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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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Determination of the C4b proteolytic activity of B. valaisiana.(A) Schematic representation of the α-, β-, and the γ-chain of C4b and the cleavage fragments of the α-chain generated by C4Bp and Factor I. (B) Degradation of C4b by an intrinsic proteolytic activity of borrelial cells (5×108) was analyzed by detection of characteristic cleavage fragments after incubation of spirochetes with (+) or without (−) purified C4Bp. B. burgdorferi LW2, B. garinii G1, B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 were incubated with C4Bp for 60 min at room temperature. After extensive washing with GVB++, C4b (1 µg/ml) and Factor I (1 µg/ml) were added and the mixture was incubated for 120 min at 37°C. Subsequently, the samples were heated to 95°C for 5 min, subjected to 10% tris/tricine SDS-PAGE and transferred onto a nitrocellulose membrane. The C4b cleavage products were visualized by Western blotting using a polyclonal goat anti-human C4 antiserum. As a positive control, purified C4b (1 µg) was incubated with C4Bp and Factor I, and as a negative control complement proteins were incubated in the absence of complement regulator C4Bp. The mobility of the α’-, β’-, and γ’-chain and the α’4 fragment is indicated. (+) incubation with all complement proteins; (−) incubation without C4Bp.
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pone-0053659-g006: Determination of the C4b proteolytic activity of B. valaisiana.(A) Schematic representation of the α-, β-, and the γ-chain of C4b and the cleavage fragments of the α-chain generated by C4Bp and Factor I. (B) Degradation of C4b by an intrinsic proteolytic activity of borrelial cells (5×108) was analyzed by detection of characteristic cleavage fragments after incubation of spirochetes with (+) or without (−) purified C4Bp. B. burgdorferi LW2, B. garinii G1, B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 were incubated with C4Bp for 60 min at room temperature. After extensive washing with GVB++, C4b (1 µg/ml) and Factor I (1 µg/ml) were added and the mixture was incubated for 120 min at 37°C. Subsequently, the samples were heated to 95°C for 5 min, subjected to 10% tris/tricine SDS-PAGE and transferred onto a nitrocellulose membrane. The C4b cleavage products were visualized by Western blotting using a polyclonal goat anti-human C4 antiserum. As a positive control, purified C4b (1 µg) was incubated with C4Bp and Factor I, and as a negative control complement proteins were incubated in the absence of complement regulator C4Bp. The mobility of the α’-, β’-, and γ’-chain and the α’4 fragment is indicated. (+) incubation with all complement proteins; (−) incubation without C4Bp.

Mentions: Although all borrelial isolates examined were unable to bind complement regulator C4Bp, we sought to determine whether spirochetes, in particular B. valaisiana ZWU3 Ny3, are able to cleave C4b in the absence of C4Bp. Thus, cells were incubated with purified C4Bp from human serum, with NHS as a natural source of C4Bp or in GVB++ buffer for 60 min before being extensively washed and subsequently purified C4b and purified Factor I were added. To test for C4b degradation, a polyclonal anti-C4 antibody was used that recognizes the 13 kDa α ´4 fragment, the 75 kDa β-chain as well as the 33 kDa γ-chain (Fig 6A). Cleavage of C4b, as detected by visualization of the characteristic α ´4 fragment, was demonstrated only for the positive control but was absent for the borrelial strains analyzed (Fig. 6B). When incubated with C4b purified from human serum alone, or in the absence of C4Bp, the 83 kDa α ´chain remained intact, using B. garinii G1, B. valaisiana Bv9, VS116, and ZWU3 Ny3 cells (Fig. 6B). In addition, no degradation was found after incubation of these strains with NHS or GVB++ buffer (data not shown). Unexpectedly, a weak signal of the C4b cleavage product corresponding to the α ´4 fragment was detectable upon incubation of serum-resistant B. burgdorferi LW2 with C4b and Factor I.


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)

Determination of the C4b proteolytic activity of B. valaisiana.(A) Schematic representation of the α-, β-, and the γ-chain of C4b and the cleavage fragments of the α-chain generated by C4Bp and Factor I. (B) Degradation of C4b by an intrinsic proteolytic activity of borrelial cells (5×108) was analyzed by detection of characteristic cleavage fragments after incubation of spirochetes with (+) or without (−) purified C4Bp. B. burgdorferi LW2, B. garinii G1, B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 were incubated with C4Bp for 60 min at room temperature. After extensive washing with GVB++, C4b (1 µg/ml) and Factor I (1 µg/ml) were added and the mixture was incubated for 120 min at 37°C. Subsequently, the samples were heated to 95°C for 5 min, subjected to 10% tris/tricine SDS-PAGE and transferred onto a nitrocellulose membrane. The C4b cleavage products were visualized by Western blotting using a polyclonal goat anti-human C4 antiserum. As a positive control, purified C4b (1 µg) was incubated with C4Bp and Factor I, and as a negative control complement proteins were incubated in the absence of complement regulator C4Bp. The mobility of the α’-, β’-, and γ’-chain and the α’4 fragment is indicated. (+) incubation with all complement proteins; (−) incubation without C4Bp.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3539980&req=5

pone-0053659-g006: Determination of the C4b proteolytic activity of B. valaisiana.(A) Schematic representation of the α-, β-, and the γ-chain of C4b and the cleavage fragments of the α-chain generated by C4Bp and Factor I. (B) Degradation of C4b by an intrinsic proteolytic activity of borrelial cells (5×108) was analyzed by detection of characteristic cleavage fragments after incubation of spirochetes with (+) or without (−) purified C4Bp. B. burgdorferi LW2, B. garinii G1, B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 were incubated with C4Bp for 60 min at room temperature. After extensive washing with GVB++, C4b (1 µg/ml) and Factor I (1 µg/ml) were added and the mixture was incubated for 120 min at 37°C. Subsequently, the samples were heated to 95°C for 5 min, subjected to 10% tris/tricine SDS-PAGE and transferred onto a nitrocellulose membrane. The C4b cleavage products were visualized by Western blotting using a polyclonal goat anti-human C4 antiserum. As a positive control, purified C4b (1 µg) was incubated with C4Bp and Factor I, and as a negative control complement proteins were incubated in the absence of complement regulator C4Bp. The mobility of the α’-, β’-, and γ’-chain and the α’4 fragment is indicated. (+) incubation with all complement proteins; (−) incubation without C4Bp.
Mentions: Although all borrelial isolates examined were unable to bind complement regulator C4Bp, we sought to determine whether spirochetes, in particular B. valaisiana ZWU3 Ny3, are able to cleave C4b in the absence of C4Bp. Thus, cells were incubated with purified C4Bp from human serum, with NHS as a natural source of C4Bp or in GVB++ buffer for 60 min before being extensively washed and subsequently purified C4b and purified Factor I were added. To test for C4b degradation, a polyclonal anti-C4 antibody was used that recognizes the 13 kDa α ´4 fragment, the 75 kDa β-chain as well as the 33 kDa γ-chain (Fig 6A). Cleavage of C4b, as detected by visualization of the characteristic α ´4 fragment, was demonstrated only for the positive control but was absent for the borrelial strains analyzed (Fig. 6B). When incubated with C4b purified from human serum alone, or in the absence of C4Bp, the 83 kDa α ´chain remained intact, using B. garinii G1, B. valaisiana Bv9, VS116, and ZWU3 Ny3 cells (Fig. 6B). In addition, no degradation was found after incubation of these strains with NHS or GVB++ buffer (data not shown). Unexpectedly, a weak signal of the C4b cleavage product corresponding to the α ´4 fragment was detectable upon incubation of serum-resistant B. burgdorferi LW2 with C4b and Factor I.

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