cells in conjunction with a His tag, a V5 epitope, and a resistance gene for hygromycin as described previously (1). Salp15 Iric-1 (GenBank accession number ABU93613) was purified using Drosophila cells expressing salp15 together with a resistance gene for blastomycin (J. W. Hovius, T. J. Schuijt, K. A. de Groot, J. T. T. H. Roelofs, A. Oei, J. A. Marquart, C. van ′t Veer, T. van der Poll, N. Ramamoorthi, E. Fikrig, and A. P. van Dam, submitted for publication). The Schneider Drosophila cells expressing the salp15 gene from I. scapularis or I. ricinus were selected with hygromycin (500 μg ml⁻¹) or blastomycin (25 μg ml⁻¹), respectively, and were grown in large spinner flasks together with penicillin and streptomycin (Invitrogen) for 3 days. The Drosophila cells were subsequently induced with copper sulfate with a final concentration of 500 mM for 4 days and centrifuged at 1,000 × g for 15 min. The supernatant was filtered using a 0.22-μm filter (Millipore). Both Salp15 Iric-1 and Iscap Salp15 were purified from the supernatant by use of the HisTrap Ni²⁺ column (GE Healthcare) and eluted with 100 mM imidazole. The eluted fractions were filtered through a 0.22-μm filter and concentrated with a 5-kDa concentrator (Vivascience) through centrifugal concentration.

The purity of the purified Salp15 was checked by silver staining (Bio-Rad) according to the manufacturer's recommendations, and the concentration was determined with the Bradford assay.

NHS.Serum samples used were derived from one donor and were checked for the absence of antibodies against B. burgdorferi by Western blot analysis. Heat inactivation of NHS was achieved by incubation of the serum samples at 56°C for 30 min.

Assays for detection of complement-mediated killing of spirochetes and Salp15 protection.Two serum-sensitive B. garinii strains, the A87S and the VSBP strains, and two intermediately resistant B. burgdorferi sensu stricto strains, VS215 and B31, were used (10⁷ spirochetes ml⁻¹). Spirochetes (2.5 × 10⁵) were preincubated with bovine serum albumin (BSA), Salp15 Iric-1, or Iscap Salp15 (80 μg/ml) for 30 min at 33°C. They were then incubated with NHS or heat-inactivated NHS and examined after 1.5 h, 4.5 h, and 24 h. The two parameters of borreliacidal effect that were recorded are immobilization and bleb formation of the spirochetes. Immotile spirochetes were considered dead (28). The percentages of immotile spirochetes for 200 spirochetes per well were assessed. In a separate titration experiment, different Salp15 concentrations, ranging from 5 μg/ml to 160 μg/ml, were also tested in the same way.

To find out if membrane-bound Salp15 protects the spirochetes against antibody-independent complement-mediated killing, the spirochetes were washed twice with BSK-H medium (4,000 × g, 10 min) after incubation with Salp15 Iric-1. After removal of unbound Salp15 by washing, the spirochetes were subjected to 12.5% NHS and examined for borreliacidal effect after 1.5 h, 4.5 h, and 24 h of incubation.

Subculture of B. garinii VSBP after incubation with I. ricinus Salp15.The serum-sensitive B. garinii VSBP strain was preincubated with Salp15 Iric-1 for 60 min at 33°C. Then, spirochetes were exposed to 50% NHS for 24 h. As described above, the surviving spirochetes were subcultured in BSK-H medium for 7 days. This selection process was repeated twice.

Binding of I. ricinus Salp15 to B. garinii VSBP in overlay assay. B. garinii VSBP lysates were obtained from 100-ml cultures that were grown to a density of 1 × 10⁸ spirochetes/ml. Lysates were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto an Immobilon-P membrane (Millipore) to have approximately similar amounts of OspC, as determined by Coomassie staining. After the membrane was incubated overnight in blocking buffer (1% BSA, 3% milk in TBS-0.05% Tween), it was incubated with 1 μg/ml purified Salp15 Iric-1 in blocking buffer for 1.5 h at room temperature, washed, and consequently incubated with a 1:5,000 dilution of horseradish peroxidase-conjugated V5 antibody (Invitrogen) in blocking buffer. Blots were developed by enhanced chemiluminescence.

Binding assay of Salp15 Iric-1 to B. garinii VSBP by immunofluorescence.Purified Salp15 Iric-1 was biotinylated by incubating 1 mg/ml Salp15 with 0.25 mg/ml sulfo-NHS-biotin (Pierce) for 45 min at 4°C. Unbound sulfo-NHS-biotin was removed by dialysis against 25 mM lysine in phosphate-buffered saline (PBS) overnight and four times against PBS for 15 min at 4°C.

Biotinylated purified Salp15 Iric-1 was incubated with B. garinii VSBP spirochetes for 30 min at 33°C. The spirochetes were washed twice with PBS-1% BSA and were resuspended in PBS-1% BSA, air dried on microscope slides overnight, and fixed in 100% methanol. Slides were incubated with bisbenzimide and streptavidine-Cy3 (Sigma) and examined with a fluorescence microscope (Axioscop 2 mot plus; Carl Zeiss).

Detection of terminal C5b-9 complement complexes. B. garinii strain VSBP was preincubated with either BSA, Iscap Salp15, or Salp15 Iric-1 for 30 min at 33°C. Then, the spirochetes were incubated in BSK-H medium containing 12.5% NHS for 30 min. The spirochetes were washed twice with PBS-1% BSA. They were resuspended in PBS-1% BSA, air dried on microscope slides overnight, and fixed in 100% methanol. Spirochetes were detected by incubation with human serum containing antibodies against B. burgdorferi, and the C5b to C9 (C5b-9; one molecule each of C5b, C6, C7, and C8 and one or more molecules of C9) complement complexes were indicated with monoclonal mouse C5b-9 antibodies (Dako). Slides were washed with PBS-1% BSA and incubated with an anti-human immunoglobulin G-fluorescein isothiocyanate-labeled antibody (BioMerieux) and an anti-mouse Cy3 antibody (Jackson). After slides were washed and were mounted with Mowiol, they were visualized by confocal microscopy using a fluorescence microscope (Axioscop 2 mot plus; Carl Zeiss). At least 100 spirochetes were counted, and the experiment was performed two times.

Statistical analysis.The protection of spirochetes against complement-mediated killing by Salp15 Iric-1 or Iscap Salp15 was compared to the protection by the control protein BSA. The chi-square test was used for the analysis of proportions, where absolute numbers of spirochetes were used in cross tabulations. Crude relative risks for surviving different circumstances were estimated as odds ratios (OR) and presented with both 95% confidence intervals (95% CI) and P values. Tests were performed using SPSS 14 software. Calculated P values of <0.05 were considered significant.

Salp15 protects serum-sensitive B. burgdorferi sensu lato isolates against complement-mediated killing.As expected, B. burgdorferi sensu lato isolates differed in their sensitivities to NHS. No motile B. garinii A87S or B. garinii VSBP organisms were seen when incubated for 24 h in 12.5% NHS (Fig. 1). In contrast, other spirochetes required greater amounts of NHS to induce partial killing: B. burgdorferi B31 was killed at 24 h by 50% NHS (Fig. 1) and B. burgdorferi VS215 was killed by 75% NHS at 24 h (data not shown). All these Borrelia isolates survived incubation for 1.5, 4.5, or 24 h with heat-inactivated NHS, demonstrating the importance of complement in serum sensitivity.

• Open in new tab
• Download powerpoint

FIG. 1.

Spirochetes are protected against complement-mediated killing in the presence of Salp15 Iric-1 or Iscap Salp15. Serum-sensitive B. garinii strains VSBP (A) and A87S (B) were incubated with 12.5% NHS, while intermediately resistant B. burgdorferi strains VS215 (C) and B31 (D) were incubated with 50% NHS, after they had been preincubated for 30 min with BSA, Iscap Salp15, or Salp15 Iric-1. As a control, spirochetes were also incubated with heat-inactivated NHS. After 1.5 h, 4.5 h, and 24 h of incubation with serum, the percentages of immotile spirochetes were determined. Two hundred spirochetes were counted.

Since Salp15 has previously been shown to enhance the capacity of spirochetes to survive in naive mice (25), we determined whether Salp15 could alter the serum sensitivity of these B. burgdorferi isolates. Indeed, Salp15 from I. scapularis ticks, Iscap Salp15, altered the serum sensitivity of Borrelia (Fig. 1). The percentage of dead spirochetes significantly decreased when Borrelia isolates were preincubated with Iscap Salp15 (Table 1). The strongest protective effect was seen with B. garinii VSBP. We had previously cloned Salp15 from I. scapularis ticks, but since the serum-sensitive Borrelia are transmitted predominantly by I. ricinus ticks, we wanted to determine whether Salp15 from these ticks was as potent as or more potent than Salp15 from I. scapularis for selected Borrelia isolates. We therefore cloned, expressed, and purified Salp15 from I. ricinus, Salp15 Iric-1, as described in Materials and Methods. Salp15 Iric-1 afforded protection against complement-mediated killing (Fig. 1; Table 1). The protective effect of Salp15 Iric-1 for all four Borrelia isolates was also greater than that of Iscap Salp15 (Table 1) and the difference in protection of serum-sensitive B. garinii spirochetes by Salp15 Iric-1 compared to that by Iscap Salp15 was most apparent (Table 2). A titration of Salp15 showed that both Iscap Salp15 and Salp15 Iric-1 had a dose-dependent protective effect (data not shown).

Salp15 Iric-1 binds to the surface of B. garinii VSBP. B. garinii VSBP was preincubated with biotinylated Salp15 Iric-1 and membrane-bound Salp15 Iric-1 was detected using streptavidin-Cy3 in the immunofluorescence assay. It has previously been shown that Iscap Salp15 binds on the surface of B. burgdorferi 297 (25) and we now show that Salp15 Iric-1 also specifically binds to the surface of B. garinii VSBP (Fig. 2A) by immunofluorescence assay. By solid-phase overlay, we demonstrated that both Iscap Salp15 and Salp15 Iric-1 are able to bind to OspC of B. garinii VSBP and B. burgdorferi N40 (Fig. 2C). All spirochetes were found to bind Salp15 Iric-1 on their surfaces.

• Open in new tab
• Download powerpoint

FIG. 2.

Salp15 Iric-1 binds to the surfaces of B. garinii VSBP spirochetes. Spirochetes were preincubated with biotinylated Salp15 Iric-1 (A) or biotinylated BSA (B). Spirochetes were detected with bisbenzimide (blue) and bound Salp15 or BSA was detected using streptavidin-Cy3 (red). (C) Both Iscap Salp15 and Salp15 Iric-1 bind B. burgdorferi N40 (1) and B. garinii VSBP (2) in the overlay binding assay. The arrow indicates Salp15 bound to OspC. M, molecular mass.

Surface-bound Salp15 protects spirochetes against complement-mediated killing.To study whether bound Salp15 protects spirochetes against complement-mediated killing, B. garinii was preincubated with Salp15 Iric-1, after which unbound Salp15 was washed away. BSA was used as a control. Spirochetes to which Salp15 had been bound were still protected against complement-mediated killing (Fig. 3), and there was a significant difference between the survivals of spirochetes after incubation with either BSA or Salp15 and subsequent washing. Notably fewer spirochetes were initially killed in the control group after washing. This could be caused by the loss of less viable spirochetes in the washing procedure. The fact that this difference is not observed among spirochetes preincubated with Salp15 suggests that all spirochetes, independently of initial viability, are potentially protected by Salp15.

• Open in new tab
• Download powerpoint

FIG. 3.

Membrane-bound Salp15 Iric-1 protects B. garinii VSBP against complement-mediated killing. Spirochetes were preincubated with BSA or Salp15 for 60 min. Subsequently, BSA or Salp15 was removed by washing as indicated in Materials and Methods, and spirochetes were exposed to 12.5% NHS. Percentages of immotile spirochetes were determined for samples and for controls in which Salp15 was still present. Standard deviations, based on duplicate countings of two independent experiments, are included. The asterisks indicate a statistically significant difference between the groups (P < 0.0001).

Spirochetes that survive in the presence of Salp15 do not have an increased capacity to bind Salp15.In the presence of Salp15 Iric-1, 2% of the B. garinii VSBP spirochetes survived incubation with 50% NHS for 24 h. The surviving spirochetes were reisolated to determine whether the “survival” phenotype could be enriched via selection. After repeating the selection assay two times, the survival rate of the VSBP subculture was compared to that of the original VSBP isolate after incubation with Salp15 and exposure to 12.5% NHS. The B. garinii VSBP subculture was not more protected by Salp15 Iric-1 than the original B. garinii VSBP culture.

C5b-9 deposition is inhibited by Salp15 Iric-1 or Iscap Salp15.To examine the influence of Salp15 against complement-mediated killing, we investigated, by use of an immunofluorescence assay, the differences in deposition of C5b-9 terminal complement complexes between spirochetes that had been incubated with Salp15 Iric-1, Iscap Salp15, or BSA (control) and then exposed to NHS. When B. garinii VSBP was first incubated with Salp15 Iric-1 or Iscap Salp15, significantly fewer (P < 0.0001) C5b-9 complement complexes were found on the membranes of the spirochetes (Fig. 4A to C). Of the spirochetes that had been incubated with BSA and 12.5% NHS, 90% had C5b-9 complement complexes on their membrane, while 52% and 15% of the spirochetes showed C5b-9 deposition after incubation with Iscap Salp15 and Salp15 Iric-1, respectively (Fig. 4D).

• Open in new tab
• Download powerpoint

FIG. 4.

Inhibition of C5b-9 deposition by Salp15. B. garinii VSBP was preincubated with BSA (A), Iscap Salp15 (B), or Salp15 Iric-1 (C) before being subjected to 12.5% NHS. Spirochetes were double labeled with antibodies specific for Borrelia spirochetes (green) and specific for C5b-9 complement complexes (red). (D) Deposition of C5b-9 complexes was strongly inhibited in the presence of Iscap Salp15 and even more by Salp15 Iric-1. Bars indicate standard deviations, based on duplicate countings of two independent experiments. One hundred spirochetes were counted, and the experiment was performed two times. The asterisk indicates a statistically significant difference (P < 0.0001) between the BSA- and Salp15 Iric-1-treated groups, the double asterisk shows a significant difference (P < 0.0001) between the BSA- and Iscap Salp15-treated groups, and the triple asterisk indicates a significant difference (P < 0.0001) between the Iscap Salp15- and Salp15 Iric-1-treated groups.