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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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Deposition of complement components C3, C6 and C5b-9 on the surface of B. garinii G1 producing CRASP-3 or CRASP-5.Complement components deposited on B. burgdorferi s.s. LW2 (control strain), transformants G1/pKFSS1, G1/pCRASP-3, and G1/pCRASP-5 were detected by indirect immunofluorescence microscopy. Spirochetes were incubated with either 25% normal human serum (NHS) or heat-inactivated NHS (hiNHS). Bound C3, C6, or C5b-9 were detected using specific antibodies against each component plus appropriate Alexa 488-conjugated secondary antibodies. For visualization of intact spirochetes, the DNA-binding dye DAPI was used. Slides were visualized at a magnification of ×1000 and the data were recorded via a DS-5Mc CCD camera (Nikon) mounted on an Olympus CX40 fluorescence microscope. Panels shown are representative of at least 20 microscope fields.
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pone-0013519-g007: Deposition of complement components C3, C6 and C5b-9 on the surface of B. garinii G1 producing CRASP-3 or CRASP-5.Complement components deposited on B. burgdorferi s.s. LW2 (control strain), transformants G1/pKFSS1, G1/pCRASP-3, and G1/pCRASP-5 were detected by indirect immunofluorescence microscopy. Spirochetes were incubated with either 25% normal human serum (NHS) or heat-inactivated NHS (hiNHS). Bound C3, C6, or C5b-9 were detected using specific antibodies against each component plus appropriate Alexa 488-conjugated secondary antibodies. For visualization of intact spirochetes, the DNA-binding dye DAPI was used. Slides were visualized at a magnification of ×1000 and the data were recorded via a DS-5Mc CCD camera (Nikon) mounted on an Olympus CX40 fluorescence microscope. Panels shown are representative of at least 20 microscope fields.

Mentions: Next deposition of complement activation products was analyzed on the bacterial surface. Following incubation in NHS, the two transformed strains G1/pCRASP-3 and G1/pCRASP-5 as well as G1/pKFSS1 and the wild-type strain G1 showed surface deposition of C3, C6 and C5b-9 (Fig. 7). Furthermore, extensive bleb formation and lack of DAPI staining suggests that cells are lysed. In contrast, bacteria incubated with heat-inactivated NHS did not show evidence of complement deposition.


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)

Deposition of complement components C3, C6 and C5b-9 on the surface of B. garinii G1 producing CRASP-3 or CRASP-5.Complement components deposited on B. burgdorferi s.s. LW2 (control strain), transformants G1/pKFSS1, G1/pCRASP-3, and G1/pCRASP-5 were detected by indirect immunofluorescence microscopy. Spirochetes were incubated with either 25% normal human serum (NHS) or heat-inactivated NHS (hiNHS). Bound C3, C6, or C5b-9 were detected using specific antibodies against each component plus appropriate Alexa 488-conjugated secondary antibodies. For visualization of intact spirochetes, the DNA-binding dye DAPI was used. Slides were visualized at a magnification of ×1000 and the data were recorded via a DS-5Mc CCD camera (Nikon) mounted on an Olympus CX40 fluorescence microscope. Panels shown are representative of at least 20 microscope fields.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958145&req=5

pone-0013519-g007: Deposition of complement components C3, C6 and C5b-9 on the surface of B. garinii G1 producing CRASP-3 or CRASP-5.Complement components deposited on B. burgdorferi s.s. LW2 (control strain), transformants G1/pKFSS1, G1/pCRASP-3, and G1/pCRASP-5 were detected by indirect immunofluorescence microscopy. Spirochetes were incubated with either 25% normal human serum (NHS) or heat-inactivated NHS (hiNHS). Bound C3, C6, or C5b-9 were detected using specific antibodies against each component plus appropriate Alexa 488-conjugated secondary antibodies. For visualization of intact spirochetes, the DNA-binding dye DAPI was used. Slides were visualized at a magnification of ×1000 and the data were recorded via a DS-5Mc CCD camera (Nikon) mounted on an Olympus CX40 fluorescence microscope. Panels shown are representative of at least 20 microscope fields.
Mentions: Next deposition of complement activation products was analyzed on the bacterial surface. Following incubation in NHS, the two transformed strains G1/pCRASP-3 and G1/pCRASP-5 as well as G1/pKFSS1 and the wild-type strain G1 showed surface deposition of C3, C6 and C5b-9 (Fig. 7). Furthermore, extensive bleb formation and lack of DAPI staining suggests that cells are lysed. In contrast, bacteria incubated with heat-inactivated NHS did not show evidence of complement deposition.

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