Borrelia valaisiana Resist Complement-Mediated Killing Independently of the Recruitment of Immune Regulators and Inactivation of Complement Components

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.


Introduction
Lyme disease, the most prevalent vector-borne anthropozoonosis in Europe and North America is caused by spirochetes belonging to the Borrelia (B.) burgdorferi sensu lato (s.l.) complex [1]. In Central Europe, B. burgdorferi, B. afzelii, B. garinii, B. spielmanii as well as B. bavariensis are the causative agents of Lyme disease while the pathogenic potential for B. valaisiana remains unclear [2][3][4][5][6]. There are several lines of evidence that B. valaisiana (formely described as genomic groups VS116 and M19) might also cause human Lyme disease even though only skin biopsies or cerebrospinal fluid samples of erythema migrans, acrodermatits chronica atrophicans, and neuroborreliosis patients were found to be positive for B. valaisiana DNA [3,4]. Complement plays an important role for the recognition, discrimination, and elimination of invading microorganisms [7]. As a central part of the human innate immune system, it is immediately activated upon entry of an intruder via the alternative or the lectin pathways and also by the classical pathway. Activation results in the cleavage of the central component C3 and the generation of active splice fragments C3a and C3b, and deposition of highly-reactive C3b molecules on activator cell surfaces leads to opsonization followed by opsonophagocytosis. In addition, progression of the cascade and formation of the lytic membrane attack complex (MAC) results in complement-mediated killing of the invading pathogen.
The potent complement regulators include C1 esterase inhibitor (C1-Inh), a highly glycosylated molecule of approximately 105 kDa [8] which regulates the initial complement activation steps of the classical and lectin pathways [9][10][11]. Interaction of C1-Inh with C3b and factor B interferes with formation of the C3 convertase and thereby also prevents alternative pathway activation [11].
Factor H (CFH) and Factor H-like protein 1 (FHL1) are the key inhibitors of the alternative pathway, both of which act as cofactors for factor I-mediated inactivation of C3b to iC3b, inhibiting the formation and accelerating the decay of the C3bBb convertase, and finally competing with factor B for binding to C3b [12][13][14][15]. CFH is composed of 20 individually folding protein domains, termed short complement regulator (SCR) [15]. The 42-kDa FHL1 protein consists of the seven Nterminal SCRs of CFH, including the complement regulatory domains and has a unique C-terminal extension of four hydrophobic amino acid residues [15]. In addition, there are six Factor H-related proteins (CFHRs) all of which belong to the human CFH family [16,17]. A common feature of these molecules is the high degree of similarity of their C-terminal SCR domains of CFH [16,18]. CFHR1 acts as a complement regulator by inhibiting the assembly and membrane insertion of the terminal membrane attack complex and inhibits C5 convertase activity [19] while the biological function(s) of CFHR2 is as yet unclear.
The key fluid-phase complement regulator of the classical pathway, C4Bp acts as cofactor for factor I-mediated degradation of C4b to C4d and facilitates dissociation of C2a from the C3 convertase C4b2a of the classical pathway. By preventing formation of the C3 convertase, the generation of C5b and further downstream activation products in the cascade are impeded [20,21].
Differences in host specificity largely correlate with the resistance/sensitivity pattern of Lyme disease spirochetes to complement and allow the various kinds of borreliae to selectively survive in diverse mammalian hosts [22,23].
To extend our analysis on complement resistance of Lyme disease spirochetes, we examined the serum susceptibility of three B. valaisiana isolates derived from feeding or questing Ixodes ricinus ticks. Here we show for the first time, that a particular B. valaisiana strain resists complement-mediated killing. Interestingly, the molecular mechanism of complement resistance differs from other borrelial species in that it is independent of the recruitment of complement regulators of the alternative (CFH, FHL1), classical (C1-Inh, C4Bp) or Lectin pathways (C1-Inh) or degradation of key complement components C3, C3b, C4, C4b and C5, suggesting that certain spirochetes may have developed alternative strategies to overcome innate immunity.

Ethics Statement
The study and the respective consent documents were approved by the ethics committee at the Johann Wolfgang Goethe-University of Frankfurt (control number 160/10). All healthy blood donors provided written informed consent.

Human Sera, Monoclonal and Polyclonal Antibodies, and Human Serum Proteins
Normal human serum (NHS) used for serum susceptibility testing and ligand affinity blotting or as a source of CFH was tested for the presence of anti-Borrelia IgM and IgG antibodies by commercially available ELISAs (EnzygnostH Borreliosis/IgM and EnzygnostH Lyme link VlsE/IgG, Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). Only sera proven to be negative for IgM or IgG anti-Borrelia antibodies were used to form the serum pool.

Molecular Genotyping of Borrelial Isolates by Restriction Fragment Length Polymorphism
Genotyping of borrelial isolates was performed by PCR amplification of the ospA gene followed by restriction fragment length polymorphism analysis as described by Michel et al. [45]. For differentiation, PCR-generated ospA fragments were digested separately with 0.5 U of restriction endonucleases BglII, SspI, HindIII (New England Biolabs, Frankfurt, Germany), Kpn21 (Fermentas, St. Leon-Rot, Germany), and SfuI (Roche Applied Science, Mannheim, Germany) overnight according to the manufacturers instruction. The digested PCR fragments were then analyzed by electrophoresis on a 2% agarose gel and visualized with ethidium bromide. Fragment size was determined by comparison to DNA fragments of a 123 bp molecular standard (Invitrogen, Karlsruhe, Germany). Genospecies' designation of the isolates was performed by analyzing the resulting genospeciesspecific PCR-RFLP patterns according to Michel et al. [45]. In addition, amplicons generated were sequenced by the dideoxy chain-termination method and the processed sequences were subjected to BLAST search (http://blast.ncbi.nlm.nih.gov).

Ligand Affinity Blot Analysis
Whole cell lysates obtained from each borrelial isolate (15 mg per lane) were subjected to 10% tris/tricine SDS-PAGE under reducing conditions and transferred to nitrocellulose as previously described [30,44].

Serum Susceptibility Testing
Serum susceptibility of borrelial strains was assessed by employing a growth inhibition assay as previously described [46,47]. Briefly, highly motile spirochetes (1.25610 7 ) diluted in a final volume of 100 ml in BSK medium containing 240 mg ml 21 phenol red were incubated with 50% NHS or 50% heat-inactivated human serum (hiNHS) in microtiter plates for 9 days at 33uC (Costar, Cambridge, MA, USA). Growth of spirochetes was monitored by measuring a color shift of the pH indicator in the medium at 562/630 nm using an ELISA reader (PowerWave HT, Bio-Tek Instruments, Winooski, VT, USA). The Gene 5 software was used to calculate the growth curves (Bio-Tek Instruments, Winooski, VT, USA).

Binding of Complement Proteins to Viable Spirochetes
Borreliae (2610 9 cells) grown to mid-log phase were gently washed and resuspended in 750 ml NHS supplemented with 34 mM EDTA (pH 8.0) to avoid complement activation or 750 ml hiNHS. After 1 h incubation at room temperature and four wash steps with PBSA (0.15 M NaCl, 0.03 M phosphate, 0.02% sodium azide, pH 7.2) containing 0.05% Tween-20, proteins bound to the borrelial surface were eluted by incubation with 100 mM glycine-HCl (pH 2.0) for 15 min. Cells were then removed by centrifugation at 14.0006g for 10 min at 4uC, and the supernatant was neutralized by adding 1 M Tris (pH 9.0). Both, the last wash and the eluate fraction were separated by Laemmli-SDS-PAGE under non-reducing conditions and binding of CFH, FHL1, CFHR1, CFHR2, and CFHR5 was analyzed by Western blotting using specific antisera [44]. For C4Bp detection, the samples were separated by 10% tris/tricine SDS-PAGE under reducing conditions.

Binding of Purified Complement Proteins to Spirochetes by ELISA
Spirochetes grown to mid-log phase were gently washed and resuspended in 500 ml Dulbecco's PBS without Ca 2+ and Mg 2+ . Microtiter plates (Polysorb, Nunc, Roskilde, Denmark) were coated with 5610 7 cells over night at 4uC. Microtiter plates were washed threefold with PBS containing 0.05% Tween 20 and treated for 1 h at RT with blocking buffer (Applichem GmbH, Darmstadt, Germany). After washing, CFH or C4Bp (500 ng each in a final volume of 100 ml) was added and incubated for 1 h at RT. Thereafter, the wells were washed with PBS containing 0.05% Tween 20 (CFH and C4Bp) and bound CFH or C4Bp was detected with either goat anti-CFH polyclonal antiserum or sheep anti-C4Bp polyclonal antiserum, respectively. After washing, bound proteins were identified using appropriate secondary horseradish peroxidase-coupled antisera. Detection was performed with 1,2-phenylenediamine dihydrochloride as substrate (OPD, Sigma-Aldrich, Taufkirchen, Germany) and absorbance was measured at 490 nm.

Determination of Intrinsic C3b and C4b Cleavage Activity of Spirochetes
Proteolytic activity of borrelial cells was analyzed in the presence and absence of complement regulators CFH and C4Bp. Cells (5610 8 ) grown to mid-log phase were gently washed and resuspended in either 100 ml hiNHS, 100 ml GVB ++ -buffer (Complement Technology, Tyler, TX, USA) containing 5 mg CFH or 100 ml GVB ++ -buffer containing 5 mg C4Bp, respectively. After incubation for 60 min at room temperature, cells were washed and resuspended in GVB ++ -buffer containing C3b (1 mg/ ml) and Factor I (250 ng/ml) or C4b (1 mg/ml) and Factor I (250 ng/ml), respectively. The reaction mixtures were incubated for 60 min at 37 uC and cells were sedimented by centrifugation at 14.0006g for 10 min at 4uC. Each supernatant was then mixed with sample buffer, subjected to SDS-PAGE under reducing conditions and transferred onto a nitrocellulose membrane. C3b and C4b degradation products were visualized by Western blotting using a polyclonal goat anti-C3 antiserum or a polyclonal goat anti-C4 antiserum.
Determination of Intrinsic C3, C4 and C5 Cleavage Activity of Spirochetes Spirochetes (5610 8 ) grown to mid-log phase were gently washed and resuspended in 20 ml GVB ++ -buffer (Complement Technology, Tyler, TX, USA) containing 1 mg C3, 1 mg C4 or 1 mg C5. After incubation for 60 min or 120 min at 37 uC with gentle agitation, cells were sedimented by centrifugation at 14.0006g for 10 min at 4uC. Each supernatant was then mixed with sample buffer, subjected to 10% tris/tricin SDS-PAGE under reducing conditions and transferred onto a nitrocellulose membrane. C3, C4 and C5 degradation products were visualized by Western blotting using a polyclonal goat anti-C3 antiserum, a polyclonal goat anti-C4 antiserum or a polyclonal goat anti-C5 antiserum.

Detection of Deposited Complement Components by Immunofluorescence Microscopy
For detection of complement components deposited on the borrelial surface after complement activation, an immunofluorescence assay was performed as previously described [29,43]. In brief, spirochetes (6610 6 ) were incubated in 25% NHS or, as a control, in 25% hiNHS for 30 min at 37uC with gentle agitation. Ten microliters of cell suspension were spotted on glass slides, allowed to air dry overnight, and fixed in methanol. After 1 h incubation at 37uC with polyclonal antibodies directed against the complement components C3, C4, and C6 or a mAb directed against the membrane attack complex (MAC) slides were washed and subsequently incubated with Alexa 488conjugated antibodies directed against either goat or mouse antibodies (Molecular Probes, Eugene, OR, USA). After washing, the slides were mounted with ProLong Gold antifade reagent (Molecular Probes, Eugene, OR, USA) containing DAPI.

Determination of the C3a and C4a Generation
Spirochetes (6610 6 ) grown to mid-log phase were gently washed and resuspended in 100 ml sample dilution buffer containing 25 ml NHS. After incubation for 5 min at 21uC (C3a) or for 5 min at 37uC (C4a), cells were sedimented by centrifugation at 14.0006g for 10 min at 4uC. As a control, NHS was also pre-incubated for 5 min at 21uC and 37uC, respectively. Supernatants were then diluted as recommend by the manufacturer and samples were applied to microtiter plates containing immobilized monoclonal antibodies against C3a and C4a, respectively. Generation of C3a was analyzed by the MicroVue C3a Plus ELISA (Quidel, San Diego, CA, USA) and for determination of C4a in NHS, the BD OptEIA TM Human C4a ELISA Kit (BD Biosciences, Heidelberg, Germany) was used.

Serum Susceptibility of B. valaisiana
To further evaluate the serum resistance pattern of B. valaisiana to human complement, three isolates collected from ticks at different geographical locations were incubated in 50% active NHS or in 50% hiNHS for up to 9 days and cell growth was monitored in a colorimetric growth survival assay [47,48]. Growth of viable spirochetes results in a continuous decrease of the absorbance values due to accumulation of secondary metabolites in the BSK-H medium. As demonstrated in figure 1, growth of B. valaisiana isolate Bv9, B. valaisiana type strain VS116, and serumsensitive control strain B. garinii G1 when challenged with 50% NHS was completely inhibited as evidenced by only minor changes of absorbance values. Under identical experimental conditions, B. valaisiana ZWU3 Ny3 as well as serum-resistant B. burgdorferi LW2 showed growth in NHS as indicated by continuous decrease of absorbance values. As expected, hiNHS did not affect growth of the five borrelial strains analyzed. In contrast to previous findings, only B. valaisiana strain ZWU3 Ny3 resist complementmediated killing by human serum.

Deposition of Activated Complement Components on the Surface of B. valaisiana
Next, we assessed deposition of activated complement components C3, C6 and the membrane attack complex (MAC) on the surface of B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 as well as serum-resistant B. burgdorferi LW2 and serum-sensitive B. garinii G1. Complement C3, C4, C6 and MAC (C5b-9) were strongly deposited on the surface of the serum-sensitive B. valaisiana Bv9 and VS116 as well as on the serum-sensitive control strain B. garinii G1 (Fig. 2). In contrast, serum-resistant strains B. valaisiana ZWU3 Ny3 and B. burgdorferi LW2 showed marginal or no fluorescent staining for all three complement components analyzed, suggesting that the complement cascade was inhibited at the level of C3 and C4 activation. Slides were also counterstained with DAPI to identify all spirochetes in a given field of view. Most of the complementpositive spirochetes showed extensive bleb formation when exposed to human serum, while a few cells stained negative for complement but were positive in the DAPI stain. Those spirochetes most likely represent ''cell ghost'' as previously described for serum susceptible B. garinii and B. lusitaniae strains [43,49]. When spirochetes were incubated with heat-inactivated NHS, the cell morphology remained completely intact and deposition of complement was not detectable by immunofluorescence microscopy (data not shown). Thus, treatment of spirochetes with active NHS leads to strong complement activation followed by robust deposition of effector components to B. valaisiana Bv9 and VS116 but not to serum-resistant isolate ZWU3 Ny3.

Identification of CFH-binding Proteins in B. valaisiana
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.

Identification of Complement Regulators Bound to the Surface of Intact Borrelial Cells
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, serumsensitive 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.
CFH-binding proteins belonging to the Erp protein family, in particular ErpA/CRASP-5, ErpC/CRASP-4, ErpP/CRASP-3 as well as OspE bound CFH only under denaturated conditions in a solid phase assay (e.g. by using purified recombinant proteins or borrelial cell lysates) but did not bind CFH when the same proteins are exposed on the surface of viable cells [44,50]. By contrast, these borrelial proteins bound strongly to other members of the CFH protein family, namely CFHR1, CFHR2 and to a lower extent to CFHR5. Elucidating binding of CFHRs to B. valaisiana Bv9 and VS116 producing the 15 kDa CFH-binding protein, the wash and eluate fractions of all borrelial isolates tested were also analyzed for CFHR1 and CFHR2 (Fig. 4A). The two glycosylated forms of CFHR1 (CFHR1a and CFHR1b) were detected in the eluate fractions of B. burgdorferi LW2 (this strain produced CFHR-binding proteins ErpP and ErpA) as well as B. valaisiana Bv9, VS116, and ZWU3 Ny3 but not in the eluate fraction of B. garinii G1. Intriguingly, the binding patterns of the 15 kDa proteins of B. valaisiana Bv9 and VS116 resemble the CFHR-binding Erp proteins ErpA/CRASP-5 and ErpP/CRASP-3 of B. burgdorferi LW2. Binding of CFHR2 and CFHR2a was only detected for B. burgdorferi LW2 and B. valaisiana Bv9. Concerning the African tick-borne relapsing fever spirochete B. duttonii LA1, binding of CFHR1a and CFHR1b could be detected for this particular borrelial strain.
Next, we sought to determine whether B. valaisiana is able to bind C4Bp and/or C1-Inh as the key complement regulators of the classical and lectin pathways. To this end, isolates were incubated with NHS-EDTA (for C4Bp binding) or heatinactivated NHS (for C1-Inh binding). As demonstrated in Figure 4, none of the Lyme disease spirochetes bound C4Bp or C1-Inh to their surface. To exclude possible limitations of our experimental setup, we also investigated B. duttonii LA1 known to bind C4Bp and C1-Inh [51,52]. Employing identical conditions for the serum adsorption assays, binding of C4Bp and C1-Inh to strain LA1 could be clearly detected by Western blotting ( Figure 4A).
Interaction of spirochetes with CFH and C4Bp was also assessed by ELISA as quantitative method for analyzing binding of distinct complement regulators. Again, none of the B. valaisiana isolates bound to purified CFH and C4Bp ( Figures 4B and C, respectively). In addition, binding of CFH and C4Bp to spirochetes were also assessed under less stringent washing conditions. Again, none of the B. valaisiana strains including ZWU3 Ny3 bound either complement regulators (data not shown). This finding strongly suggests that isolate ZWU3 Ny3 overcomes complement-mediated killing by a different mechanism that is independent of binding of host-derived complement regulators.

Determination of the Potential Intrinsic Proteolytic Activity of B. valaisiana for Various Complement Components
To clarify our somewhat paradoxical observation that serumresistant B. valaisiana ZWU3 Ny3 failed to bind any of the tested complement regulators to its surface, a degradation assay was employed to detect intrinsic proteolytic cleavage capacity of spirochetes for C3b (Fig. 5). To this end, spirochetes were incubated with or without purified CFH for 60 min. After extensive washing, Factor I and C3b were added to each reaction mixture and following incubation, the reaction mixtures were separated by SDS-PAGE and C3b degradation products were then analyzed by Western blotting. CFH bound to serumresistant B. burgdorferi LW2 retained its cofactor activity as shown by the appearance of the characteristic cleavage pattern of C3b (68 and 43 kDa a-chain). In contrast, C3b remained intact when B. garinii G1 and B. valaisiana isolates Bv9, VS116 as well as ZWU3 Ny3 were investigated, in the presence or absence of CFH as evidenced by the presence of the 110 kDa achain and the 75 kDa b-chain (Fig. 5B). Thus, none of the B. valaisiana isolates analyzed exhibit an intrinsic proteolytic activity to allow inactivation of the complement component C3b. No complement C3 cleavage products could be detected after incubation with the respective borrelial strains, except for B. burgdorferi LW2, showing a fragment that correspond to the 41 kDa achain ( Figure S1).
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 a4 fragment, the 75 kDa b-chain as well as the 33 kDa c-chain (Fig 6A). Cleavage of C4b, as detected by visualization of the characteristic a4 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 achain 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 a4 fragment was detectable upon incubation of serum-resistant B. burgdorferi LW2 with C4b and Factor I.
Investigating degradation of complement component C4, we found that neither of the Lyme disease spirochetes tested, nor the relapsing fever B. duttonii strain LA1 displayed any proteolytic capacity to cleave C4 in either the presence or absence of Factor I ( Figure S1).
In order to test the inactivation capacity on complement component C5, spirochetes were incubated with purified C5 for 60 and 120 min. After centrifugation, supernatants were subjected to SDS-PAGE and C5 cleavage products were analyzed by Western blotting using a goat anti-C5 antibody. No degradation fragments of the 118-kDa a-chain or of the 74-kDa b-chain were detected indicating that spirochetes lacked proteolytic activity for the cleavage of C5 (Fig. 7).
Next, to exclude proteolytic degradation of complement components C3, C3b, C4, C4b, and C5 by secreted proteases we also analyzed supernatants collected from late logarithmic borrelial cultures. Spirochete-free supernatants were incubated with 2 mg of purified complement components for 180 min at 37uC. After termination of the reaction, a portion of the reaction mixtures were applied to SDS-PAGE and cleavage products were detected by Western blotting. As shown in Figure S2, no degradation fragments could be visualized, suggesting that spirochetes did not secrete proteases into the culture medium.
Taken together, these findings strongly suggest that serumresistant B. valaisiana ZWU3 Ny3 displayed no intrinsic proteolytic activity that would have resulted in inhibition of complement by cleavage of C3, C3b, C4, C4b, and C5.

Determination of the C3a and C4a Generation by B. valaisiana
To further evaluate the ability of serum-resistant B. burgdorferi LW2 and B. valaisiana ZWU3 Ny3 to inhibit complement activation at the level of C3 and C4 we sought to quantify the generation of C3a and C4a after incubation of spirochetes with human serum by ELISA. As demonstrated in figure 8, C3 activation was significantly reduced for B. burgdorferi LW2 (p-value ,0.001) as well as for B. valaisiana ZWU3 Ny3 (p-value 0.05) while, in contrast, a stronger C3a generation could be detected for serum-sensitive B. garinii G1 as well as B. valaisiana Bv9 and VS116. Of note, pre-incubation of NHS for 5 min at 21uC resulted in a somewhat higher C3a generation compared to untreated NHS samples. Nevertheless, the data collected support the notion that ZWU3 Ny3 inhibits C3 activation to some extent.
To investigate C4a generation, spirochetes were incubated with 2% or 25% human serum for 5 min at 37uC and C4a was measured using ELISA. However, no significant differences could be observed between the serum-resistant and serum-sensitive strains analyzed (data not shown). was analyzed by detection of characteristic cleavage fragments after incubation of spirochetes with (+) or without (2) purified CFH. B. burgdorferi LW2, B. garinii G1, B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 were incubated with CFH for 60 min at room temperature. After extensive washing with PBS, C3b (10 ng/ml) and Factor I (20 ng/ml) were added and the mixture was incubated for 30 min at 37uC. Subsequently, samples were heated to 95uC for 5 min, subjected to 12.5% Laemmli-SDS-PAGE and transferred onto a nitrocellulose membrane. The C3b degradation products were visualized by Western blotting using a polyclonal goat anti-human C3 antiserum. As a positive control, purified CFH (50 ng) was incubated with C3b and Factor I, and as a negative control complement proteins were incubated in the absence of CFH. The mobility of the a'and the b-chain of C3b and the cleavage products of the a'-chain (a'-68 and a'-43) is indicated. (+) incubation with all complement proteins; (2) incubation without CFH. doi:10.1371/journal.pone.0053659.g005

Discussion
Recruitment of complement regulators of the alternative pathway, CFH and FHL1 by serum-resistant B. burgdorferi, B. afzelii, and B. spielmanii enable these spirochetes to inhibit complement activation at the central step of the cascade (C3 level), and thereby overcome complement-mediated killing [24,25,29,31,36]. Here, we demonstrate that B. valaisiana isolates differ in their susceptibility to human serum. Similar to the serum-sensitive B. garinii control strain G1, B. valaisiana isolate Bv9 and B. valaisiana type strain VS116 strongly activate complement and large amounts of complement components were deposited on their surface, whereas isolate ZWU3 Ny3 resists complement-mediated killing and showed limited or no deposition of destructive complement components. Irrespective of whether they displayed a serum-sensitive or resistant phenotype, all three B. valaisiana isolates acquired CFHR-1 and strain Bv9, in addition, also bound CFHR2 from human serum. Despite binding of these complement regulators, Bv9 and VS116 failed to regulate complement activation, thus permitting bacteriolysis as previously shown for B. lusitaniae [49]. More interestingly, isolate ZWU3 Ny3 bound neither complement regulators of the alternative pathway CFH and FHL1, nor of the classical and lectin pathways, C4Bp and C1-Inh, respectively. We also showed that none of the B. valaisiana isolates displayed an intrinsic proteolytic capacity to inactivate C3, C3b, C4, C4b, and C5. These findings strongly suggest that B. valaisiana ZWU3 Ny3 resist complement-mediated killing by a novel mechanism of immune evasion. 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 mg/ml) and Factor I (1 mg/ml) were added and the mixture was incubated for 120 min at 37uC. Subsequently, the samples were heated to 95uC 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 mg) 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 a'-, b'-, and c'-chain and the a'4 fragment is indicated. (+) incubation with all complement proteins; (2) incubation without C4Bp. doi:10.1371/journal.pone.0053659.g006 It has recently been shown that spirochetes of the B. burgdorferi sensu lato-complex exhibit a high diversity in their susceptibility pattern to human complement [23,29,46,47,49,[53][54][55][56]. Concerning serum susceptibility of B. valaisiana, the isolates investigated so far showed a serum-sensitive phenotype [53,56,57]. Due to the fragmentary information available and the limited number of isolates (n = 5) tested so far, the question of whether this particular genospecies, in general, exhibits a serum-sensitive phenotype, as observed for B. garinii and B. lusitaniae [46,47,49,53,56], has not been adequately addressed. Here, we show for the first time that a particular isolate of B. valaisiana ZWU3 Ny3 may resist complement-mediated killing even though the majority of isolates are susceptible to human serum. Hence, it is tempting to speculate that complement-resistant cells are protected from innate immune defenses and qua argumentum e contrario can more efficiently establish an infection and survive in the human host. Although B. valaisiana has not been successfully isolated from patients yet, there is some evidence that this particular genospecies is an, albeit infrequent, cause of Lyme disease [3,4,6]. However, to which extent serumresistant B. valaisiana are responsible for establishing human infections remains unclear and necessitates cultivation of this particular genospecies from Lyme disease patients.
To evade complement-mediated killing, serum-resistant spirochetes acquire complement regulators CFH and FHL1 which in turn leads to an efficient inactivation of deposited C3b [24][25][26]29,53,55]. In addition, no or inadequate binding of CFH and/ or FHL1 is correlated with an excess of complement activation and deposition of harmful complement components on the borrelial surface as demonstrated for B. garinii and B. lusitaniae [24,46,47,49,53,56]. To our knowledge, binding of human complement regulators to viable B. valaisiana cells has not been reported so far, although a proposed CFH-binding protein has recently been identified using cell lysates of isolate VS116 [58]. Here, we show for the first time that a member of the B. valaisiana genospecies did not bind the key complement regulators of the alternative, classical, and lectin pathways (CFH, FHL1, C4Bp as well as C1-Inh) ( Figure 4). Interestingly, all B. valaisiana isolates were able to bind CFHR1 and isolate Bv9 also bound CFHR2, irrespective of their serum susceptibility. More recently, we showed that surface-bound CFHR1 and CFHR2 were insufficient to completely prevent complement activation, and thus failed to protect spirochetes from complement-mediated killing [44,49,50]. In line with this assumption, large amounts of C3, C4, C6 and MAC were detectable on the spirochetal surface following incubation of serum-sensitive B. valaisiana VS116 and Bv9 with active human serum ( Figure 2). In contrast, the majority of cells of B. valaisiana ZWU3 Ny3 did not show strong deposition of lethal complement components akin to the serum-resistant B. burgdorferi control strain LW2, indicating that this particular isolate uses other mechanisms to combat complement activation. By investigating the borreliacidal effect of complement on B. valaisiana, we observed a complete loss of cell mobility after serum incubation and excessive bleb formation for isolates VS116 and Bv9, (Figure 1 and 2) which is in complete agreement with previous findings showing that B. valaisiana is highly sensitive to human complement [53,56]. In contrast, the serum-resistant isolate B. valaisiana ZWU3 Ny3 and the control strain B. burgdorferi LW2 showed continuous growth in human serum and limited complement deposition on their surface. Our observation of the borreliacidal effect of human complement on spirochetes is in accordance with previous findings that serum-sensitive B. valaisiana, B. garinii, and B. lusitaniae were strongly affected by complement [47,49,56].
Serum resistance of spirochetes is primarily mediated by the interaction of CFH and FHL1 with two distinct borrelial outer surface proteins, namely CspA and CspZ [26][27][28][29][30]35,36]. Additionally, spirochetes that lack the CspA or CspZ encoding genes through loss of the respective plasmids or by inactivation of the cspA gene become serum-sensitive [41][42][43]. Binding of CFHR1 and CFHR2 also necessitates interacting ligands, which have previously been identified to be ErpP (CRASP-3), ErpC (CRASP-4) and ErpA (CRASP-5), all of which belong to the polymorphic OspE protein family [28,30,44,50,59]. Due to the considerable degree of identity between the C-terminal domains of CFHR1 (100 and 97%) and CFHR2 (89 and 61%) to the C-terminal SCRs 19 and 20 of CFH [16], ErpP (CRASP-3), ErpC (CRASP-4), ErpA (CRASP-5) as well as OspE orthologs of B. burgdorferi also bind complement regulator CFH [28]. Further analyses revealed that whereas recombinant CRASP-3, CRASP-4, and CRASP-5 readily bind CFH, the native proteins predominately interacted with CFHR1 and CFHR2 and failed to bind CFH [44,50]. Employing ligand affinity blotting, we identified potential CFH/CFHR1binding proteins of B. valaisiana isolates Bv9 and VS116 with an estimated molecular weight of ,15 kDa ( Figure 3) which in regard to their CFH/CFHR1-binding capabilities strongly resemble the OspE orthologs of B. lusitaniae and B. spielmanii, as well as a ,17 kDa protein recently described for B. valaisiana VS116 [49,58,60]. While the denaturated ,17 kDa protein of B. valaisiana VS116 described by Bhide et al. [58] potentially bind CFH in a ligand affinity blot, the authors did not analyzed binding of CFH to viable spirochetes, strongly suggesting that the native protein displays a different binding capability to this complement regulator. In conclusion, it is apparent, that the findings from our binding analysis with viable cells better serve to explain the serum susceptible phenotype of a given borrelial isolate than do conclusions based on ligand affinity blots.
Pathogenic microorganisms develop different strategies to counteract complement [61][62][63][64]. Apart from the acquisition of CFH and FHL1, Lyme disease spirochetes appear to bind the negative regulator of the classical pathway, C4Bp [65]. Employing different methodologies and conditions, we were unable to detect binding of this regulator to B. valaisiana, B. burgdorferi, and B. garinii ( Figure 4). Under identical experimental conditions, binding of C4Bp could be demonstrated for the relapsing fever B. duttonii strain LA1 which is in line with previously published data [52,66]. However, binding of C4Bp failed to protect serum-sensitive B. garinii from the destructive attack of complement, thus the biological impact of the interaction of this regulator with Lyme disease spirochetes for complement evasion is elusive.
Serum-resistant B. valaisiana ZWU3 Ny3 deposited limited amounts of C3 and C4 on the surface (Figure 2) suggesting that complement activation was influenced at very early steps of the cascade. Complement inhibition should be accompanied by a reduced release of potent bioactive mediators C3a and C4a upon activation. The activation assays investigated ( Figure 8) showed a statistically significant reduction of C3 activation upon pre-incubation of NHS with serum-resistant B. valaisiana ZWU3-Ny3 (p-value,0.05) and serum-resistant B. burgdorferi LW2 (pvalue ,0.001) while no reduction could be observed concerning the generation of C4a. The high amount of C4a generated by preincubation of NHS might account for the similar C4a values calculated for all borrelial strains and, seemingly, did not mirror  6610 6 ) were incubated with 25% of NHS for 5 min at 21uC and activation of C3 was then analyzed by the MicroVue C3a Plus ELISA. Generated C3a was detected using a monoclonal anti-C3a capture antibody and a HRP-conjugated polyclonal anti-C3 antiserum. All experiments were performed three times with at least three replicates, obtaining very similar results. For clarity, data of a representative experiment are shown. Error bars represent 6 SD. Raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction. ***p,0.001; *p,0.05. doi:10.1371/journal.pone.0053659.g008 the finding obtained by immunofluorescence microscopy. Thus, in light of these experiments it is not entirely clear how this particular B. valaisiana strain negatively influences complement activation at the level of C4.
Excluding CFH, FHL1, and C4Bp as potential interacting molecules responsible for mediating serum resistance of ZWU3 Ny3, we also investigated binding of C1-Inh as an additional promising candidate. By analyzing binding of serum-derived regulators, we detected binding of C1-Inh exclusively to the tickborne relapsing fever B. duttonii strain LA1, but not to Lyme disease spirochetes, including isolate ZWU3 Ny3 (Figure 4), indicating that this specific isolate exerts different means to resist complement-mediated killing.
Concerning degradation of key complement components, diverse microorganisms secrete to inactivate complement [67][68][69][70][71]. Investigating degradation of C3, C3b, C4, C4b as well as C5, none of the key components of the complement cascade were cleaved by serum-resistant B. valaisiana ZWU3 Ny3 or could be detected in the supernatant of this strain (Figures 5, 6, and 7, and Figures S1 and S2), suggesting that this isolate lacks potent proteolytic enzymes to inactivate complement.
In conclusion, we demonstrated that B. valaisiana isolates differ in their susceptibility to human serum, thus providing some evidence that in particular serum-resistant isolates might cause Lyme disease. Contrary to our expectations, certain B. valaisiana isolates appear to possess different molecular mechanism(s) to inhibit complement activation, independently of the recruitment of complement regulators or by inactivation of central complement components. Even though that we are currently unable to decipher the precise molecular mechanism, it is tempting to speculate that B. valaisiana ZWU3 Ny3 expresses an outer surface protein that directly interacts with components of the complement system to inhibit complement activation. Further investigation is required to identify potential complement inhibitory protein(s) of this particular borrelial strain. Figure S1 Determination of the C3 and C4 proteolytic activity of B. valaisiana. Degradation of C3 (A) and C4 (B) by an intrinsic proteolytic activity of borrelial cells (5610 8 ) was analyzed by detection of characteristic cleavage fragments after incubation of spirochetes. B. burgdorferi LW2, B. garinii G1, B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 were incubated with C3 or C4 for 120 min at 37uC. Subsequently, the samples were heated to 95uC for 5 min, subjected to 10% tris/tricine SDS-PAGE under reducing conditions. The C3 and C4 cleavage products were visualized by Western blotting using a polyclonal goat anti-human C3 and a polyclonal goat anti-human C4 antiserum, respectively. As a control, purified C3 or C4 (1 mg each) were incubated under identical conditions. The mobility of the C3 and C4 cleavage fragments are indicated and the mobility of the protein standard is indicated on the left. The mobility of the marker proteins is indicated on the left. (TIF) Figure S2 Determination of complement degradation by external proteases. Supernatants obtained from late logarithmic cultures (10 ml each), as well as BSK medium (negative control) were incubated with 2 mg of purified complement components for 180 min at 37 uC. As a further control, purified C3, C3b, C4, C4b, and C5 were incubated under identical conditions.The reaction mixtures were terminated by adding SDS sample buffer and one fourth of the reactions were subjected to 10% tris/tricin SDS-PAGE under reducing conditions. After transfer to nitrocellulose membranes, complement components were detected by using the appropriate polyclonal antisera as described in the Material & Method section. The mobility of the marker proteins is indicated on the left. For detection of complement components in culture medium, same amounts of BSK were subjected to 10% tris/tricine SDS-PAGE and proteins were transferred to nitrocellulose membranes. Complement components were then detected using appropriate polyclonal antibodies. The mobility of marker proteins is indicated on the left. (TIF)