A genome-wide association study identifies key modulators of complement factor H binding to malondialdehyde-epitopes.

Genetic variants within complement factor H (CFH), a major alternative complement pathway regulator, are associated with the development of age-related macular degeneration (AMD) and other complementopathies. This is explained with the reduced binding of CFH or its splice variant factor H-like protein 1 (FHL-1) to self-ligands or altered self-ligands (e.g., malondialdehyde [MDA]-modified molecules) involved in homeostasis, thereby causing impaired complement regulation. Considering the critical role of CFH in inhibiting alternative pathway activation on MDA-modified surfaces, we performed an unbiased genome-wide search for genetic variants that modify the ability of plasma CFH to bind MDA in 1,830 individuals and characterized the mechanistic basis and the functional consequences of this. In a cohort of healthy individuals, we identified rs1061170 in CFH and the deletion of CFHR3 and CFHR1 as dominant genetic variants that modify CFH/FHL-1 binding to MDA. We further demonstrated that FHR1 and FHR3 compete with CFH for binding to MDA-epitopes and that FHR1 displays the highest affinity toward MDA-epitopes compared to CFH and FHR3. Moreover, FHR1 bound to MDA-rich areas on necrotic cells and prevented CFH from mediating its cofactor activity on MDA-modified surfaces, resulting in enhanced complement activation. These findings provide a mechanistic explanation as to why the deletion of CFHR3 and CFHR1 is protective in AMD and highlight the importance of genetic variants within the CFH/CFHR3/CFHR1 locus in the recognition of altered-self in tissue homeostasis.

The Protease SplB of Staphylococcus aureus Targets Host Complement Components and Inhibits Complement-Mediated Bacterial Opsonophagocytosis.

Staphylococcus aureus is an opportunistic pathogen that can cause life-threatening infections, particularly in immunocompromised individuals. The high-level virulence of S. aureus largely relies on its diverse and variable collection of virulence factors and immune evasion proteins, including the six serine protease-like proteins SplA to SplF. Spl proteins are expressed by most clinical isolates of S. aureus, but little is known about the molecular mechanisms by which these proteins modify the host's immune response for the benefit of the bacteria. Here, we identify SplB as a protease that inactivates central human complement proteins, i.e., C3, C4, and the activation fragments C3b and C4b, by preferentially cleaving their α-chains. SplB maintained its proteolytic activity in human serum, degrading C3 and C4. SplB further cleaved the components of the terminal complement pathway, C5, C6, C7, C8, and C9. In contrast, the important soluble human complement regulators factor H and C4b-binding protein (C4BP), as well as C1q, were left intact. Thereby, SplB reduced C3b-mediated opsonophagocytosis by human neutrophils as well as C5b-9 deposition on the bacterial surface. In conclusion, we identified the first physiological substrates of the S. aureus extracellular protease SplB. This enzyme inhibits all three complement pathways and blocks opsonophagocytosis. Thus, SplB can be considered a novel staphylococcal complement evasion protein. IMPORTANCE The success of bacterial pathogens in immunocompetent humans depends on the control and inactivation of host immunity. S. aureus, like many other pathogens, efficiently blocks host complement attack early in infection. Aiming to understand the role of the S. aureus-encoded orphan proteases of the Spl operon, we asked whether these proteins play a role in immune escape. We found that SplB inhibits all three complement activation pathways as well as the lytic terminal complement pathway. This blocks the opsonophagocytosis of the bacteria by neutrophils. We also clarified the molecular mechanisms: SplB cleaves the human complement proteins C3, C4, C5, C6, C7, C8, and C9 as well as factor B but not the complement inhibitors factor H and C4BP. Thus, we identify the first physiological substrates of the extracellular protease SplB of S. aureus and characterize SplB as a novel staphylococcal complement evasion protein.

Acquisition of human plasminogen facilitates complement evasion by the malaria parasite Plasmodium falciparum.

We show that the intraerythrocytic stages of the malaria parasite Plasmodium falciparum bind plasminogen and mediate its conversion into plasmin to inactivate parasite-bound C3b. This complement evasion mechanism counteracts terminal complex formation and hence promotes parasite survival in human blood.

Complement catalyzing glomerular diseases.

Complement is an evolutionarily conserved system which is important in the defense against microorganisms and also in the elimination of modified or necrotic elements of the body. Complement is activated in a cascade type manner and activation and all steps of cascade progression are tightly controlled and regulatory interleaved with many processes of inflammatory machinery. Overshooting of the complement system due to dysregulation can result in the two prototypes of primary complement mediated renal diseases: C3 glomerulopathy and thrombotic microangiopathy. Apart from these, complement also is highly activated in many other inflammatory native kidney diseases, such as membranous nephropathy, ANCA-associated necrotizing glomerulonephritis, and IgA nephropathy. Moreover, it likely plays an important role also in the transplant setting, such as in antibody-mediated rejection or in hematopoietic stem cell transplant associated thrombotic microangiopathy. In this review, these glomerular disorders are discussed with regard to the role of complement in their pathogenesis. The consequential, respective clinical trials for complement inhibitory therapy strategies for these diseases are described.

CFHR Gene Variations Provide Insights in the Pathogenesis of the Kidney Diseases Atypical Hemolytic Uremic Syndrome and C3 Glomerulopathy.

Sequence and copy number variations in the human CFHR-Factor H gene cluster comprising the complement genes CFHR1, CFHR2, CFHR3, CFHR4, CFHR5, and Factor H are linked to the human kidney diseases atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy. Distinct genetic and chromosomal alterations, deletions, or duplications generate hybrid or mutant CFHR genes, as well as hybrid CFHR-Factor H genes, and alter the FHR and Factor H plasma repertoire. A clear association between the genetic modifications and the pathologic outcome is emerging: CFHR1, CFHR3, and Factor H gene alterations combined with intact CFHR2, CFHR4, and CFHR5 genes are reported in atypical hemolytic uremic syndrome. But alterations in each of the five CFHR genes in the context of an intact Factor H gene are described in C3 glomerulopathy. These genetic modifications influence complement function and the interplay of the five FHR proteins with each other and with Factor H. Understanding how mutant or hybrid FHR proteins, Factor H::FHR hybrid proteins, and altered Factor H, FHR plasma profiles cause pathology is of high interest for diagnosis and therapy.

Cutting Edge: FHR-1 Binding Impairs Factor H-Mediated Complement Evasion by the Malaria Parasite Plasmodium falciparum.

Human complement is the first line of defense against invading pathogens, including the malaria parasite Plasmodium falciparum We previously demonstrated that human complement represents a particular threat for the clinically relevant blood stages of the parasite. To evade complement-mediated destruction, the parasites acquire factor H (FH) via specific receptors. We now report that the FH-related protein FHR-1 competes with FH for binding to the parasites. FHR-1, which is composed of five complement control protein domains with variable homology to FH but lacks C3b regulatory activity, accumulates on the surfaces of intraerythrocytic schizonts and free merozoites. Although binding of FH to schizont-infected RBCs and merozoites is increased in FHR-1-deficient human serum, the addition of recombinant FHR-1 decreases FH binding. The presence of FHR-1 consequently impairs C3b inactivation and parasite viability. We conclude that FHR-1 acts as a protective factor in human immunity by counteracting FH-mediated microbial complement evasion.

FHR5 Binds to Laminins, Uses Separate C3b and Surface-Binding Sites, and Activates Complement on Malondialdehyde-Acetaldehyde Surfaces.

Factor H related-protein 5 (CFHR5) is a surface-acting complement activator and variations in the CFHR5 gene are linked to CFHR glomerulonephritis. In this study, we show that FHR5 binds to laminin-521, the major constituent of the glomerular basement membrane, and to mesangial laminin-211. Furthermore, we identify malondialdehyde-acetaldehyde (MAA) epitopes, which are exposed on the surface of human necrotic cells (Homo sapiens), as new FHR5 ligands. Using a set of novel deletion fragments, we show that FHR5 binds to laminin-521, MAA epitopes, heparin, and human necrotic cells (HUVECs) via the middle region [short consensus repeats (SCRs) 5-7]. In contrast, surface-bound FHR5 contacts C3b via the C-terminal region (SCRs8-9). Thus, FHR5 uses separate domains for C3b binding and cell surface interaction. MAA epitopes serve as a complement-activating surface by recruiting FHR5. The complement activator FHR5 and the complement inhibitor factor H both bind to oxidation-specific MAA epitopes and FHR5 competes with factor H for binding. The C3 glomerulopathy-associated FHR21-2-FHR5 hybrid protein is more potent in MAA epitope binding and activation compared with wild-type FHR5. The implications of these results for pathology of CFHR glomerulonephritis are discussed. In conclusion, we identify laminins and oxidation-specific MAA epitopes as novel FHR5 ligands and show that the surface-binding site of FHR5 (SCRs5-7) is separated from the C3b binding site (SCRs8-9). Furthermore, FHR5 competes with factor H for binding to MAA epitopes and activates complement on these modified structures.

Long-term data on two sisters with C3GN due to an identical, homozygous CFH mutation and autoantibodies.

C3 glomerulonephritis (C3GN) is a rare but severe form of kidney disease caused by fluid-phase dysregulation of the alternative complement pathway. Causative mutations in complement regulating genes as well as auto-immune forms of C3GN have been described. However, therapy and prognosis in individual patients remain a matter of debate and long-term data are scarce. This also applies for the management of transplant patients as disease recurrence post-transplant is frequent. Here, we depict the clinical courses of two sisters with the unique combination of an identical, homozygous mutation in the complement factor H (CFH) gene as well as autoantibodies with a clinical follow-up of more than 20 years. Interestingly, the sisters presented with discordant clinical courses of C3GN with normal kidney function in one (patient A) and end-stage kidney disease in the other sister (patient B). In patient B, eculizumab was administered immediately prior to and in the course after kidney transplantation, with the result of a stable graft function without any signs of disease recurrence. Comprehensive genetic work-up revealed no further disease-causing mutation in both sisters. Intriguingly, the auto-antibody profile substantially differed in both sisters: autoantibodies in patient A reduced the C3b deposition, while the antibodies identified in patient B increased complement activation and deposition of split products. This study underlines the concept of a personalized-medicine approach in complement-associated diseases after thorough evaluation of the individual risk profile in each patient.

The MFHR1 Fusion Protein Is a Novel Synthetic Multitarget Complement Inhibitor with Therapeutic Potential.

The complement system is essential for host defense, but uncontrolled complement system activation leads to severe, mostly renal pathologies, such as atypical hemolytic uremic syndrome or C3 glomerulopathy. Here, we investigated a novel combinational approach to modulate complement activation by targeting C3 and the terminal pathway simultaneously. The synthetic fusion protein MFHR1 links the regulatory domains of complement factor H (FH) with the C5 convertase/C5b-9 inhibitory fragment of the FH-related protein 1. In vitro, MFHR1 showed cofactor and decay acceleration activity and inhibited C5 convertase activation and C5b-9 assembly, which prevented C3b deposition and reduced C3a/C5a and C5b-9 generation. Furthermore, this fusion protein showed the ability to escape deregulation by FH-related proteins and form multimeric complexes with increased inhibitory activity. In addition to substantially inhibiting alternative and classic pathway activation, MFHR1 blocked hemolysis mediated by serum from a patient with aHUS expressing truncated FH. In FH-/- mice, MFHR1 administration augmented serum C3 levels, reduced abnormal glomerular C3 deposition, and ameliorated C3 glomerulopathy. Taking the unique design of MFHR1 into account, we suggest that the combination of proximal and terminal cascade inhibition together with the ability to form multimeric complexes explain the strong inhibitory capacity of MFHR1, which offers a novel basis for complement therapeutics.

Streptococcus pneumoniae From Patients With Hemolytic Uremic Syndrome Binds Human Plasminogen via the Surface Protein PspC and Uses Plasmin to Damage Human Endothelial Cells.

Pneumococcal hemolytic uremic syndrome (HUS) in children is caused by infections with Streptococcus pneumoniae. Because endothelial cell damage is a hallmark of HUS, we studied how HUS-inducing pneumococci derived from infant HUS patients during the acute phase disrupt the endothelial layer. HUS pneumococci efficiently bound human plasminogen. These clinical isolates of HUS pneumococci efficiently bound human plasminogen via the bacterial surface proteins Tuf and PspC. When activated to plasmin at the bacterial surface, the active protease degraded fibrinogen and cleaved C3b. Here, we show that PspC is a pneumococcal plasminogen receptor and that plasmin generated on the surface of HUS pneumococci damages endothelial cells, causing endothelial retraction and exposure of the underlying matrix. Thus, HUS pneumococci damage endothelial cells in the blood vessels and disturb local complement homeostasis. Thereby, HUS pneumococci promote a thrombogenic state that drives HUS pathology.

FHR3 Blocks C3d-Mediated Coactivation of Human B Cells.

The autoimmune renal disease deficient for complement factor H-related (CFHR) genes and autoantibody-positive form of hemolytic uremic syndrome is characterized by the presence of autoantibodies specific for the central complement regulator, factor H, combined with a homozygous deficiency, mostly in CFHR3 and CFHR1 Because FHR3 and FHR1 bind to C3d and inactivated C3b, which are ligands for complement receptor type 2 (CR2/CD21), the aim of the current study was to examine whether FHR3-C3d or FHR1-C3d complexes modulate B cell activation. Laser-scanning microscopy and automated image-based analysis showed that FHR3, but not FHR1 or factor H, blocked B cell activation by the BCR coreceptor complex (CD19/CD21/CD81). FHR3 bound to C3d, thereby inhibiting the interaction between C3d and CD21 and preventing colocalization of the coreceptor complex with the BCR. FHR3 neutralized the adjuvant effect of C3d on B cells, as shown by inhibited intracellular CD19 and Akt phosphorylation in Raji cells, as well as Ca(2+) release in peripheral B cells. In cases of CFHR3/CFHR1 deficiency, the FHR3 binding sites on C3d are occupied by factor H, which lacks B cell-inhibitory functions. These data provide evidence that FHR3, which is absent in patients with the autoimmune form of hemolytic uremic syndrome, is involved in B cell regulation.

BGA66 and BGA71 facilitate complement resistance of Borrelia bavariensis by inhibiting assembly of the membrane attack complex.

Borrelia (B.) bavariensis exhibits a marked tropism for nervous tissues and frequently causes neurological manifestations in humans. The molecular mechanism by which B. bavariensis overcomes innate immunity, in particular, complement remains elusive. In contrast to other serum-resistant spirochetes, none of the B. bavariensis isolates investigated bound complement regulators of the alternative (AP) and classical pathway (CP) or proteolytically inactivated complement components. Focusing on outer surface proteins BGA66 and BGA71, we demonstrated that both molecules either inhibit AP, CP and terminal pathway (TP) activation, or block activation of the CP and TP respectively. Both molecules bind complement components C7, C8 and C9, and thereby prevent assembly of the terminal complement complex. This inhibitory activity was confirmed by the introduction of the BGA66 and BGA71 encoding genes into a serum-sensitive B. garinii strain. Transformed spirochetes producing either BGA66 or BGA71 overcome complement-mediated killing, thus indicating that both proteins independently facilitate serum resistance of B. bavariensis. The generation of C-terminally truncated proteins as well as a chimeric BGA71 protein lead to the localization of the complement-interacting binding site within the N-terminus. Collectively, our data reveal a novel immune evasion strategy of B. bavariensis that is directed against the activation of the TP.

Age-related macular degeneration associated polymorphism rs10490924 in ARMS2 results in deficiency of a complement activator.

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. The polymorphism rs10490924 in the ARMS2 gene is highly associated with AMD and linked to an indel mutation (del443ins54), the latter inducing mRNA instability. At present, the function of the ARMS2 protein, the exact cellular sources in the retina and the biological consequences of the rs10490924 polymorphism are unclear. METHODS: Recombinant ARMS2 was expressed in Pichia pastoris, and protein functions were studied regarding cell surface binding and complement activation in human serum using fluoresence-activated cell sorting (FACS) as well as laser scanning microscopy (LSM). Biolayer interferometry defined protein interactions. Furthermore, endogenous ARMS2 gene expression was studied in human blood derived monocytes and in human induced pluripotent stem cell-derived microglia (iPSdM) by PCR and LSM. The ARMS2 protein was localized in human genotyped retinal sections and in purified monocytes derived from AMD patients without the ARMS2 risk variant by LSM. ARMS2 expression in monocytes under oxidative stress was determined by Western blot analysis. RESULTS: Here, we demonstrate for the first time that ARMS2 functions as surface complement regulator. Recombinant ARMS2 binds to human apoptotic and necrotic cells and initiates complement activation by recruiting the complement activator properdin. ARMS2-properdin complexes augment C3b surface opsonization for phagocytosis. We also demonstrate for the first time expression of ARMS2 in human monocytes especially under oxidative stress and in microglia cells of the human retina. The ARMS2 protein is absent in monocytes and also in microglia cells, derived from patients homozygous for the ARMS2 AMD risk variant (rs10490924). CONCLUSIONS: ARMS2 is likely involved in complement-mediated clearance of cellular debris. As AMD patients present with accumulated proteins and lipids on Bruch's membrane, ARMS2 protein deficiency due to the genetic risk variant might be involved in drusen formation.

The Plasmodium falciparum blood stages acquire factor H family proteins to evade destruction by human complement.

The acquisition of regulatory proteins is a means of blood-borne pathogens to avoid destruction by the human complement. We recently showed that the gametes of the human malaria parasite Plasmodium falciparum bind factor H (FH) from the blood meal of the mosquito vector to assure successful sexual reproduction, which takes places in the mosquito midgut. While these findings provided a first glimpse of a complex mechanism used by Plasmodium to control the host immune attack, it is hitherto not known, how the pathogenic blood stages of the malaria parasite evade destruction by the human complement. We now show that the human complement system represents a severe threat for the replicating blood stages, particularly for the reinvading merozoites, with complement factor C3b accumulating on the surfaces of the intraerythrocytic schizonts as well as of free merozoites. C3b accumulation initiates terminal complement complex formation, in consequence resulting in blood stage lysis. To inactivate C3b, the parasites bind FH as well as related proteins FHL-1 and CFHR-1 to their surface, and FH binding is trypsin-resistant. Schizonts acquire FH via two contact sites, which involve CCP modules 5 and 20. Blockage of FH-mediated protection via anti-FH antibodies results in significantly impaired blood stage replication, pointing to the plasmodial complement evasion machinery as a promising malaria vaccine target.

Complement factor H binds malondialdehyde epitopes and protects from oxidative stress.

Oxidative stress and enhanced lipid peroxidation are linked to many chronic inflammatory diseases, including age-related macular degeneration (AMD). AMD is the leading cause of blindness in Western societies, but its aetiology remains largely unknown. Malondialdehyde (MDA) is a common lipid peroxidation product that accumulates in many pathophysiological processes, including AMD. Here we identify complement factor H (CFH) as a major MDA-binding protein that can block both the uptake of MDA-modified proteins by macrophages and MDA-induced proinflammatory effects in vivo in mice. The CFH polymorphism H402, which is strongly associated with AMD, markedly reduces the ability of CFH to bind MDA, indicating a causal link to disease aetiology. Our findings provide important mechanistic insights into innate immune responses to oxidative stress, which may be exploited in the prevention of and therapy for AMD and other chronic inflammatory diseases.

Complement Factor H-Related 5-Hybrid Proteins Anchor Properdin and Activate Complement at Self-Surfaces.

C3 glomerulopathy (C3G) is a severe kidney disease for which no specific therapy exists. The causes of C3G are heterogeneous, and defective complement regulation is often linked to C3G pathogenesis. Copy number variations in the complement factor H-related (CFHR) gene cluster on chromosome 1q32 and CFHR5 mutant proteins associate with this disease. Here, we identified CFHR5 as a pattern recognition protein that binds to damaged human endothelial cell surfaces and to properdin, the human complement activator. We found the two N-terminal short consensus repeat domains of CFHR5 contact properdin and mediate dimer formation. These properdin-binding segments are duplicated in two mutant CFHR5 proteins, CFHR2-CFHR5Hyb from German patients with C3G and CFHR5Dup from Cypriot patients with C3G. Each of these mutated proteins assembled into large multimeric complexes and, compared to CFHR5, bound damaged human cell surfaces and properdin with greater intensity and exacerbated local complement activation. This enhanced surface binding and properdin recruitment was further evidenced in the mesangia of a transplanted and explanted kidney from a German patient with a CFHR2-CFHR5Hyb protein. Enhanced properdin staining correlated with local complement activation with C3b and C5b-9 deposition on the mesangial cell surface in vitro This gain of function in complement activation for two disease-associated CFHR5 mutants describes a new disease mechanism of C3G, which is relevant for defining appropriate treatment options for this disorder.

Activation of endogenously expressed ion channels by active complement in the retinal pigment epithelium.

Defective regulation of the alternative pathway of the complement system is believed to contribute to damage of retinal pigment epithelial (RPE) cells in age-related macular degeneration. Thus we investigated the effect of complement activation on the RPE cell membrane by analyzing changes in membrane conductance via patch-clamp techniques and Ca(2+) imaging. Exposure of human ARPE-19 cells to complement-sufficient normal human serum (NHS) (25 %) resulted in a biphasic increase in intracellular free Ca(2+) ([Ca(2+)]i); an initial peak followed by sustained Ca(2+) increase. C5- or C7-depleted sera did not fully reproduce the signal generated by NHS. The initial peak of the Ca(2+) response was reduced by sarcoplasmic Ca(2+)-ATPase inhibitor thapsigargin, L-type channel blockers (R)-(+)-BayK8644 and isradipine, transient-receptor-potential (TRP) channel blocker ruthenium-red and ryanodine receptor blocker dantrolene. The sustained phase was carried by CaV1.3 L-type channels via tyrosine-phosphorylation. Changes in [Ca(2+)]I were accompanied by an abrupt hyperpolarization, resulting from a transient increase in membrane conductance, which was absent under extracellular Ca(2+)- or K(+)-free conditions and blocked by (R)-(+)-BayK8644 or paxilline, a maxiK channel inhibitor. Single-channel recordings confirmed the contribution of maxiK channels. Primary porcine RPE cells responded to NHS in a comparable manner. Pre-incubation with NHS reduced H2O2-induced cell death. In summary, in a concerted manner, C3a, C5a and sC5b-9 increased [Ca(2+)]i by ryanodine-receptor-dependent activation of L-type channels in addition to maxi-K channels and TRP channels absent from any insertion of a lytic pore.

Genetic influences on plasma CFH and CFHR1 concentrations and their role in susceptibility to age-related macular degeneration.

It is a longstanding puzzle why non-coding variants in the complement factor H (CFH) gene are more strongly associated with age-related macular degeneration (AMD) than functional coding variants that directly influence the alternative complement pathway. The situation is complicated by tight genetic associations across the region, including the adjacent CFH-related genes CFHR3 and CFHR1, which may themselves influence the alternative complement pathway and are contained within a common deletion (CNP147) which is associated with protection against AMD. It is unclear whether this association is mediated through a protective effect of low plasma CFHR1 concentrations, high plasma CFH or both. We examined the triangular relationships of CFH/CFHR3/CFHR1 genotype, plasma CFH or CFHR1 concentrations and AMD susceptibility in combined case-control (1256 cases, 1020 controls) and cross-sectional population (n = 1004) studies and carried out genome-wide association studies of plasma CFH and CFHR1 concentrations. A non-coding CFH SNP (rs6677604) and the CNP147 deletion were strongly correlated both with each other and with plasma CFH and CFHR1 concentrations. The plasma CFH-raising rs6677604 allele and raised plasma CFH concentration were each associated with AMD protection. In contrast, the protective association of the CNP147 deletion with AMD was not mediated by low plasma CFHR1, since AMD-free controls showed increased plasma CFHR1 compared with cases, but it may be mediated by the association of CNP147 with raised plasma CFH concentration. The results are most consistent with a regulatory locus within a 32 kb region of the CFH gene, with a major effect on plasma CFH concentration and AMD susceptibility.

BBA70 of Borrelia burgdorferi is a novel plasminogen-binding protein.

The Lyme disease spirochete Borrelia burgdorferi lacks endogenous, surface-exposed proteases. In order to efficiently disseminate throughout the host and penetrate tissue barriers, borreliae rely on recruitment of host proteases, such as plasmin(ogen). Here we report the identification of a novel plasminogen-binding protein, BBA70. Binding of plasminogen is dose-dependent and is affected by ionic strength. The BBA70-plasminogen interaction is mediated by lysine residues, primarily located in a putative C-terminal α-helix of BBA70. These lysine residues appear to interact with the lysine-binding sites in plasminogen kringle domain 4 because a deletion mutant of plasminogen lacking that domain was unable to bind to BBA70. Bound to BBA70, plasminogen activated by urokinase-type plasminogen activator was able to degrade both a synthetic chromogenic substrate and the natural substrate fibrinogen. Furthermore, BBA70-bound plasmin was able to degrade the central complement proteins C3b and C5 and inhibited the bacteriolytic effects of complement. Consistent with these functional activities, BBA70 is located on the borrelial outer surface. Additionally, serological evidence demonstrated that BBA70 is produced during mammalian infection. Taken together, recruitment and activation of plasminogen could play a beneficial role in dissemination of B. burgdorferi in the human host and may possibly aid the spirochete in escaping the defense mechanisms of innate immunity.

Versatile roles of CspA orthologs in complement inactivation of serum-resistant Lyme disease spirochetes.

CspA of the Lyme disease spirochete Borrelia burgdorferi represents a key molecule in immune evasion, protecting borrelial cells from complement-mediated killing. As previous studies focused almost exclusively on CspA of B. burgdorferi, here we investigate the different binding capacities of CspA orthologs of Borrelia burgdorferi, B. afzelii, and B. spielmanii for complement regulator factor H and plasminogen and their ability to inhibit complement activation by either binding these host-derived plasma proteins or independently by direct interaction with components involved in formation of the lethal, pore-like terminal complement complex. To further examine their function in serum resistance in vivo, a serum-sensitive B. garinii strain was used to generate spirochetes, ectopically producing functional CspA orthologs. Irrespective of their species origin, all three CspA orthologs impart resistance to complement-mediated killing when produced in a serum-sensitive B. garinii surrogate strain. To analyze the inhibitory effect on complement activation and to assess the potential to inactivate C3b by binding of factor H and plasminogen, recombinant CspA orthologs were also investigated. All three CspA orthologs simultaneously bound factor H and plasminogen but differed in regard to their capacity to inactivate C3b via bound plasmin(ogen) and inhibit formation of the terminal complement complex. CspA of B. afzelii binds plasmin(ogen) and inhibits the terminal complement complex more efficiently than CspA of B. burgdorferi and B. spielmanii. Taken together, CspA orthologs of serum-resistant Lyme disease spirochetes act as multifunctional evasion molecules that inhibit complement on two central activation levels, C3b generation and assembly of the terminal complement complex.