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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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Identification of CRASP-3 and CRASP-5 ligands present in human serum.Recombinant, polyhistidine-tagged CRASP-3 or CRASP-5 proteins were immobilized onto magnetic beads and incubated with normal human serum. Empty beads were also incubated under the same conditions and used as a control to identify nonspecific binding of serum proteins. After extensive washing, bound proteins were eluted with 100 mM glycine-HCl and the eluate fractions were separated by Glycine-SDS-PAGE, following silver staining. Protein bands indicated were cut from stained gels and proteins were identified by mass spectrometry. Mobilities of molecular mass standards are indicated to the left.
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pone-0013519-g001: Identification of CRASP-3 and CRASP-5 ligands present in human serum.Recombinant, polyhistidine-tagged CRASP-3 or CRASP-5 proteins were immobilized onto magnetic beads and incubated with normal human serum. Empty beads were also incubated under the same conditions and used as a control to identify nonspecific binding of serum proteins. After extensive washing, bound proteins were eluted with 100 mM glycine-HCl and the eluate fractions were separated by Glycine-SDS-PAGE, following silver staining. Protein bands indicated were cut from stained gels and proteins were identified by mass spectrometry. Mobilities of molecular mass standards are indicated to the left.

Mentions: To elucidate whether CRASP-3 and CRASP-5 bind several human proteins, the recombinant his-tagged CRASP-3 and CRASP-5 proteins were immobilized to magnetic beads. Following incubation with NHS, beads were extensively washed and the recombinant proteins along with bound serum proteins were eluted. Eluates were separated by Glycine-SDS-PAGE and analyzed by silver staining (Fig. 1). In the elute fraction of CRASP-3- and CRASP-5-coupled beads, proteins with apparent molecular mass of 180-, 55-, 37-, 32-, 29- and 24-kDa were detected. Two additional proteins of 25- and 20-kDa were found in the elute fraction of CRASP-3-coupled beads, while an 18-kDa protein was detected only in the elute fraction of CRASP-5-coupled beads. Several proteins in the 60- to 80-kDa range that also attach to uncoated beads were excluded from further analysis. A very faint band of 25-kDa could also be found in the control lane. All eluted proteins were subjected to in-gel trypsin digestion and peptides were analyzed using MALDI-TOF. Obtained spectra were searched against the NCBI.fasta protein database and a score >80 was defined to be significant (p<0.05). The 180-kDa band found in both elution fractions yield a high protein score of >300 of a number of the tryptic peptides representing the complement regulator CFH. Peptides of the 55-kDa protein were identified as fragments of CFHR5 and tryptic peptides of the 37- and 32-kDa protein represented CFHR1β and CFHR1α, the two different glycosylated forms of CFHR1 [39]. The 29- and the 24-kDa band were identified as CFHR2α and CFHR2, respectively. The 25-kDa and 20-kDa bands in the eluate fraction of CRASP-3-coupled beads corresponded to CRASP-3 itself. Likewise, the 18-kDa band in the eluate fraction of CRASP-5-coupled beads was identified as CRASP-5. Thus, CRASP-3 and CRASP-5 bind several members of the human CFH protein family including CFHR1, CFHR2, and CFHR5.


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)

Identification of CRASP-3 and CRASP-5 ligands present in human serum.Recombinant, polyhistidine-tagged CRASP-3 or CRASP-5 proteins were immobilized onto magnetic beads and incubated with normal human serum. Empty beads were also incubated under the same conditions and used as a control to identify nonspecific binding of serum proteins. After extensive washing, bound proteins were eluted with 100 mM glycine-HCl and the eluate fractions were separated by Glycine-SDS-PAGE, following silver staining. Protein bands indicated were cut from stained gels and proteins were identified by mass spectrometry. Mobilities of molecular mass standards are indicated to the left.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0013519-g001: Identification of CRASP-3 and CRASP-5 ligands present in human serum.Recombinant, polyhistidine-tagged CRASP-3 or CRASP-5 proteins were immobilized onto magnetic beads and incubated with normal human serum. Empty beads were also incubated under the same conditions and used as a control to identify nonspecific binding of serum proteins. After extensive washing, bound proteins were eluted with 100 mM glycine-HCl and the eluate fractions were separated by Glycine-SDS-PAGE, following silver staining. Protein bands indicated were cut from stained gels and proteins were identified by mass spectrometry. Mobilities of molecular mass standards are indicated to the left.
Mentions: To elucidate whether CRASP-3 and CRASP-5 bind several human proteins, the recombinant his-tagged CRASP-3 and CRASP-5 proteins were immobilized to magnetic beads. Following incubation with NHS, beads were extensively washed and the recombinant proteins along with bound serum proteins were eluted. Eluates were separated by Glycine-SDS-PAGE and analyzed by silver staining (Fig. 1). In the elute fraction of CRASP-3- and CRASP-5-coupled beads, proteins with apparent molecular mass of 180-, 55-, 37-, 32-, 29- and 24-kDa were detected. Two additional proteins of 25- and 20-kDa were found in the elute fraction of CRASP-3-coupled beads, while an 18-kDa protein was detected only in the elute fraction of CRASP-5-coupled beads. Several proteins in the 60- to 80-kDa range that also attach to uncoated beads were excluded from further analysis. A very faint band of 25-kDa could also be found in the control lane. All eluted proteins were subjected to in-gel trypsin digestion and peptides were analyzed using MALDI-TOF. Obtained spectra were searched against the NCBI.fasta protein database and a score >80 was defined to be significant (p<0.05). The 180-kDa band found in both elution fractions yield a high protein score of >300 of a number of the tryptic peptides representing the complement regulator CFH. Peptides of the 55-kDa protein were identified as fragments of CFHR5 and tryptic peptides of the 37- and 32-kDa protein represented CFHR1β and CFHR1α, the two different glycosylated forms of CFHR1 [39]. The 29- and the 24-kDa band were identified as CFHR2α and CFHR2, respectively. The 25-kDa and 20-kDa bands in the eluate fraction of CRASP-3-coupled beads corresponded to CRASP-3 itself. Likewise, the 18-kDa band in the eluate fraction of CRASP-5-coupled beads was identified as CRASP-5. Thus, CRASP-3 and CRASP-5 bind several members of the human CFH protein family including CFHR1, CFHR2, and CFHR5.

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