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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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Identification of CRASPs among B. valaisiana.Cell lysates (30 µg each) obtained from control strains B. burgdorferi LW2 and B. garinii G1 as well as from B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 were subjected to 10% tris/tricine SDS-PAGE and transferred to nitrocellulose. The membranes were then incubated with either NHS as source for CFH and CFHR1 or with cell culture supernatant containing recombinant FHL1. Potential CFH/FHL1/CFHR1-binding proteins were detected using a polyclonal anti-CFH serum, a polyclonal anti-CFHR1 serum or a polyclonal anti-SCR1-4 antiserum. Monoclonal antibody L41 1C11 recognizing the FlaB protein was used to show equal loading of bacterial lysates. The identified CRASP proteins, CspA, CspZ, ErpP, and ErpA of B. burgdorferi LW2, the CFH-binding protein of B. valaisiana Bv9 and VS116, and the CFHR1-binding protein of B. valaisiana Bv9 are indicated on the right and the mobility of the marker proteins is indicated on the left.
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pone-0053659-g003: Identification of CRASPs among B. valaisiana.Cell lysates (30 µg each) obtained from control strains B. burgdorferi LW2 and B. garinii G1 as well as from B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 were subjected to 10% tris/tricine SDS-PAGE and transferred to nitrocellulose. The membranes were then incubated with either NHS as source for CFH and CFHR1 or with cell culture supernatant containing recombinant FHL1. Potential CFH/FHL1/CFHR1-binding proteins were detected using a polyclonal anti-CFH serum, a polyclonal anti-CFHR1 serum or a polyclonal anti-SCR1-4 antiserum. Monoclonal antibody L41 1C11 recognizing the FlaB protein was used to show equal loading of bacterial lysates. The identified CRASP proteins, CspA, CspZ, ErpP, and ErpA of B. burgdorferi LW2, the CFH-binding protein of B. valaisiana Bv9 and VS116, and the CFHR1-binding protein of B. valaisiana Bv9 are indicated on the right and the mobility of the marker proteins is indicated on the left.

Mentions: It is well-established that resistance of B. burgdorferi, B. afzelii, and B. spielmanii to complement-mediated lysis directly correlates with binding of complement regulators CFH and FHL1 [24], [25], [29], [31], [36]. For this reason, we sought to identify potential CFH/FHL1-binding proteins produced by serum-resistant B. valaisiana ZWU3 Ny3 using ligand affinity blotting. Whole cell lysates obtained from all three B. valaisiana isolates and the two control strains were separated by SDS-PAGE and proteins were transferred to nitrocellulose membranes. Following incubation with NHS as a source for CFH and a polyclonal anti-CFH antiserum, a very faint signal corresponding to a CFH-binding protein of approximately 15 kDa could be detected within serum-sensitive B. valaisiana isolate Bv9 and VS116 but not in serum-resistant ZWU3 Ny3 (Fig. 3). Using a polyclonal anti-CFHR1 antiserum, Bv9 but neither VS116 nor ZWU3 Ny3 showed a 15 kDa CFHR1-binding protein. For comparison, a cell lysate obtained from serum-resistant B. burgdorferi LW2 showed four CFH-binding CRASP proteins (CspA, CspZ, ErpP, and ErpA), three proteins that strongly bound CFHR-1 (CspA, ErpP and ErpA) and two FHL1-binding proteins (CspA and CspZ). Furthermore, FHL1-binding proteins could not be detected within the three B. valaisiana isolates. In agreement with our previous data, serum-sensitive B. garinii G1 did not produce any CFH/FHL1-binding proteins under in vitro cultivation [25], [29], [43]. Taken together, distinct B. valaisiana isolates are able to produce a 15 kDa protein that possesses potential CFH/CFHR1-binding capability, but apparently did not contribute to resistance to complement.


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)

Identification of CRASPs among B. valaisiana.Cell lysates (30 µg each) obtained from control strains B. burgdorferi LW2 and B. garinii G1 as well as from B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 were subjected to 10% tris/tricine SDS-PAGE and transferred to nitrocellulose. The membranes were then incubated with either NHS as source for CFH and CFHR1 or with cell culture supernatant containing recombinant FHL1. Potential CFH/FHL1/CFHR1-binding proteins were detected using a polyclonal anti-CFH serum, a polyclonal anti-CFHR1 serum or a polyclonal anti-SCR1-4 antiserum. Monoclonal antibody L41 1C11 recognizing the FlaB protein was used to show equal loading of bacterial lysates. The identified CRASP proteins, CspA, CspZ, ErpP, and ErpA of B. burgdorferi LW2, the CFH-binding protein of B. valaisiana Bv9 and VS116, and the CFHR1-binding protein of B. valaisiana Bv9 are indicated on the right and the mobility of the marker proteins is indicated on the left.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3539980&req=5

pone-0053659-g003: Identification of CRASPs among B. valaisiana.Cell lysates (30 µg each) obtained from control strains B. burgdorferi LW2 and B. garinii G1 as well as from B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 were subjected to 10% tris/tricine SDS-PAGE and transferred to nitrocellulose. The membranes were then incubated with either NHS as source for CFH and CFHR1 or with cell culture supernatant containing recombinant FHL1. Potential CFH/FHL1/CFHR1-binding proteins were detected using a polyclonal anti-CFH serum, a polyclonal anti-CFHR1 serum or a polyclonal anti-SCR1-4 antiserum. Monoclonal antibody L41 1C11 recognizing the FlaB protein was used to show equal loading of bacterial lysates. The identified CRASP proteins, CspA, CspZ, ErpP, and ErpA of B. burgdorferi LW2, the CFH-binding protein of B. valaisiana Bv9 and VS116, and the CFHR1-binding protein of B. valaisiana Bv9 are indicated on the right and the mobility of the marker proteins is indicated on the left.
Mentions: It is well-established that resistance of B. burgdorferi, B. afzelii, and B. spielmanii to complement-mediated lysis directly correlates with binding of complement regulators CFH and FHL1 [24], [25], [29], [31], [36]. For this reason, we sought to identify potential CFH/FHL1-binding proteins produced by serum-resistant B. valaisiana ZWU3 Ny3 using ligand affinity blotting. Whole cell lysates obtained from all three B. valaisiana isolates and the two control strains were separated by SDS-PAGE and proteins were transferred to nitrocellulose membranes. Following incubation with NHS as a source for CFH and a polyclonal anti-CFH antiserum, a very faint signal corresponding to a CFH-binding protein of approximately 15 kDa could be detected within serum-sensitive B. valaisiana isolate Bv9 and VS116 but not in serum-resistant ZWU3 Ny3 (Fig. 3). Using a polyclonal anti-CFHR1 antiserum, Bv9 but neither VS116 nor ZWU3 Ny3 showed a 15 kDa CFHR1-binding protein. For comparison, a cell lysate obtained from serum-resistant B. burgdorferi LW2 showed four CFH-binding CRASP proteins (CspA, CspZ, ErpP, and ErpA), three proteins that strongly bound CFHR-1 (CspA, ErpP and ErpA) and two FHL1-binding proteins (CspA and CspZ). Furthermore, FHL1-binding proteins could not be detected within the three B. valaisiana isolates. In agreement with our previous data, serum-sensitive B. garinii G1 did not produce any CFH/FHL1-binding proteins under in vitro cultivation [25], [29], [43]. Taken together, distinct B. valaisiana isolates are able to produce a 15 kDa protein that possesses potential CFH/CFHR1-binding capability, but apparently did not contribute to resistance to complement.

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