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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 susceptibility of transformed B. garinii G1.A growth inhibition assay was used to investigate susceptibility to human serum of B. burgdorferi s.s. strain LW2 (A), and B. garinii strains G1 (B), G1/pKFSS1 (C), G1/pCRASP-3 (D), and G1/pCRASP-5 (E). Spirochetes were incubated in either 50% NHS (filled diamonds) or 50% heat-inactivated NHS (open diamonds) over a cultivation period of 8 days at 33°C, respectively. Color changes were monitored by measurement of the absorbance at 562/630 nm. All experiments were performed three times in which each test was done at least threefold with very similar results. For clarity only data from representative experiments are shown. Error bars represent ± SD.
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pone-0013519-g006: Serum susceptibility of transformed B. garinii G1.A growth inhibition assay was used to investigate susceptibility to human serum of B. burgdorferi s.s. strain LW2 (A), and B. garinii strains G1 (B), G1/pKFSS1 (C), G1/pCRASP-3 (D), and G1/pCRASP-5 (E). Spirochetes were incubated in either 50% NHS (filled diamonds) or 50% heat-inactivated NHS (open diamonds) over a cultivation period of 8 days at 33°C, respectively. Color changes were monitored by measurement of the absorbance at 562/630 nm. All experiments were performed three times in which each test was done at least threefold with very similar results. For clarity only data from representative experiments are shown. Error bars represent ± SD.

Mentions: Having demonstrated binding of CFHRs to intact borrelial cells, the role of CFHR for complement resistance was assayed under more physiological conditions. B. garinii strain G1 is sensitive to complement and does not survive in NHS while wild-type B. burgdorferi LW2 resist complement-mediated killing and survives even in high concentrations of NHS [7], [43]. Therefore, survival and growth of CRASP-3 and CRASP-5 expressing spirochetes in NHS was assayed. Neither of the CRASP-3 or CRASP-5 expressing transformants grew in the presence of NHS (Fig. 6D and E) suggesting that binding of CFHR1, CFHR2, and CFHR5 alone is not sufficient for complement resistance. The serum-resistant strain LW2 grew equally well in medium supplemented with NHS or heat-inactivated NHS (Fig. 6A) while both isolates G1 and G1/pKFSS1 survived only when heat-inactivated NHS was used (Fig. 6B and C).


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 susceptibility of transformed B. garinii G1.A growth inhibition assay was used to investigate susceptibility to human serum of B. burgdorferi s.s. strain LW2 (A), and B. garinii strains G1 (B), G1/pKFSS1 (C), G1/pCRASP-3 (D), and G1/pCRASP-5 (E). Spirochetes were incubated in either 50% NHS (filled diamonds) or 50% heat-inactivated NHS (open diamonds) over a cultivation period of 8 days at 33°C, respectively. Color changes were monitored by measurement of the absorbance at 562/630 nm. All experiments were performed three times in which each test was done at least threefold with very similar results. For clarity only data from representative experiments are shown. Error bars represent ± SD.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958145&req=5

pone-0013519-g006: Serum susceptibility of transformed B. garinii G1.A growth inhibition assay was used to investigate susceptibility to human serum of B. burgdorferi s.s. strain LW2 (A), and B. garinii strains G1 (B), G1/pKFSS1 (C), G1/pCRASP-3 (D), and G1/pCRASP-5 (E). Spirochetes were incubated in either 50% NHS (filled diamonds) or 50% heat-inactivated NHS (open diamonds) over a cultivation period of 8 days at 33°C, respectively. Color changes were monitored by measurement of the absorbance at 562/630 nm. All experiments were performed three times in which each test was done at least threefold with very similar results. For clarity only data from representative experiments are shown. Error bars represent ± SD.
Mentions: Having demonstrated binding of CFHRs to intact borrelial cells, the role of CFHR for complement resistance was assayed under more physiological conditions. B. garinii strain G1 is sensitive to complement and does not survive in NHS while wild-type B. burgdorferi LW2 resist complement-mediated killing and survives even in high concentrations of NHS [7], [43]. Therefore, survival and growth of CRASP-3 and CRASP-5 expressing spirochetes in NHS was assayed. Neither of the CRASP-3 or CRASP-5 expressing transformants grew in the presence of NHS (Fig. 6D and E) suggesting that binding of CFHR1, CFHR2, and CFHR5 alone is not sufficient for complement resistance. The serum-resistant strain LW2 grew equally well in medium supplemented with NHS or heat-inactivated NHS (Fig. 6A) while both isolates G1 and G1/pKFSS1 survived only when heat-inactivated NHS was used (Fig. 6B and C).

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