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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: 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.

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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Binding of CFH, CFHR-1 and PLG to HcpA.(A) Purified recombinant HcpA protein and various deletion mutants were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were incubated with a monoclonal anti-His-tag mAb (upper panel). CFH and CFHR-1 binding capabilities were analyzed by ligand affinity blotting utilizing normal human serum and goat anti-CFH immune serum (middle panel) or the CFHR-1 specific mAb JHD8 (lower panel). (B) Microtiter plates were coated with full-length recombinant HcpA and the indicated deletion mutants, respectively, and incubated with CFH, normal human serum (as source for CFHR-1) or PLG. Binding was detected using goat anti-CFH, the CFHR-1 specific mAb JHD8 or goat anti-PLG immune serum followed by the respective peroxidase-conjugated IgGs. (C) Diagrammatic representation of native and expressed recombinant HcpA proteins and their binding characteristics for serum proteins CFH, CFHR-1 and PLG as determined by ligand affinity blot analysis and ELISA. Numbers refer to amino acid residues. (D) Dose dependent binding of CFH and PLG by HcpA. A competition inhibition assay was performed, adding different amounts of PLG or CFH to inhibit the binding of 2 µg/ml CFH (left panel) or 10 µg/ml PLG (right panel) to immobilized HcpA. Bound CFH and PLG was detected using goat anti-CFH (dashed line) and goat anti-PLG Ab (solid line), respectively, followed by peroxidase-conjugated secondary antibody.
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pone-0004858-g003: Binding of CFH, CFHR-1 and PLG to HcpA.(A) Purified recombinant HcpA protein and various deletion mutants were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were incubated with a monoclonal anti-His-tag mAb (upper panel). CFH and CFHR-1 binding capabilities were analyzed by ligand affinity blotting utilizing normal human serum and goat anti-CFH immune serum (middle panel) or the CFHR-1 specific mAb JHD8 (lower panel). (B) Microtiter plates were coated with full-length recombinant HcpA and the indicated deletion mutants, respectively, and incubated with CFH, normal human serum (as source for CFHR-1) or PLG. Binding was detected using goat anti-CFH, the CFHR-1 specific mAb JHD8 or goat anti-PLG immune serum followed by the respective peroxidase-conjugated IgGs. (C) Diagrammatic representation of native and expressed recombinant HcpA proteins and their binding characteristics for serum proteins CFH, CFHR-1 and PLG as determined by ligand affinity blot analysis and ELISA. Numbers refer to amino acid residues. (D) Dose dependent binding of CFH and PLG by HcpA. A competition inhibition assay was performed, adding different amounts of PLG or CFH to inhibit the binding of 2 µg/ml CFH (left panel) or 10 µg/ml PLG (right panel) to immobilized HcpA. Bound CFH and PLG was detected using goat anti-CFH (dashed line) and goat anti-PLG Ab (solid line), respectively, followed by peroxidase-conjugated secondary antibody.

Mentions: To isolate and further characterize HcpA, whole cell lysates of B. recurrentis A1 were incubated with CFH and subsequently treated with a goat anti-CFH immune serum. A resulting co-precipitated protein of 17 kDa was analyzed by mass spectrometry and peptides generated matched an open reading frame of 525 bp on the genome of B. recurrentis A1 (unpublished data), designated hcpA. Due to the presence of a spirochetal lipobox at its N-terminus HcpA represents a putative outer surface lipoprotein [33]. The deduced amino acid sequence exhibits 54% similarity with the recently identified BhCRASP-1 of B. hermsii HS1 (Fig. 2) [8]. A BLAST search detected another protein with significant homology in the genome of B. turicatae, indicating that this protein is to be found in other Borrelia species. To further elucidate the binding properties of HcpA for complement regulators CFH and CFHR-1, various N- and C-terminal deletion mutants of HcpA were generated. Variants of the encoding hcpA gene lacking the hydrophobic leader peptide and the indicated N- or C-terminal regions were cloned and expressed as His-tagged fusion proteins in E. coli (Fig. 3). Expression of each protein was confirmed by immunoblot analysis using anti-His antiserum (Fig. 3A).


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)

Binding of CFH, CFHR-1 and PLG to HcpA.(A) Purified recombinant HcpA protein and various deletion mutants were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were incubated with a monoclonal anti-His-tag mAb (upper panel). CFH and CFHR-1 binding capabilities were analyzed by ligand affinity blotting utilizing normal human serum and goat anti-CFH immune serum (middle panel) or the CFHR-1 specific mAb JHD8 (lower panel). (B) Microtiter plates were coated with full-length recombinant HcpA and the indicated deletion mutants, respectively, and incubated with CFH, normal human serum (as source for CFHR-1) or PLG. Binding was detected using goat anti-CFH, the CFHR-1 specific mAb JHD8 or goat anti-PLG immune serum followed by the respective peroxidase-conjugated IgGs. (C) Diagrammatic representation of native and expressed recombinant HcpA proteins and their binding characteristics for serum proteins CFH, CFHR-1 and PLG as determined by ligand affinity blot analysis and ELISA. Numbers refer to amino acid residues. (D) Dose dependent binding of CFH and PLG by HcpA. A competition inhibition assay was performed, adding different amounts of PLG or CFH to inhibit the binding of 2 µg/ml CFH (left panel) or 10 µg/ml PLG (right panel) to immobilized HcpA. Bound CFH and PLG was detected using goat anti-CFH (dashed line) and goat anti-PLG Ab (solid line), respectively, followed by peroxidase-conjugated secondary antibody.
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getmorefigures.php?uid=PMC2654920&req=5

pone-0004858-g003: Binding of CFH, CFHR-1 and PLG to HcpA.(A) Purified recombinant HcpA protein and various deletion mutants were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were incubated with a monoclonal anti-His-tag mAb (upper panel). CFH and CFHR-1 binding capabilities were analyzed by ligand affinity blotting utilizing normal human serum and goat anti-CFH immune serum (middle panel) or the CFHR-1 specific mAb JHD8 (lower panel). (B) Microtiter plates were coated with full-length recombinant HcpA and the indicated deletion mutants, respectively, and incubated with CFH, normal human serum (as source for CFHR-1) or PLG. Binding was detected using goat anti-CFH, the CFHR-1 specific mAb JHD8 or goat anti-PLG immune serum followed by the respective peroxidase-conjugated IgGs. (C) Diagrammatic representation of native and expressed recombinant HcpA proteins and their binding characteristics for serum proteins CFH, CFHR-1 and PLG as determined by ligand affinity blot analysis and ELISA. Numbers refer to amino acid residues. (D) Dose dependent binding of CFH and PLG by HcpA. A competition inhibition assay was performed, adding different amounts of PLG or CFH to inhibit the binding of 2 µg/ml CFH (left panel) or 10 µg/ml PLG (right panel) to immobilized HcpA. Bound CFH and PLG was detected using goat anti-CFH (dashed line) and goat anti-PLG Ab (solid line), respectively, followed by peroxidase-conjugated secondary antibody.
Mentions: To isolate and further characterize HcpA, whole cell lysates of B. recurrentis A1 were incubated with CFH and subsequently treated with a goat anti-CFH immune serum. A resulting co-precipitated protein of 17 kDa was analyzed by mass spectrometry and peptides generated matched an open reading frame of 525 bp on the genome of B. recurrentis A1 (unpublished data), designated hcpA. Due to the presence of a spirochetal lipobox at its N-terminus HcpA represents a putative outer surface lipoprotein [33]. The deduced amino acid sequence exhibits 54% similarity with the recently identified BhCRASP-1 of B. hermsii HS1 (Fig. 2) [8]. A BLAST search detected another protein with significant homology in the genome of B. turicatae, indicating that this protein is to be found in other Borrelia species. To further elucidate the binding properties of HcpA for complement regulators CFH and CFHR-1, various N- and C-terminal deletion mutants of HcpA were generated. Variants of the encoding hcpA gene lacking the hydrophobic leader peptide and the indicated N- or C-terminal regions were cloned and expressed as His-tagged fusion proteins in E. coli (Fig. 3). Expression of each protein was confirmed by immunoblot analysis using anti-His antiserum (Fig. 3A).

Bottom Line: 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.

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