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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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Binding of serum molecules by B. garinii transformants.B. garinii strains G1, G1/pKFSS1, G1/pCRASP-3, and G1/pCRASP-5 were incubated in NHS plus EDTA to prevent complement activation, washed extensively, and then bound proteins were eluted using 0.1 M glycine (pH 2.0). Both the last wash (w) and the eluate (e) fractions obtained from each strain were separated by Glycine-SDS-PAGE and transferred to nitrocellulose. Membranes were probed with either (A) MAb VIG8, which recognizes the C-terminus of CFH and CFHR proteins, or (B) mAb JHD 7.10, which recognizes CFHR1 and CFHR2, but not CFH. Probable identities of protein bands (confirmed by data shown in Fig. 1) are indicated to the right of each panel. Mobilities of molecular mass standards are shown to the left of the panels.
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pone-0013519-g004: Binding of serum molecules by B. garinii transformants.B. garinii strains G1, G1/pKFSS1, G1/pCRASP-3, and G1/pCRASP-5 were incubated in NHS plus EDTA to prevent complement activation, washed extensively, and then bound proteins were eluted using 0.1 M glycine (pH 2.0). Both the last wash (w) and the eluate (e) fractions obtained from each strain were separated by Glycine-SDS-PAGE and transferred to nitrocellulose. Membranes were probed with either (A) MAb VIG8, which recognizes the C-terminus of CFH and CFHR proteins, or (B) mAb JHD 7.10, which recognizes CFHR1 and CFHR2, but not CFH. Probable identities of protein bands (confirmed by data shown in Fig. 1) are indicated to the right of each panel. Mobilities of molecular mass standards are shown to the left of the panels.

Mentions: We next examined whether transformed strains G1/pCRASP-3 and G1/pCRASP-5 bind human complement regulators. Spirochetes incubated in EDTA-treated NHS were washed extensively and bound proteins were eluted. The final wash and the elute fraction were separated by Glycine-SDS-PAGE and after transfer to nitrocellulose, presence of CFH was analyzed by immunoblot with a specific mAb. CRASP-3 and CRASP-5 expressing transformants bound low amounts of CFH (Fig. 4A). In addition, four prominent bands with an mobility or apparent mass of 43-, 37-, 29- and 24-kDa were present in elute fractions of G1/pCRASP-3 and G1/pCRASP-5. In contrast, wild-type strain G1 and transformant G1/pKFSS1 did not bind CFH and CFH-related proteins. Based on the reactivity with the mAb VIG8 which reacts with CFH, CFHR1 and CFHR2 [41] and based on the mobility, the 43- and 37-kDa proteins correspond the two glycosylated forms CFHR1α and CFHR1β. Similarly, the 29- and 24-kDa bands represent the non-glycosylated and the glycosylated form of CFHR2.


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)

Binding of serum molecules by B. garinii transformants.B. garinii strains G1, G1/pKFSS1, G1/pCRASP-3, and G1/pCRASP-5 were incubated in NHS plus EDTA to prevent complement activation, washed extensively, and then bound proteins were eluted using 0.1 M glycine (pH 2.0). Both the last wash (w) and the eluate (e) fractions obtained from each strain were separated by Glycine-SDS-PAGE and transferred to nitrocellulose. Membranes were probed with either (A) MAb VIG8, which recognizes the C-terminus of CFH and CFHR proteins, or (B) mAb JHD 7.10, which recognizes CFHR1 and CFHR2, but not CFH. Probable identities of protein bands (confirmed by data shown in Fig. 1) are indicated to the right of each panel. Mobilities of molecular mass standards are shown to the left of the panels.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2958145&req=5

pone-0013519-g004: Binding of serum molecules by B. garinii transformants.B. garinii strains G1, G1/pKFSS1, G1/pCRASP-3, and G1/pCRASP-5 were incubated in NHS plus EDTA to prevent complement activation, washed extensively, and then bound proteins were eluted using 0.1 M glycine (pH 2.0). Both the last wash (w) and the eluate (e) fractions obtained from each strain were separated by Glycine-SDS-PAGE and transferred to nitrocellulose. Membranes were probed with either (A) MAb VIG8, which recognizes the C-terminus of CFH and CFHR proteins, or (B) mAb JHD 7.10, which recognizes CFHR1 and CFHR2, but not CFH. Probable identities of protein bands (confirmed by data shown in Fig. 1) are indicated to the right of each panel. Mobilities of molecular mass standards are shown to the left of the panels.
Mentions: We next examined whether transformed strains G1/pCRASP-3 and G1/pCRASP-5 bind human complement regulators. Spirochetes incubated in EDTA-treated NHS were washed extensively and bound proteins were eluted. The final wash and the elute fraction were separated by Glycine-SDS-PAGE and after transfer to nitrocellulose, presence of CFH was analyzed by immunoblot with a specific mAb. CRASP-3 and CRASP-5 expressing transformants bound low amounts of CFH (Fig. 4A). In addition, four prominent bands with an mobility or apparent mass of 43-, 37-, 29- and 24-kDa were present in elute fractions of G1/pCRASP-3 and G1/pCRASP-5. In contrast, wild-type strain G1 and transformant G1/pKFSS1 did not bind CFH and CFH-related proteins. Based on the reactivity with the mAb VIG8 which reacts with CFH, CFHR1 and CFHR2 [41] and based on the mobility, the 43- and 37-kDa proteins correspond the two glycosylated forms CFHR1α and CFHR1β. Similarly, the 29- and 24-kDa bands represent the non-glycosylated and the glycosylated form of CFHR2.

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