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Complement factor H-related proteins CFHR2 and CFHR5 represent novel ligands for the infection-associated CRASP proteins of Borrelia burgdorferi.

Siegel C, Hallström T, Skerka C, Eberhardt H, Uzonyi B, Beckhaus T, Karas M, Wallich R, Stevenson B, Zipfel PF, Kraiczy P - PLoS ONE (2010)

Bottom Line: In the present study we elucidate the role of the infection-associated CRASP-3 and CRASP-5 protein to serve as ligands for additional complement regulatory proteins as well as for complement resistance of B. burgdorferi.Both CRASPs expressed on the B. garinii outer surface bound CFH as well as CFHR1 and CFHR2 in ELISA.In the absence of CFH and the presence of CFHR1, CFHR2 and CFHR5, assembly and integration of the membrane attack complex was not efficiently inhibited indicating that CFH in co-operation with CFHR1, CFHR2 and CFHR5 supports complement evasion of B. burgdorferi.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology and Infection Control, University Hospital of Frankfurt, Frankfurt/Main, Germany.

ABSTRACT

Background: One virulence property of Borrelia burgdorferi is its resistance to innate immunity, in particular to complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASP) which interact with complement regulator factor H (CFH) and factor H-like protein 1 (FHL1) or factor H-related protein 1 (CFHR1). In the present study we elucidate the role of the infection-associated CRASP-3 and CRASP-5 protein to serve as ligands for additional complement regulatory proteins as well as for complement resistance of B. burgdorferi.

Methodology/principal findings: To elucidate whether CRASP-5 and CRASP-3 interact with various human proteins, both borrelial proteins were immobilized on magnetic beads. Following incubation with human serum, bound proteins were eluted and separated by Glycine-SDS-PAGE. In addition to CFH and CFHR1, complement regulators CFHR2 and CFHR5 were identified as novel ligands for both borrelial proteins by employing MALDI-TOF. To further assess the contributions of CRASP-3 and CRASP-5 to complement resistance, a serum-sensitive B. garinii strain G1 which lacks all CFH-binding proteins was used as a valuable model for functional analyses. Both CRASPs expressed on the B. garinii outer surface bound CFH as well as CFHR1 and CFHR2 in ELISA. In contrast, live B. garinii bound CFHR1, CFHR2, and CFHR5 and only miniscute amounts of CFH as demonstrated by serum adsorption assays and FACS analyses. Further functional analysis revealed that upon NHS incubation, CRASP-3 or CRASP-5 expressing borreliae were killed by complement.

Conclusions/significance: In the absence of CFH and the presence of CFHR1, CFHR2 and CFHR5, assembly and integration of the membrane attack complex was not efficiently inhibited indicating that CFH in co-operation with CFHR1, CFHR2 and CFHR5 supports complement evasion of B. burgdorferi.

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Serum proteins display differential binding capabilities to CRASP-3 and CRASP-5.(A) The binding capabilites of serum proteins to B. garinii strains G1/pKFSS1, G1/pCRASP-3, and G1/pCRASP-5 were assessed in the presence of increasing salt concentrations. Spirochetes were incubated in NHS plus EDTA, washed fourfold with PBSA containing 0.05% Tween20. Cells were then resuspended in PBSA containing 450 mM NaCl, incubated for 15 min at room temperature, and sedimented by centrifugation. The steps were repeated with increasing concentrations of NaCl. Strong binding proteins were finally eluted using 0.1 M glycine buffer (e). The supernatants obtained from the last wash fraction (150 mM NaCl), fractions from the incubation reactions (450, 750, 1050, 1350 mM NaCl), and the eluate fraction were then separated by Glycine-SDS-PAGE and transferred to nitrocellulose. Membranes were probed with polyclonal anti-CFHR1 antiserum to detect CFH and CFHR proteins. Mobilities of molecular mass standards are shown to the left of the panels. (B) The binding capability of CFH to G1/pCRASP-3 and G1/pCRASP-5 was further analyzed by flow cytometry. The binding of CFH to G1/pCRASP-3 and G1/pCRASP-5 is shown by the solid line while the grey shaded histogramm represents the binding of control strain G1/pKFSS1 (control). Borrelial cells were incubated with 4 µg CFH. The x-acis shows the fluorescence on a log10 scale and the the y-acis represents the numbers of events. The isotype control (no CFH added) has been omitted for easier visualization.
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pone-0013519-g005: Serum proteins display differential binding capabilities to CRASP-3 and CRASP-5.(A) The binding capabilites of serum proteins to B. garinii strains G1/pKFSS1, G1/pCRASP-3, and G1/pCRASP-5 were assessed in the presence of increasing salt concentrations. Spirochetes were incubated in NHS plus EDTA, washed fourfold with PBSA containing 0.05% Tween20. Cells were then resuspended in PBSA containing 450 mM NaCl, incubated for 15 min at room temperature, and sedimented by centrifugation. The steps were repeated with increasing concentrations of NaCl. Strong binding proteins were finally eluted using 0.1 M glycine buffer (e). The supernatants obtained from the last wash fraction (150 mM NaCl), fractions from the incubation reactions (450, 750, 1050, 1350 mM NaCl), and the eluate fraction were then separated by Glycine-SDS-PAGE and transferred to nitrocellulose. Membranes were probed with polyclonal anti-CFHR1 antiserum to detect CFH and CFHR proteins. Mobilities of molecular mass standards are shown to the left of the panels. (B) The binding capability of CFH to G1/pCRASP-3 and G1/pCRASP-5 was further analyzed by flow cytometry. The binding of CFH to G1/pCRASP-3 and G1/pCRASP-5 is shown by the solid line while the grey shaded histogramm represents the binding of control strain G1/pKFSS1 (control). Borrelial cells were incubated with 4 µg CFH. The x-acis shows the fluorescence on a log10 scale and the the y-acis represents the numbers of events. The isotype control (no CFH added) has been omitted for easier visualization.

Mentions: To define the effect of NaCl on the interaction of the human serum proteins to CRASP expressed on transformed borrelial strains G1/pCRASP-3, G1/pCRASP-5 and G1/pKFSS1 was assayed. To this end spirochetes were washed extensively with a buffer containing 150 mM NaCl and cell-bound proteins were subsequently eluted with increasing concentrations of salt (450 mM to 1350 mM). The elute fractions were then separated by Glycine-SDS-PAGE and after transfer to nitrocellulose, human serum proteins were detected with a polyclonal anti-CFHR1 antiserum (Fig. 5A). Concerning G1/pCRASP-3, neither CFH nor CFHR2 and CFHR5 could be detected in the fractions containing increasing salt concentrations (Fig. 5A, middle panel). In contrast, CFHR1 and CFHR2 as well as their glycosylated forms were readily detectable in the final glycine fraction suggesting that these serum proteins possess a stronger binding capacity to surface-exposed CRASP-3. As depicted in Figure 5A (right panel), the faint band of 150-kDa corresponding to CFH detected after incubation of G1/pCRASP-5 with up to 450 mM NaCl and which signal decreased at higher salt concentrations, suggests that CFH is relatively loosely attached to the borrelial surface. The 150-kDa band was also hardly visible in the respective fractions of the control strain G1/pKFSS1, thus argueing for a non-specific binding of CFH to the borrelial surface. In contrast, CFHR1 was detectable in all fractions of transformants expressing CRASP-3 or CRASP-5 even in the highest concentration of NaCl as well as in the final glycine fraction. CFHR2 was only detected in the final glycine fraction, suggesting a stronger binding capacity of CFHR2 to CRASP-3 and CRASP-5. However, CFHR5 was not detected by this assay. No CFHRs proteins were identified in the control transformed strain G1/pKFSS1 (Fig. 5A, left panel).


Complement factor H-related proteins CFHR2 and CFHR5 represent novel ligands for the infection-associated CRASP proteins of Borrelia burgdorferi.

Siegel C, Hallström T, Skerka C, Eberhardt H, Uzonyi B, Beckhaus T, Karas M, Wallich R, Stevenson B, Zipfel PF, Kraiczy P - PLoS ONE (2010)

Serum proteins display differential binding capabilities to CRASP-3 and CRASP-5.(A) The binding capabilites of serum proteins to B. garinii strains G1/pKFSS1, G1/pCRASP-3, and G1/pCRASP-5 were assessed in the presence of increasing salt concentrations. Spirochetes were incubated in NHS plus EDTA, washed fourfold with PBSA containing 0.05% Tween20. Cells were then resuspended in PBSA containing 450 mM NaCl, incubated for 15 min at room temperature, and sedimented by centrifugation. The steps were repeated with increasing concentrations of NaCl. Strong binding proteins were finally eluted using 0.1 M glycine buffer (e). The supernatants obtained from the last wash fraction (150 mM NaCl), fractions from the incubation reactions (450, 750, 1050, 1350 mM NaCl), and the eluate fraction were then separated by Glycine-SDS-PAGE and transferred to nitrocellulose. Membranes were probed with polyclonal anti-CFHR1 antiserum to detect CFH and CFHR proteins. Mobilities of molecular mass standards are shown to the left of the panels. (B) The binding capability of CFH to G1/pCRASP-3 and G1/pCRASP-5 was further analyzed by flow cytometry. The binding of CFH to G1/pCRASP-3 and G1/pCRASP-5 is shown by the solid line while the grey shaded histogramm represents the binding of control strain G1/pKFSS1 (control). Borrelial cells were incubated with 4 µg CFH. The x-acis shows the fluorescence on a log10 scale and the the y-acis represents the numbers of events. The isotype control (no CFH added) has been omitted for easier visualization.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958145&req=5

pone-0013519-g005: Serum proteins display differential binding capabilities to CRASP-3 and CRASP-5.(A) The binding capabilites of serum proteins to B. garinii strains G1/pKFSS1, G1/pCRASP-3, and G1/pCRASP-5 were assessed in the presence of increasing salt concentrations. Spirochetes were incubated in NHS plus EDTA, washed fourfold with PBSA containing 0.05% Tween20. Cells were then resuspended in PBSA containing 450 mM NaCl, incubated for 15 min at room temperature, and sedimented by centrifugation. The steps were repeated with increasing concentrations of NaCl. Strong binding proteins were finally eluted using 0.1 M glycine buffer (e). The supernatants obtained from the last wash fraction (150 mM NaCl), fractions from the incubation reactions (450, 750, 1050, 1350 mM NaCl), and the eluate fraction were then separated by Glycine-SDS-PAGE and transferred to nitrocellulose. Membranes were probed with polyclonal anti-CFHR1 antiserum to detect CFH and CFHR proteins. Mobilities of molecular mass standards are shown to the left of the panels. (B) The binding capability of CFH to G1/pCRASP-3 and G1/pCRASP-5 was further analyzed by flow cytometry. The binding of CFH to G1/pCRASP-3 and G1/pCRASP-5 is shown by the solid line while the grey shaded histogramm represents the binding of control strain G1/pKFSS1 (control). Borrelial cells were incubated with 4 µg CFH. The x-acis shows the fluorescence on a log10 scale and the the y-acis represents the numbers of events. The isotype control (no CFH added) has been omitted for easier visualization.
Mentions: To define the effect of NaCl on the interaction of the human serum proteins to CRASP expressed on transformed borrelial strains G1/pCRASP-3, G1/pCRASP-5 and G1/pKFSS1 was assayed. To this end spirochetes were washed extensively with a buffer containing 150 mM NaCl and cell-bound proteins were subsequently eluted with increasing concentrations of salt (450 mM to 1350 mM). The elute fractions were then separated by Glycine-SDS-PAGE and after transfer to nitrocellulose, human serum proteins were detected with a polyclonal anti-CFHR1 antiserum (Fig. 5A). Concerning G1/pCRASP-3, neither CFH nor CFHR2 and CFHR5 could be detected in the fractions containing increasing salt concentrations (Fig. 5A, middle panel). In contrast, CFHR1 and CFHR2 as well as their glycosylated forms were readily detectable in the final glycine fraction suggesting that these serum proteins possess a stronger binding capacity to surface-exposed CRASP-3. As depicted in Figure 5A (right panel), the faint band of 150-kDa corresponding to CFH detected after incubation of G1/pCRASP-5 with up to 450 mM NaCl and which signal decreased at higher salt concentrations, suggests that CFH is relatively loosely attached to the borrelial surface. The 150-kDa band was also hardly visible in the respective fractions of the control strain G1/pKFSS1, thus argueing for a non-specific binding of CFH to the borrelial surface. In contrast, CFHR1 was detectable in all fractions of transformants expressing CRASP-3 or CRASP-5 even in the highest concentration of NaCl as well as in the final glycine fraction. CFHR2 was only detected in the final glycine fraction, suggesting a stronger binding capacity of CFHR2 to CRASP-3 and CRASP-5. However, CFHR5 was not detected by this assay. No CFHRs proteins were identified in the control transformed strain G1/pKFSS1 (Fig. 5A, left panel).

Bottom Line: In the present study we elucidate the role of the infection-associated CRASP-3 and CRASP-5 protein to serve as ligands for additional complement regulatory proteins as well as for complement resistance of B. burgdorferi.Both CRASPs expressed on the B. garinii outer surface bound CFH as well as CFHR1 and CFHR2 in ELISA.In the absence of CFH and the presence of CFHR1, CFHR2 and CFHR5, assembly and integration of the membrane attack complex was not efficiently inhibited indicating that CFH in co-operation with CFHR1, CFHR2 and CFHR5 supports complement evasion of B. burgdorferi.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology and Infection Control, University Hospital of Frankfurt, Frankfurt/Main, Germany.

ABSTRACT

Background: One virulence property of Borrelia burgdorferi is its resistance to innate immunity, in particular to complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASP) which interact with complement regulator factor H (CFH) and factor H-like protein 1 (FHL1) or factor H-related protein 1 (CFHR1). In the present study we elucidate the role of the infection-associated CRASP-3 and CRASP-5 protein to serve as ligands for additional complement regulatory proteins as well as for complement resistance of B. burgdorferi.

Methodology/principal findings: To elucidate whether CRASP-5 and CRASP-3 interact with various human proteins, both borrelial proteins were immobilized on magnetic beads. Following incubation with human serum, bound proteins were eluted and separated by Glycine-SDS-PAGE. In addition to CFH and CFHR1, complement regulators CFHR2 and CFHR5 were identified as novel ligands for both borrelial proteins by employing MALDI-TOF. To further assess the contributions of CRASP-3 and CRASP-5 to complement resistance, a serum-sensitive B. garinii strain G1 which lacks all CFH-binding proteins was used as a valuable model for functional analyses. Both CRASPs expressed on the B. garinii outer surface bound CFH as well as CFHR1 and CFHR2 in ELISA. In contrast, live B. garinii bound CFHR1, CFHR2, and CFHR5 and only miniscute amounts of CFH as demonstrated by serum adsorption assays and FACS analyses. Further functional analysis revealed that upon NHS incubation, CRASP-3 or CRASP-5 expressing borreliae were killed by complement.

Conclusions/significance: In the absence of CFH and the presence of CFHR1, CFHR2 and CFHR5, assembly and integration of the membrane attack complex was not efficiently inhibited indicating that CFH in co-operation with CFHR1, CFHR2 and CFHR5 supports complement evasion of B. burgdorferi.

Show MeSH
Related in: MedlinePlus