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Borrelia recurrentis employs a novel multifunctional surface protein with anti-complement, anti-opsonic and invasive potential to escape innate immunity.

Grosskinsky S, Schott M, Brenner C, Cutler SJ, Kraiczy P, Zipfel PF, Simon MM, Wallich R - PLoS ONE (2009)

Bottom Line: Cell surface bound factor H was found to retain its activity and to confer resistance to complement attack.Furthermore, HcpA-bound plasminogen/plasmin endows B. recurrentis with the potential to resist opsonization and to degrade extracellular matrix components.Together, the present study underscores the high virulence potential of B. recurrentis.

View Article: PubMed Central - PubMed

Affiliation: Infectious Immunology, Institute for Immunology, University of Heidelberg, Heidelberg, Germany.

ABSTRACT
Borrelia recurrentis, the etiologic agent of louse-borne relapsing fever in humans, has evolved strategies, including antigenic variation, to evade immune defence, thereby causing severe diseases with high mortality rates. Here we identify for the first time a multifunctional surface lipoprotein of B. recurrentis, termed HcpA, and demonstrate that it binds human complement regulators, Factor H, CFHR-1, and simultaneously, the host protease plasminogen. Cell surface bound factor H was found to retain its activity and to confer resistance to complement attack. Moreover, ectopic expression of HcpA in a B. burgdorferi B313 strain, deficient in Factor H binding proteins, protected the transformed spirochetes from complement-mediated killing. Furthermore, HcpA-bound plasminogen/plasmin endows B. recurrentis with the potential to resist opsonization and to degrade extracellular matrix components. Together, the present study underscores the high virulence potential of B. recurrentis. The elucidation of the molecular basis underlying the versatile strategies of B. recurrentis to escape innate immunity and to persist in human tissues, including the brain, may help to understand the pathological processes underlying louse-borne relapsing fever.

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Ectopic expression of HcpA in serum-sensitive B. burgdorferi B313.(A) Expression of HcpA by transformed B. burgdorferi B313 was assessed using immunoblot analysis. Whole cell lysates were separated by SDS-PAGE, transferred to nitrocellulose and probed with mAb BR-1 (upper panel) or analyzed for CFH binding by incubation with NHS and a CFH-specific mAb (JHD7, middle panel) followed by peroxidase conjugated IgG. For control, a flagellin-specific antibody (LA21) was used (lower panel). (B) Surface expression of HcpA as analyzed by whole cell ELISA using mAb BR-1. As control, a flagellin-specific mAb LA21 was employed. (C) C3b deposition on the surface of B. burgdorferi B313/pBR cells incubated with 10% NHS was determined using a whole cell ELISA as described above. Values represent the mean of triplicates±SEM. *, P<0.0001. To investigate serum susceptibility to human serum B. recurrentis A1 (D), B.burgdorferi B313 and transformed B. burgdorferi B313/pBR cells (E) were incubated with the indicated concentrations of NHS (dashed line) or heat-inactivated serum (solid line) at 30°C for 3 days. Cells were stained with a nucleic acid dye and the relative growth was determined by measurement of the fluorescence intensities. Values represent the mean±SEM of a single experiment performed in triplicate that is representative of three independent experiments. *, P<0.01; **, P<0.001; ***P<0.0001.
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pone-0004858-g009: Ectopic expression of HcpA in serum-sensitive B. burgdorferi B313.(A) Expression of HcpA by transformed B. burgdorferi B313 was assessed using immunoblot analysis. Whole cell lysates were separated by SDS-PAGE, transferred to nitrocellulose and probed with mAb BR-1 (upper panel) or analyzed for CFH binding by incubation with NHS and a CFH-specific mAb (JHD7, middle panel) followed by peroxidase conjugated IgG. For control, a flagellin-specific antibody (LA21) was used (lower panel). (B) Surface expression of HcpA as analyzed by whole cell ELISA using mAb BR-1. As control, a flagellin-specific mAb LA21 was employed. (C) C3b deposition on the surface of B. burgdorferi B313/pBR cells incubated with 10% NHS was determined using a whole cell ELISA as described above. Values represent the mean of triplicates±SEM. *, P<0.0001. To investigate serum susceptibility to human serum B. recurrentis A1 (D), B.burgdorferi B313 and transformed B. burgdorferi B313/pBR cells (E) were incubated with the indicated concentrations of NHS (dashed line) or heat-inactivated serum (solid line) at 30°C for 3 days. Cells were stained with a nucleic acid dye and the relative growth was determined by measurement of the fluorescence intensities. Values represent the mean±SEM of a single experiment performed in triplicate that is representative of three independent experiments. *, P<0.01; **, P<0.001; ***P<0.0001.

Mentions: To further verify the significance of HcpA of B. recurrentis for complement resistance and removal of C3b from the bacterial surface the serum-sensitive B. burgdorferi B313 strain, lacking CFH and CFHL-1 binding proteins was transformed with the shuttle vector pBR containing the complete hcpA gene (B. burgdorferi B313/pBR). Expression of HcpA was determined by Western blot analysis using an HcpA-specific mAb, BR-1. B. recurrentis A1 and the transformed B313/pBR isolate, but not the CFH and CFHL-1-deficient B. burgdorferi B313 mutant, expressed HcpA (Fig. 9A, upper panel). HcpA expression on the cell surface of transformed B. burgdorferi B313 strains was also detected by whole cell ELISA (Fig. 4B). Moreover, the expressed HcpA protein bound CFH as confirmed by ligand affinity blotting (Fig. 9A, middle panel). In addition, we demonstrate that HcpA expressed on the surface of transformed B. burgdorferi B313/pBR cells promotes degradation of deposited C3b (Fig. 9C). To compare the susceptibility of B. recurrentis A1, B313 and B313/pBR to complement-mediated killing, the three strains were subjected to a human serum sensitivity assay. Accordingly, B. burgdorferi B313/pBR, B. recurrentis A1, and B. burgdorferi B313 were incubated in NHS or heat-inactivated serum for up to three days. Spirochetal growth was monitored by uptake of a nucleic acid dye. As shown in Figure 9D, B. recurrentis A1 readily multiplied during the 72 h time interval in normal human serum, demonstrating the pronounced resistance to human serum of louse-borne relapsing fever spirochetes. Serum-sensitive B. burgdorferi B313 as well as B313 spirochetes containing the shuttle vector alone (data not shown) did not grow under similar conditions, suggesting their susceptibility to complement-mediated lysis (Fig. 9E). In contrast, B313/pBR expressing HcpA survived and multiplied in human serum. B. burgdorferi B313 and the transformed B313/pBR isolate showed similar growth rates when cultured in heat-inactivated human serum. These findings strongly suggest that HcpA is required for resistance of B. recurrentis to complement-mediated killing.


Borrelia recurrentis employs a novel multifunctional surface protein with anti-complement, anti-opsonic and invasive potential to escape innate immunity.

Grosskinsky S, Schott M, Brenner C, Cutler SJ, Kraiczy P, Zipfel PF, Simon MM, Wallich R - PLoS ONE (2009)

Ectopic expression of HcpA in serum-sensitive B. burgdorferi B313.(A) Expression of HcpA by transformed B. burgdorferi B313 was assessed using immunoblot analysis. Whole cell lysates were separated by SDS-PAGE, transferred to nitrocellulose and probed with mAb BR-1 (upper panel) or analyzed for CFH binding by incubation with NHS and a CFH-specific mAb (JHD7, middle panel) followed by peroxidase conjugated IgG. For control, a flagellin-specific antibody (LA21) was used (lower panel). (B) Surface expression of HcpA as analyzed by whole cell ELISA using mAb BR-1. As control, a flagellin-specific mAb LA21 was employed. (C) C3b deposition on the surface of B. burgdorferi B313/pBR cells incubated with 10% NHS was determined using a whole cell ELISA as described above. Values represent the mean of triplicates±SEM. *, P<0.0001. To investigate serum susceptibility to human serum B. recurrentis A1 (D), B.burgdorferi B313 and transformed B. burgdorferi B313/pBR cells (E) were incubated with the indicated concentrations of NHS (dashed line) or heat-inactivated serum (solid line) at 30°C for 3 days. Cells were stained with a nucleic acid dye and the relative growth was determined by measurement of the fluorescence intensities. Values represent the mean±SEM of a single experiment performed in triplicate that is representative of three independent experiments. *, P<0.01; **, P<0.001; ***P<0.0001.
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Related In: Results  -  Collection

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pone-0004858-g009: Ectopic expression of HcpA in serum-sensitive B. burgdorferi B313.(A) Expression of HcpA by transformed B. burgdorferi B313 was assessed using immunoblot analysis. Whole cell lysates were separated by SDS-PAGE, transferred to nitrocellulose and probed with mAb BR-1 (upper panel) or analyzed for CFH binding by incubation with NHS and a CFH-specific mAb (JHD7, middle panel) followed by peroxidase conjugated IgG. For control, a flagellin-specific antibody (LA21) was used (lower panel). (B) Surface expression of HcpA as analyzed by whole cell ELISA using mAb BR-1. As control, a flagellin-specific mAb LA21 was employed. (C) C3b deposition on the surface of B. burgdorferi B313/pBR cells incubated with 10% NHS was determined using a whole cell ELISA as described above. Values represent the mean of triplicates±SEM. *, P<0.0001. To investigate serum susceptibility to human serum B. recurrentis A1 (D), B.burgdorferi B313 and transformed B. burgdorferi B313/pBR cells (E) were incubated with the indicated concentrations of NHS (dashed line) or heat-inactivated serum (solid line) at 30°C for 3 days. Cells were stained with a nucleic acid dye and the relative growth was determined by measurement of the fluorescence intensities. Values represent the mean±SEM of a single experiment performed in triplicate that is representative of three independent experiments. *, P<0.01; **, P<0.001; ***P<0.0001.
Mentions: To further verify the significance of HcpA of B. recurrentis for complement resistance and removal of C3b from the bacterial surface the serum-sensitive B. burgdorferi B313 strain, lacking CFH and CFHL-1 binding proteins was transformed with the shuttle vector pBR containing the complete hcpA gene (B. burgdorferi B313/pBR). Expression of HcpA was determined by Western blot analysis using an HcpA-specific mAb, BR-1. B. recurrentis A1 and the transformed B313/pBR isolate, but not the CFH and CFHL-1-deficient B. burgdorferi B313 mutant, expressed HcpA (Fig. 9A, upper panel). HcpA expression on the cell surface of transformed B. burgdorferi B313 strains was also detected by whole cell ELISA (Fig. 4B). Moreover, the expressed HcpA protein bound CFH as confirmed by ligand affinity blotting (Fig. 9A, middle panel). In addition, we demonstrate that HcpA expressed on the surface of transformed B. burgdorferi B313/pBR cells promotes degradation of deposited C3b (Fig. 9C). To compare the susceptibility of B. recurrentis A1, B313 and B313/pBR to complement-mediated killing, the three strains were subjected to a human serum sensitivity assay. Accordingly, B. burgdorferi B313/pBR, B. recurrentis A1, and B. burgdorferi B313 were incubated in NHS or heat-inactivated serum for up to three days. Spirochetal growth was monitored by uptake of a nucleic acid dye. As shown in Figure 9D, B. recurrentis A1 readily multiplied during the 72 h time interval in normal human serum, demonstrating the pronounced resistance to human serum of louse-borne relapsing fever spirochetes. Serum-sensitive B. burgdorferi B313 as well as B313 spirochetes containing the shuttle vector alone (data not shown) did not grow under similar conditions, suggesting their susceptibility to complement-mediated lysis (Fig. 9E). In contrast, B313/pBR expressing HcpA survived and multiplied in human serum. B. burgdorferi B313 and the transformed B313/pBR isolate showed similar growth rates when cultured in heat-inactivated human serum. These findings strongly suggest that HcpA is required for resistance of B. recurrentis to complement-mediated killing.

Bottom Line: Cell surface bound factor H was found to retain its activity and to confer resistance to complement attack.Furthermore, HcpA-bound plasminogen/plasmin endows B. recurrentis with the potential to resist opsonization and to degrade extracellular matrix components.Together, the present study underscores the high virulence potential of B. recurrentis.

View Article: PubMed Central - PubMed

Affiliation: Infectious Immunology, Institute for Immunology, University of Heidelberg, Heidelberg, Germany.

ABSTRACT
Borrelia recurrentis, the etiologic agent of louse-borne relapsing fever in humans, has evolved strategies, including antigenic variation, to evade immune defence, thereby causing severe diseases with high mortality rates. Here we identify for the first time a multifunctional surface lipoprotein of B. recurrentis, termed HcpA, and demonstrate that it binds human complement regulators, Factor H, CFHR-1, and simultaneously, the host protease plasminogen. Cell surface bound factor H was found to retain its activity and to confer resistance to complement attack. Moreover, ectopic expression of HcpA in a B. burgdorferi B313 strain, deficient in Factor H binding proteins, protected the transformed spirochetes from complement-mediated killing. Furthermore, HcpA-bound plasminogen/plasmin endows B. recurrentis with the potential to resist opsonization and to degrade extracellular matrix components. Together, the present study underscores the high virulence potential of B. recurrentis. The elucidation of the molecular basis underlying the versatile strategies of B. recurrentis to escape innate immunity and to persist in human tissues, including the brain, may help to understand the pathological processes underlying louse-borne relapsing fever.

Show MeSH
Related in: MedlinePlus