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Borrelia valaisiana resist complement-mediated killing independently of the recruitment of immune regulators and inactivation of complement components.

Schwab J, Hammerschmidt C, Richter D, Skerka C, Matuschka FR, Wallich R, Zipfel PF, Kraiczy P - PLoS ONE (2013)

Bottom Line: Among the investigated isolates, growth of ZWU3 Ny3 was not affected while growth of VS116 and Bv9 was strongly inhibited in the presence of 50% human serum.In contrast, no surface-deposited components and no aberrations in cell morphology were detected for serum-resistant ZWU3 Ny3.Taken together, these findings suggest that certain B. valaisiana isolates differ in their capability to resist complement-mediating killing by human serum.

View Article: PubMed Central - PubMed

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

ABSTRACT
Spirochetes belonging to the Borrelia (B.) burgdorferi sensu lato complex differ in their resistance to complement-mediated killing, particularly in regard to human serum. In the present study, we elucidate the serum and complement susceptibility of B. valaisiana, a genospecies with the potential to cause Lyme disease in Europe as well as in Asia. Among the investigated isolates, growth of ZWU3 Ny3 was not affected while growth of VS116 and Bv9 was strongly inhibited in the presence of 50% human serum. Analyzing complement activation, complement components C3, C4 and C6 were deposited on the surface of isolates VS116 and Bv9, and similarly the membrane attack complex was formed on their surface. In contrast, no surface-deposited components and no aberrations in cell morphology were detected for serum-resistant ZWU3 Ny3. While further investigating the protective role of bound complement regulators in mediating complement resistance, we discovered that none of the B. valaisiana isolates analyzed bound complement regulators Factor H, Factor H-like protein 1, C4b binding protein or C1 esterase inhibitor. In addition, B. valaisiana also lacked intrinsic proteolytic activity to degrade complement components C3, C3b, C4, C4b, and C5. Taken together, these findings suggest that certain B. valaisiana isolates differ in their capability to resist complement-mediating killing by human serum. The molecular mechanism utilized by B. valaisiana to inhibit bacteriolysis appears not to involve binding of the key host complement regulators of the alternative, classical, and lectin pathways as already known for serum-resistant Lyme disease or relapsing fever borreliae.

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Binding of complement regulators to intact spirochetes.(A) Ligand affinity blotting was performed to detect binding of complement regulators. B. burgdorferi LW2, B. garinii G1, B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 as well as B. duttonii LA1 were incubated in NHS-EDTA to prevent complement activation, washed extensively, and bound proteins were eluted using 100 mM glycine-HCl (pH 2.0). Both the last wash (W) and the eluate (E) fractions obtained from each strain were separated by 12.5% Laemmli-SDS-PAGE (detection of CFH, FHL1, FHR1, FHR2, and C1-Inh) or by 10% tris/tricine SDS-PAGE (C4Bp detection) and transferred to nitrocellulose. Membranes were probed either with a polyclonal anti-SCR1-4 antiserum for the detection of both CFH and FHL1, a polyclonal anti-CFHR1 antiserum for the detection of CFHR1 and CFHR2, a polyclonal anti-C4Bp antiserum, or with a polyclonal anti-C1-Inh antiserum. Binding of CFH (B) or C4Bp (C) to spirochetes (5×107 cells) was assessed by ELISA. Bound CFH was detected with a polyclonal goat anti-CFH antiserum and C4Bp was detected with a polyclonal sheep anti-C4Bp antiserum. Data represent means and SD from three separate experiments, each performed at least in triplicate. ***p<0.001; **p<0.01. Raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction.
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pone-0053659-g004: Binding of complement regulators to intact spirochetes.(A) Ligand affinity blotting was performed to detect binding of complement regulators. B. burgdorferi LW2, B. garinii G1, B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 as well as B. duttonii LA1 were incubated in NHS-EDTA to prevent complement activation, washed extensively, and bound proteins were eluted using 100 mM glycine-HCl (pH 2.0). Both the last wash (W) and the eluate (E) fractions obtained from each strain were separated by 12.5% Laemmli-SDS-PAGE (detection of CFH, FHL1, FHR1, FHR2, and C1-Inh) or by 10% tris/tricine SDS-PAGE (C4Bp detection) and transferred to nitrocellulose. Membranes were probed either with a polyclonal anti-SCR1-4 antiserum for the detection of both CFH and FHL1, a polyclonal anti-CFHR1 antiserum for the detection of CFHR1 and CFHR2, a polyclonal anti-C4Bp antiserum, or with a polyclonal anti-C1-Inh antiserum. Binding of CFH (B) or C4Bp (C) to spirochetes (5×107 cells) was assessed by ELISA. Bound CFH was detected with a polyclonal goat anti-CFH antiserum and C4Bp was detected with a polyclonal sheep anti-C4Bp antiserum. Data represent means and SD from three separate experiments, each performed at least in triplicate. ***p<0.001; **p<0.01. Raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction.

Mentions: Serum-resistant B. burgdorferi, B. afzelii, and B. spielmanii bind complement regulators CFH and FHL1 to circumvent complement-mediated lysis [24], [25], [29]. To further examine whether B. valaisiana is able to recruit complement regulators from human serum, borrelial cells were incubated in NHS-EDTA (to avoid complement activation) (Fig. 4). Following extensive washing, bound proteins were eluted from the spirochetal surface using 0.1 M glycine-HCl (pH 2.0). The last wash and the final eluate fraction were collected, separated by SDS-PAGE and subjected to Western blotting using different polyclonal antisera for the detection of complement regulators CFH, FHL1, CFHR1, CFHR2, CFHR5, C4Bp, and C1-Inh. CFH and FHL1, the key regulators of the alternative pathway could be detected in the eluate fraction obtained from serum-resistant control strains B. burgdorferi LW2 and B. duttonii LA1, but not in the appropriate fractions of serum-sensitive B. valaisiana Bv9 and VS116 as well as serum-resistant B. valaisiana ZWU3 Ny3. As expected, serum-sensitive control strain B. garinii G1 did not bind CFH and FHL1 at all. Of note, the faint band above CFH detected represents cross-reacting immunoglobulins derived from the BSK culture medium.


Borrelia valaisiana resist complement-mediated killing independently of the recruitment of immune regulators and inactivation of complement components.

Schwab J, Hammerschmidt C, Richter D, Skerka C, Matuschka FR, Wallich R, Zipfel PF, Kraiczy P - PLoS ONE (2013)

Binding of complement regulators to intact spirochetes.(A) Ligand affinity blotting was performed to detect binding of complement regulators. B. burgdorferi LW2, B. garinii G1, B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 as well as B. duttonii LA1 were incubated in NHS-EDTA to prevent complement activation, washed extensively, and bound proteins were eluted using 100 mM glycine-HCl (pH 2.0). Both the last wash (W) and the eluate (E) fractions obtained from each strain were separated by 12.5% Laemmli-SDS-PAGE (detection of CFH, FHL1, FHR1, FHR2, and C1-Inh) or by 10% tris/tricine SDS-PAGE (C4Bp detection) and transferred to nitrocellulose. Membranes were probed either with a polyclonal anti-SCR1-4 antiserum for the detection of both CFH and FHL1, a polyclonal anti-CFHR1 antiserum for the detection of CFHR1 and CFHR2, a polyclonal anti-C4Bp antiserum, or with a polyclonal anti-C1-Inh antiserum. Binding of CFH (B) or C4Bp (C) to spirochetes (5×107 cells) was assessed by ELISA. Bound CFH was detected with a polyclonal goat anti-CFH antiserum and C4Bp was detected with a polyclonal sheep anti-C4Bp antiserum. Data represent means and SD from three separate experiments, each performed at least in triplicate. ***p<0.001; **p<0.01. Raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3539980&req=5

pone-0053659-g004: Binding of complement regulators to intact spirochetes.(A) Ligand affinity blotting was performed to detect binding of complement regulators. B. burgdorferi LW2, B. garinii G1, B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 as well as B. duttonii LA1 were incubated in NHS-EDTA to prevent complement activation, washed extensively, and bound proteins were eluted using 100 mM glycine-HCl (pH 2.0). Both the last wash (W) and the eluate (E) fractions obtained from each strain were separated by 12.5% Laemmli-SDS-PAGE (detection of CFH, FHL1, FHR1, FHR2, and C1-Inh) or by 10% tris/tricine SDS-PAGE (C4Bp detection) and transferred to nitrocellulose. Membranes were probed either with a polyclonal anti-SCR1-4 antiserum for the detection of both CFH and FHL1, a polyclonal anti-CFHR1 antiserum for the detection of CFHR1 and CFHR2, a polyclonal anti-C4Bp antiserum, or with a polyclonal anti-C1-Inh antiserum. Binding of CFH (B) or C4Bp (C) to spirochetes (5×107 cells) was assessed by ELISA. Bound CFH was detected with a polyclonal goat anti-CFH antiserum and C4Bp was detected with a polyclonal sheep anti-C4Bp antiserum. Data represent means and SD from three separate experiments, each performed at least in triplicate. ***p<0.001; **p<0.01. Raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction.
Mentions: Serum-resistant B. burgdorferi, B. afzelii, and B. spielmanii bind complement regulators CFH and FHL1 to circumvent complement-mediated lysis [24], [25], [29]. To further examine whether B. valaisiana is able to recruit complement regulators from human serum, borrelial cells were incubated in NHS-EDTA (to avoid complement activation) (Fig. 4). Following extensive washing, bound proteins were eluted from the spirochetal surface using 0.1 M glycine-HCl (pH 2.0). The last wash and the final eluate fraction were collected, separated by SDS-PAGE and subjected to Western blotting using different polyclonal antisera for the detection of complement regulators CFH, FHL1, CFHR1, CFHR2, CFHR5, C4Bp, and C1-Inh. CFH and FHL1, the key regulators of the alternative pathway could be detected in the eluate fraction obtained from serum-resistant control strains B. burgdorferi LW2 and B. duttonii LA1, but not in the appropriate fractions of serum-sensitive B. valaisiana Bv9 and VS116 as well as serum-resistant B. valaisiana ZWU3 Ny3. As expected, serum-sensitive control strain B. garinii G1 did not bind CFH and FHL1 at all. Of note, the faint band above CFH detected represents cross-reacting immunoglobulins derived from the BSK culture medium.

Bottom Line: Among the investigated isolates, growth of ZWU3 Ny3 was not affected while growth of VS116 and Bv9 was strongly inhibited in the presence of 50% human serum.In contrast, no surface-deposited components and no aberrations in cell morphology were detected for serum-resistant ZWU3 Ny3.Taken together, these findings suggest that certain B. valaisiana isolates differ in their capability to resist complement-mediating killing by human serum.

View Article: PubMed Central - PubMed

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

ABSTRACT
Spirochetes belonging to the Borrelia (B.) burgdorferi sensu lato complex differ in their resistance to complement-mediated killing, particularly in regard to human serum. In the present study, we elucidate the serum and complement susceptibility of B. valaisiana, a genospecies with the potential to cause Lyme disease in Europe as well as in Asia. Among the investigated isolates, growth of ZWU3 Ny3 was not affected while growth of VS116 and Bv9 was strongly inhibited in the presence of 50% human serum. Analyzing complement activation, complement components C3, C4 and C6 were deposited on the surface of isolates VS116 and Bv9, and similarly the membrane attack complex was formed on their surface. In contrast, no surface-deposited components and no aberrations in cell morphology were detected for serum-resistant ZWU3 Ny3. While further investigating the protective role of bound complement regulators in mediating complement resistance, we discovered that none of the B. valaisiana isolates analyzed bound complement regulators Factor H, Factor H-like protein 1, C4b binding protein or C1 esterase inhibitor. In addition, B. valaisiana also lacked intrinsic proteolytic activity to degrade complement components C3, C3b, C4, C4b, and C5. Taken together, these findings suggest that certain B. valaisiana isolates differ in their capability to resist complement-mediating killing by human serum. The molecular mechanism utilized by B. valaisiana to inhibit bacteriolysis appears not to involve binding of the key host complement regulators of the alternative, classical, and lectin pathways as already known for serum-resistant Lyme disease or relapsing fever borreliae.

Show MeSH
Related in: MedlinePlus