B. burgdorferi B314, a high-passage derivative of reference strain B31 (19) was cultured until mid-exponential phase (5 x 10⁷ cells per ml) at 33°C in Barbour-Stoenner-Kelly (BSK-H) medium (Bio&SELL, Feucht, Germany) supplemented with 7.4% heat-inactivated rabbit serum (Merck, Darmstadt, Germany). Hexahistidine His-tagged proteins were produced in Escherichia (E.) coli BL21 (DE3) (New England Biolabs, Frankfurt, Germany), E. coli M15 (Qiagen, Hilden, Germany) or E. coli C43 (20) grown in yeast tryptone (YT) broth supplemented with ampicillin (50 µg/ml) at 37°C.

Non-immune human serum (NHS) was collected from healthy volunteers and initially tested for the presence of anti-Borrelia IgM and IgG antibodies as described previously (21). Only seronegative samples were combined to form the serum pool and complement activity (CH50) was tested by applying the WIESLAB^® Complement Alternative Pathway kit (SVAR Life Science, Malmö, Sweden) according to the manufacturer instructions. Complement factor C1r was purchased from Complement Technology (Tyler, TX, USA), the goat anti-C1r antibody was obtained by bio-techne (Minneapolis, MN, USA), the neoepitope-specific monoclonal anti-C5b-9 antibody was from Quidel (San Diego, CA, USA). Human glu-plasminogen was purchased from Prolytix (Essex Junction, VT, USA) and urokinase plasminogen activator (uPA) (Merck, Darmstadt, Germany) was used for the activation of plasminogen to plasmin. Fibronectin, the anti-fibronectin antibody, chromogenic substrate S-2251 (D-Val-Leu-Lys p-nitroanilide dihydrochloride), Zymosan A, and bovine serum albumin (BSA) were obtained from Merck. The polyclonal anti-plasminogen antibody was from Acris Antibodies (Herford, Germany) and gelatin was from Applichem (Darmstadt, Germany). The mouse anti-His antiserum was obtained from Novagen (Merck, Darmstadt, Germany) and Qiagen (Hilden, Germany), and the horseradish peroxidase (HRP)-conjugated immunoglobulins were purchased from Dako (Hamburg, Germany).

The generation of His-tagged CihC orthologs from B. recurrentis A17, B. hermsii FRO, B. parkeri, and B. turicatae as well as the N- and C-terminal fragment of CihC of B. recurrentis A17 and the N-terminal fragment of the CihC ortholog of B. hermsii HS1 were described previously (10, 22). The recombinant proteins used herein consisted the following amino acid residues: CihC of B. recurrentis (aa 20-356), FbpC of B. hermsii FRO (aa 21-374), FbpC ortholog of B. parkeri (aa 25-349), FbpC ortholog of B. turicatae (aa 21-363), CihC-N of B. recurrentis A17 (aa 20-194), CihC-C of B. recurrentis (aa 195-356), and FbpC-N from B. hermsii (aa 21-203). CspA and BBA70 of B. burgdorferi B31 as well as the C-terminal fragment of BBK32 originated from B. burgdorferi B31 were used as controls (21, 23, 24).

To produce recombinant proteins, E. coli cultures (500 ml) were grown in ampicillin-supplemented TB medium at an OD₆₀₀ of 0.5 to 0.7 and then induced with 200 µM isopropyl-β-D-thiogalactopyranoside (Carl Roth, Karlsruhe, Germany). Cultures were then incubated for 4 h at RT. Following sedimentation, bacterial cells were lysed in buffer containing 300 mM NaCl, 50 mM NaH₂PO₄, 10 mM imidazole, and 1 mg/ml lysozyme (Merck, Darmstadt, Germany) (pH 8.0) with a MICCRA D-9 dispersion device (Art Prozess- & Labortechnik, Müllheim, Germany). The cell suspension was thereafter sonicated six times for 30 s at 4°C with a Sonifier 450 (Branson, Danbury, CT, USA). Cell debris were removed by centrifugation and His-tagged proteins were purified in the presence of cOmplete™ protease inhibitor (Roche Diagnostics GmbH, Mannheim, Germany) by affinity chromatography using NEBExpress^® Ni Resin (New England Biolabs, Frankfurt, Germany). Increasing concentrations of imidazole (50 to 250 mM) were used for eluation and small amounts of each fraction were loaded on 10% Tris/Tricine SDS-PAGE followed by silver staining. Eluates containing highly purified protein were combined and dialyzed several times against 50 mM Tris buffer and then concentrated by using Protein Concentrator PES (Thermo Scientific, Rockford, IL, USA). The bicinchoninic acid protein assay (Thermo Scientific, Rockford, IL, USA) was used to determine the protein concentration.

To ensure that no protein degradation occurs during the purification process, His-tagged proteins (500 ng each) were separated by 10% Tris/Tricine SDS-PAGE and visualized by either silver staining or by Western blotting as described previously (23). Briefly, after separation and transfer of the proteins, nitrocellulose membranes were blocked with 5% nonfat dry milk in TBS containing 0.1% Tween 20 (TBS-T1). Following three wash steps with TBS-T1, membranes were incubated with a mixture of monoclonal anti-His antibodies (1:1000) for 1 h at RT. The membranes were washed three times with TBS containing 0.2% Tween 20 (TBS-T2) and incubated with a polyclonal horseradish peroxidase (HRP)-conjugated anti-mouse antibody (1:1000). After washing with TBS-T2, protein complexes were visualized by adding tetramethylbenzidine (TMB) (Mikrogen Diagnostics, Neuried, Germany) as substrate. For Far-Western blotting, membranes were blocked, washed and incubated with fibronectin (5 µg/ml) for 1 h at RT. After washing with TBS-T1, membranes were incubated with an anti-fibronectin antibody (1 h at RT) following incubation with a polyclonal HRP-conjugated anti-mouse antibody. Protein-protein complexes were detected by using TMB.

ELISA was performed to assess binding of complement components C1r, plasminogen or fibronectin to purified His-tagged proteins. Microtiter plates (MaxiSorp, Nunc) were coated with purified recombinant proteins, BSA and gelatin, respectively (5 µg/ml each) at 4°C overnight as described previously (25, 26). Briefly, following blocking with PBS-T containing 0.2% gelatin, C1r (5 µg/ml), plasminogen (10 µg/ml) or fibronectin (10 µg/ml) were added and the plates were incubated for 1 h at RT. Wells were then incubated for 1 h at RT with specific antisera (each diluted 1:1000) followed by an incubation with HRP-conjugated anti-rabbit or anti-goat antisera (1:1000) for 1 h at RT. Afterwards, o-phenylenediamine (Merck, Darmstadt, Germany) was added to the wells and the absorbance was measured at 490 nm (PowerWave HT, Bio-Tek Instruments, Winooski, VT, USA). In addition, CihC/FbpC orthologs were immobilized and incubated with increasing amounts of plasminogen (0 to 1.5 µM) to determine dose-dependency of the binding and to calculate the dissociation constant.

Conversion of protein-bound plasminogen to proteolytically active plasmin was assayed by cleavage of the chromogenic substrate D-Val-Leu-Lys-p-nitroanilide dihydrochloride as described previously (24, 25). In brief, microtiter plates coated with purified His-tagged proteins or BSA (5 µg/ml each) in immobilization buffer PBS were blocked with PBS-T-1% BSA for 2 h at RT, washed and then incubated with 10 µg/ml of glu-plasminogen for 1 h at RT. After washing, wells were incubated with TBS-T (50 mM Tris/HCl, pH 7.5, 300 mM NaCl, 0.003% Triton X-100) containing the chromogenic substrate S-2251 (0.3 mg/ml). Finally, 4 µl of 2.5 µg/ml urokinase plasminogen activator (uPA) were added to each well to activate protein-bound plasminogen. Plates were then placed in a spectrophotometer (PowerWave HT) and incubated at 37°C for 24 h. Kinetic measurements were conducted by reading the reactions every 30 minutes at 405 nm. In addition, control reactions containing 50 mM tranexamic acid or in which plasminogen or uPA have been omitted were also measured in parallel.

The inhibitory capacity of CihC/FbpC orthologs on the classical (CP) and alternative pathway (AP) was assessed by employing an ELISA-based approach as described previously (26, 27). Microtiter plates were coated with either human IgM (30 ng/ml) (Merck, Darmstadt, Germany) for the CP and Zymosan A (10 µg/µl) (Merck, Darmstadt, Germany) for the AP at 4°C overnight. Following three wash steps with TBS containing 0.05% Triton X-100 (TBS-T), wells were blocked with PBS containing 0.05% Tween20 and 1% BSA for 1 h at RT. NHS (1% for the CP and 15% for the AP) was pre-incubated with increasing concentrations of His-tagged proteins for 15 min at 37°C before added to the wells to initiate complement activation. After washing with TBS-T, the anti-C5b-9 antibody (1:500) (Quidel, Athens, OH; USA) was added to determine assembly of the pore-forming membrane attack complex. Following incubation for 1 h at RT, wells were washed thoroughly with TBS-T and incubated with HRP-conjugated anti-mouse IgG (1:1000) at RT for 1 h. After adding o-phenylenediamine, the absorbance values were measured at 490 nm.

Protection of spirochetes from complement-mediated killing by soluble CihC/FbpC orthologs was assessed by a modified serum protection assay as described previously (23). Initially, human serum was incubated with 5 µM of purified CihC/FbpC orthologs or the C-terminal BBK32 fragment as control for 15 min at 37°C with gentle agitation. The reaction mixtures were then adjusted to 100 µl with BSK-H medium. As additional control reactions, native NHS (not pre-treated), heat-inactivated NHS (each 30%) were also included. In parallel, 1 × 10⁷ spirochetes of the serum-sensitive B. burgdorferi strain B314 were sedimented by centrifugation and resuspended in the protein-treated serum samples as well as the controls. Spirochetes were then incubated for 4 h at 33°C. For the determination of motile cells, the number of spirochetes was adjusted by various dilutions to 5 up to 25 cells per grid in a single chamber. For every reaction and time point (0, 2, and 4 h), spirochetes in nine grids were counted, and the average was used for the calculation of the total number of cells. The percentage of motile cells was determined by dark field microscopy at time point 0, 2, and 4 h. Biological replicates were conducted at least three times.

The data collected represent means from at least three independent experiments, and error bars indicate SD. For statistical analyses, one-way ANOVA with Dunnett´s multiple comparison post-hoc test (95% confidence interval) was employed for statistical analysis using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA). Results were deemed statistically significant for the following p values: *, P ≤ 0.05, ** P ≤ 0.01 ***, P ≤ 0.001, and ****, P ≤ 0.0001.

The protein sequence of CihC of B. recurrentis, FbpC (BHA007) of B. hermsii HS1, FbpA (FbpC) of B. hermsii FRO, FbpA (FbpC) of B. parkeri, and FbpA (FbpC) of B. turicatae were used for structural prediction (NCBI accession numbers FN552439, KJ599624.1, HE983605, HE983608) (10–12). After removal of the unfolded N-terminal region, based on Clustal Omega (28) amino acid sequence alignments and the existing structures of B. miyamotoi FbpB (7RPS) and FbpA (7RPR), the remaining C-terminal protein sequence was submitted on the AlphaFold2 advanced interface (29) with the default settings using the optional “Refine structures with Amber-Relax” option. Results were exported to UCSF ChimeraX 1.2.5 (30), and modeled using the Matchmaker function for the overlays.

Collection of blood samples and consent documents were approved by the ethics committee at the University Hospital of Frankfurt (control number 160/10 and 222/14), Goethe University of Frankfurt am Main. All healthy blood donors provided written informed consent in accordance with the Declaration of Helsinki.

Previous investigations revealed interaction of FbpA (hereafter re-designated as FbpC) of B. hermsii strains YOR and FRO to fibronectin (10). In situ analyses using bioinformatics suggested that FbpC orthologs of B. parkeri and B. turicatae might also interact with fibronectin, but the authors did not characterize this protein-protein interaction experimentally. In addition, no data are available so far demonstrating binding of fibronectin to CihC of B. recurrentis as well. To extend on these previous observations and close the knowledge gap, we conducted ELISA to gather more detailed information on the fibronectin-binding capability of diverse FbpC orthologs including the N- and C-terminal CihC fragments of B. recurrentis (CihC-N_Br and CihC-C_Br) and an N-terminal FbpC fragment of B. hermsii HS1 (CihC/FbpC-N_Bh-HS1). Hence, FbpC orthologs share high homology to CihC (54%) (10, 11), these molecules were hereafter collectively termed as CihC/FbpC orthologs to avoid misunderstanding due to different nomenclatures used in the literature (see Supplementary Table 1 ).

To investigate interaction with fibronectin, microtiter plates were coated with purified proteins and binding was detected. As shown in Figure 1A , all CihC/FbpC orthologs and the N-terminal CihC fragments of B. hermsii and B. recurrentis clearly bound human fibronectin. In contrast, the C-terminal CihC fragment of B. recurrentis lacking the predicted binding motif did not interact with fibronectin. To further confirm the data obtained with ELISA, a Far-Western blot analysis was conducted. Again, binding of fibronectin could clearly be demonstrated for all CihC/FbpC orthologs but not to the C-terminal CihC fragment ( Figure 1B ). In addition, silver staining ( Figure 1C ) and Western blotting using anti-His antibodies ( Figure 1D ) were also conducted to demonstrate equal loading and purity of the analyzed His-tagged CihC/FbpC orthologs and variants. The findings gathered from the ELISA and Far-Western blotting are in agreement with previous data demonstrating that the central part of CihC/FbpC of B. hermsii HS1 and FRO forms the proposed fibronectin-binding region. In addition, the sequence comparison conducted also revealed that the middle region of the CihC/FbpC orthologs participate in binding of fibronectin but not the C-terminus ( Figure 1E ) (10).

It is well known that a number of complement-interacting proteins of Lyme and relapsing fever borreliae act as ligands for the host serine protease plasminogen, allowing spirochetes to disseminate and penetrate to deeper tissues by degrading the key complement factor C3b, as well as extracellular matrix components such as fibrinogen (reviewed in (33–35)). Thus, we examined the ability of CihC/FbpC orthologs to bind human plasminogen by employing ELISA. After immobilization of the proteins, plasminogen was added and protein-protein complexes were detected. The plasminogen-binding protein BBA70 of B. burgdorferi (24) and BSA served as positive and negative controls, respectively. Although all proteins including the N-terminal truncated fragments of CihC/FbpC of B. hermsii HS1 and CihC of B. recurrentis significantly bound to plasminogen when compared to BSA, the truncated proteins appeared to bind to this host protein to a lower extent ( Figure 2 ). Overall, our results suggest that plasminogen interacts with CihC/FbpC orthologs at different regions as previously demonstrated for the Factor H-binding proteins BpcA of B. parkeri, BtcA of B. turicatae, HcpA of B. recurrentis, BhCRASP-1 of B. hermsii, and CbiA of B. miyamotoi (25, 36–38).

Having demonstrated binding to plasminogen, we next sought to examine the interaction of CihC/FbpC orthologs of B. parkeri, B. turicatae, B. hermsii, and CihC of B. recurrentis with plasminogen in more detail. As depicted in Figure 3 , a dose-dependent binding of plasminogen to all proteins investigated was apparent whereas CihC/FbpC of B. hermsii FRO showed the strongest binding capacity with a calculated apparent dissociation constant of K_d = 24.2 nM ( ± 2.5 nM) following CihC/FbpC of B. parkeri [K_d = 62.7 nM ( ± 4.5 nM)], CihC of B. recurrentis [K_d = 93.1 nM ( ± 8.9 nM)], and CihC/FbpC of B. turicatae [K_d = 284.5 nM (± 46.8 nM)].

In circulation, plasminogen is activated by either urokinase-type (uPA) or tissue-type plasminogen activator (tPA) (39). Upon binding, an accessible cleavage site is a prerequisite for plasmin to maintain its proteolytic activity. To demonstrate whether plasminogen bound to CihC/FbpC orthologs is converted to plasmin, proteins were immobilized to microtiter plates and incubated with plasminogen. After adding uPA, cleavage of the plasmin-specific chromogenic substrate D-Val-Leu-Lys-p-nitroanilide dihydrochloride (S-2251) was continuously monitored for up to 24 h. As expected, the strongest proteolytic activity against S-2251 was achieved with purified plasminogen after conversion to plasmin in the presence of uPA ( Figure 4A ). No cleavage occurred when uPA was omitted from the reaction mixtures. By employing CihC/FbpC orthologs, degradation of the chromogenic substrate could be detected by a constant increase of the absorbance values, thus indicating that protein-bound plasminogen was converted to active plasmin ( Figures 4B–E ). A significant increase was also evident for the interaction of plasminogen with BBA70 of B. burgdorferi, known to exhibit a strong affinity to plasminogen (55.1 nM) (24) ( Figure 4F ). In contrast, no degradation of the chromogenic substrate occurred when BSA was conducted ( Figure 4G ) or when plasminogen or uPA was omitted to the reaction mixtures ( Figures 4A–G ). Cleavage of the substrate after 24 h was considered statistically significant for CihC/FbpC orthologs of B. parkeri and B. hermsii FRO, CihC of B. recurrentis and the control protein BBA70 (P ≤ 0.0001), as well as for CihC/FbpC orthologs of B. turicatae (P = ≤ 0.0001) ( Figure 4H ).

Considering the participation of lysine residues in the interaction of CihC/FbpC orthologs with plasminogen, tranexamic acid as a lysine analog (40) was added to each reaction mixture. While tranexamic acid strongly inhibited cleavage of the chromogenic substrate when CihC/FbpC orthologs of B. parkeri, B. turicatae, and B. hermsii was assayed, a reduced proteolytic activity of plasmin was observed when CihC of B. recurrentis was examined ( Figures 4B–G ). These findings disclose that plasminogen is immediately accessible to uPA and converted to active plasmin upon binding to CihC/FbpC orthologs. Moreover, our data emphasize a prominent role of lysine residues for binding of plasminogen to distinct CihC/FbpC orthologs but also revealed notable differences regarding their interaction with this nonspecific serine protease.

Previous studies revealed CihC of B. recurrentis and FbpC orthologs of B. miyamotoi and B. hermsii as complement-inhibiting proteins that primarily target the CP at the initial activation step by interacting with complement regulator C1-Inh and complement component C1r, respectively (11, 13). To obtain a closer view in the complement inactivation capacity of these borrelial molecules analyzed, comparative ELISA were performed. As expected, none of the CihC/FbpC proteins affected the AP ( Figure 5A ). In contrast, all CihC/FbpC orthologs and the C-terminal CihC fragment significantly terminated activation of the CP ( Figure 5B ) whereby the N-terminal fragments of B. recurrentis and B. hermsii as well as BSA (used as control) did not impair complement activation at all. Also, a strong inhibition could be observed when the C-terminal BBK32 fragment of B. burgdorferi (16) was employed. Our findings indicate that all CihC/FbpC orthologs displayed similar inactivation capacity toward this particular complement pathway. Next, we sought to elucidate potential differences in the efficacy of these borrelial proteins to inactivate the CP. Our analyses demonstrated that all CihC/FbpC orthologs inhibited this pathway with calculated IC₅₀ values ranging between 56 ± 5 nM and 95 ± 11 nM ( Figures 6A–F and Table 1 ) whereas the C-terminal CihC fragment was less potent in inactivating the CP (272 ± 36 nM).

As FbpC of B. miyamotoi and B. hermsii of RF spirochetes and BBK32 of B. burgdorferi are known to bind to C1r (13, 16), we wanted to close the gap of knowledge towards the interaction of C1r with CihC of B. recurrentis and the CihC/FbpC orthologs of B. parkeri and B. turicatae. By conducting ELISA, all proteins analyzed significantly bound C1r but the N-terminal CihC/FbpC fragments of B. hermsii and B. recurrentis did not ( Figure 7 ). Our findings are in full agreement with previous data demonstrating that the C-terminal region mediates binding to C1r and also facilitate inactivation of the CP. Moreover, structural comparison of the CihC/FbpC orthologs of B. hermsii, B. parkeri, B. turicatae, and B. recurrentis using AlphaFold2 predictions suggest that they adopt a very similar fold, forming an anti-parallel 4-helix bundle ( Figure 8 and Supplementary Figures 3, 4 ). However, despite the strong similarities in the overall folding of the CihC/FbpC proteins from different Borrelia strains, their putative C1r binding regions, located in the loop connecting the α1 and α2 helices, has different distributions of charged residues and hydrophobic patches as a result of sequence variation, which may explain differences in binding affinities.

Previous analyses demonstrated that ectopic expression of CihC of B. recurrentis, CihC/FbpC of B. hermsii HS1 as well as BBK32 of B. burgdorferi in serum-sensitive spirochetes facilitate resistance of these gain-of-function Borrelia strains to complement-mediated killing (11, 13). Bactericidal activity of complement could also be efficiently abrogated by pre-incubation of human serum with purified borrelial proteins displaying complement-inactivating properties that leads to the survival of categorically vulnerable spirochetes (13, 16, 18, 23). To further assess the protective nature of the analyzed CihC/FbpC orthologs towards complement, a serum protection assay was conducted by employing all full-length CihC/FbpC orthologs as well as the N- and C-terminal truncated fragments. Initially, native NHS was pre-incubated with purified proteins (5 µM) and then added to viable spirochetes for a final incubation period of 4 h. Almost all CihC/FbpC orthologs including the C-terminal CihC fragment of B. recurrentis and the control protein CspA of B. burgdorferi conferred protection of susceptible B314 cells from bacteriolysis by human complement ( Figure 9 ). However, CihC/FbpC ortholog of B. turicatae lacked sufficient inhibitory capacity under these experimental conditions. As expected, heat-inactivated NHS did not influence viability of spirochetes over the whole incubation period. By contrast, spirochetes were efficiently killed in the presence of native NHS or when serum was pre-incubated with the N-terminal CihC/FbpC fragment of B. hermsii HS1 and B. recurrentis, BSA or with Tris/HCl as buffer control indicating that complement activation was affected by CihC/FbpC orthologs originated from different Borrelia species causing relapsing fever.