Analysis of the OspE determinants involved in binding of factor H and OspE-targeting antibodies elicited during Borrelia burgdorferi infection in mice

Immune evasion by Lyme spirochetes is a multifactorial process involving numerous mechanisms. The OspE protein family undergoes antigenic variation during infection and binds factor H (fH) and possibly FHL-1/reconectin. In Borrelia burgdorferi B31MI, the OspE family consists of three paralogs: BBL39 (ErpA), BBP38, and BBN38 (ErpP). BBL39 and BBP38 are identical and therefore are referred to here as BBL39. The goals of this study were to assess the specificity of the antibody (Ab) response to the OspE paralogs and to identify the domains or determinants of OspE that are required for the binding of fH and OspE-targeting Abs that develop during infection. Here we demonstrate that at least some of the anti-OspE Abs produced during infection are paralog specific and that Ab binding requires conformational determinants whose formation requires both the N- and C-terminal domains of OspE. The binding of fH to OspE was also found to be dependent on conformational determinants. It is also demonstrated here that all of the OspE paralogs expressed by B. burgdorferi B31MI are capable of binding fH. The binding of fH to members of the OspF protein family was also assessed. In contrast to an earlier report, no binding of BBO39 or BBR42 to human fH was detected. Lastly, a series of competitive binding enzyme-linked immunosorbent assay analyses, designed to determine if fH and infection serum Abs bind to the same sites on OspE, revealed that these ligands interact with different regions of OspE.     

Assessment of the regions within complement regulator-acquiring surface protein (CRASP)-2 of Borrelia burgdorferi required for interaction with host immune regulators FHL-1 and factor H

The ability of Borrelia burgdorferi sensu lato to survive and persist in a variety of vertebrate hosts is a multifactorial process. Several potential mechanisms of immune evasion have been identified. We have shown that some Borrelia species bind host-derived fluid-phase immune regulators FHL-1 and factor H to their surface via complement regulator-acquiring surface proteins (CRASPs). Factor H and FHL-1 serve as cofactors for factor I, a serine protease that cleaves C3b directly on the cell surface and thereby confers resistance of spirochetes to complement-mediated lysis. Among the CRASP molecules produced by B. burgdorferi, BbCRASP-2 represents a novel FHL-1 and factor H binding protein that is distinct from other borrelial CRASP molecules and is predominantly expressed by serum-resistant Borrelia strains. The aim of this study was to identify BbCRASP-2 determinants required for FHL-1 and factor H binding. A number of recombinant BbCRASP-2 mutants were generated by in vitro mutagenesis and tested for factor H and FHL-1 binding employing ELISA. Up to 8 amino acid substitutions in the proposed binding regions 2 and 3 of BbCRASP-2 resulted in reduced or complete loss of FHL-1 and/or factor H binding. These results suggest that the factor H/FHL-1 binding regions are discontinuous and long distance interaction is involved in binding of both immune regulators. Furthermore, putative coiled-coil structural elements as recently discussed to be important in the interaction of BbCRASP-1 with factor H seem to play a subordinate role for binding of BbCRASP-2 to FHL-1 and factor H. The elucidation of host–pathogen interactions will help to develop novel therapeutic strategies against Lyme disease/borreliosis.     

Characterization of genetic predisposition and autoantibody profile in atypical haemolytic–uraemic syndrome

We previously reported that Indian paediatric patients with atypical haemolytic–uraemic syndrome (aHUS) showed high frequencies of anti‐complement factor H (FH) autoantibodies that are correlated with homozygous deletion of the genes for FH‐related proteins 1 and 3 (FHR1 and FHR3) (FHR1/3^–/–). We now report that Indian paediatric aHUS patients without anti‐FH autoantibodies also showed modestly higher frequencies of the FHR1/3^–/– genotype. Further, when we characterized epitope specificities and binding avidities of anti‐FH autoantibodies in aHUS patients, most anti‐FH autoantibodies were directed towards the FH cell‐surface anchoring polyanionic binding site‐containing C‐terminal short conservative regions (SCRs) 17–20 with higher binding avidities than for native FH. FH SCR17–20‐binding anti‐FH autoantibodies also bound the other cell‐surface anchoring polyanionic binding site‐containing region FH SCR5–8, at lower binding avidities. Anti‐FH autoantibody avidities correlated with antibody titres. These anti‐FH autoantibody characteristics did not differ between aHUS patients with or without the FHR1/3^–/– genotype. Our data suggest a complex matrix of interactions between FHR1‐FHR3 deletion, immunomodulation and anti‐FH autoantibodies in the aetiopathogenesis of aHUS.     

The OspE-related proteins inhibit complement deposition and enhance serum resistance of Borrelia burgdorferi, the lyme disease spirochete

Borrelia burgdorferi, the Lyme disease spirochete, binds the host complement inhibitors factor H (FH) and FH-like protein 1 (FHL-1). Binding of FH/FHL-1 by the B. burgdorferi proteins CspA and the OspE-related proteins is thought to enhance resistance to serum-mediated killing. While previous reports have shown that CspA confers serum resistance in B. burgdorferi, it is unclear whether the OspE-related proteins are relevant in B. burgdorferi serum resistance when OspE is expressed on the borrelial surface. To assess the role of the OspE-related proteins, we overexpressed them in a serum-sensitive CspA mutant strain. OspE overexpression enhanced serum resistance of the CspA-deficient organisms. Furthermore, FH was more efficiently bound to the B. burgdorferi surface when OspE was overexpressed. Deposition of complement components C3 and C5b-9 (the membrane attack complex), however, was reduced on the surface of the OspE-overexpressing strain compared to that on the CspA mutant strain. These data demonstrate that OspE proteins expressed on the surface of B. burgdorferi bind FH and protect the organism from complement deposition and subsequent serum-mediated destruction.     

Selective binding of Borrelia burgdorferi OspE paralogs to factor H and serum proteins from diverse animals: possible expansion of the role of OspE in Lyme disease pathogenesis

The binding of Borrelia burgdorferi OspE, OspF, and family 163 (Elp) proteins to factor H/factor H-like protein 1 (FHL-1) and other serum proteins from different animals was assessed. OspE paralogs bound factor H and unidentified serum proteins from a subset of animals, while OspF and Elp proteins did not. These data advance our understanding of factor H binding, the host range of the Lyme spirochetes, and the expanding role of OspE in pathogenesis.     

FHR-1, an additional human plasma protein,binds to complement regulator-acquiring surface proteins of Borrelia burgdorferi

The Borrelia burgdorferi strain LW2, a causative agent of Lyme disease, expresses up to five distinct complement regulator-acquiring surface proteins (CRASPs) that bind the human immune regulators factor H and/or FHL-1. In the present study, we identify FHR-1, a member of the human factor H protein family, as an additional ligand for CRASP-3, CRASP-4, and CRASP-5 but not for CRASP-1 and CRASP-2. A comparative analysis of the binding characteristics revealed unique and distinct binding profiles of the three host immune regulators to CRASPs. FHR-1 binds to CRASP-3, CRASP-4, and CRASP-5; factor H binds to all five CRASPs, and FHL-1 binds preferentially to CRASP-1 and CRASP-2. On the pathogen site, CRASP-3 interacts predominantly with factor H; CRASP-4 shows a preference for FHR-1, and CRASP-5 binds strongly and with equal intensity FHR-1 and factor H. Thus, expression of several CRASPs with distinct binding properties for host proteins allows the pathogen to attach functionally distinct host proteins to its surface.     

Persistence of borrelial DNA in the joints of Borrelia burgdorferi‐infected mice after ceftriaxone treatment

Yrjänäinen H, Hytönen J, Hartiala P, Oksi J, Viljanen MK. Persistence of borrelial DNA in the joints of Borrelia burgdorferi‐infected mice after ceftriaxone treatment. APMIS 2010; 118: 665–73. We have earlier shown that Borrelia burgdorferi‐infected and ceftriaxone‐treated mice have viable spirochetes in their body, since immunosuppressive treatment allows B. burgdorferi to be detected by culture. However, the niche of the persisting spirochetes remained unknown. In the present study, we analyzed the tissues of B. burgdorferi‐infected and ceftriaxone‐treated mice by culture and PCR to reveal the foci of persisting spirochetes. C3H/HeN mice were infected via intradermal needle injection with B. burgdorferi s.s. N40. The mice were treated as follows: (i) short (5 days) and (ii) long (18 days) course of ceftriaxone at 2 weeks of infection and killed after either 10 or 30 weeks, or (iii) the mice received ceftriaxone for 5 days at 18 weeks of infection and were killed 21 weeks after the treatment. All samples of ceftriaxone‐treated mice were culture negative, whereas all untreated controls were culture positive. Importantly, B. burgdorferi DNA was detected in the joints of 30–100% of the treated mice. In conclusion, these results combined with earlier results suggest that the joint or a tissue adjacent to the joint is the niche of persisting B. burgdorferi in ceftriaxone‐treated mice.     

Expression of complement factor H binding immunoevasion proteins in Borrelia garinii isolated from patients with neuroborreliosis

The Lyme disease-pathogen Borrelia burgdorferi binds the complement inhibitor factor H (FH) to its outer surface protein E- (OspE) and BbA68-families of lipoproteins. In earlier studies, only serum-resistant strains of the genospecies B. burgdorferi sensu stricto or B. afzelii, but not serum-sensitive B. garinii strains, have been shown to bind FH. Since B. garinii often causes neuroborreliosis in man, we have readdressed the interactions of B. garinii with FH. B. garinii 50/97 strain did not express FH-binding proteins. By transforming the B. garinii 50/97 strain with an OspE-encoding gene from complement-resistant B. burgdorferi (ospE-297), its resistance to serum killing could be increased. OspE genes were detected and cloned from the B. garinii BITS, Pistoia and 40/97 strains by PCR and sequencing. The deduced amino acid sequences differed in an N-terminal lysine-rich FH-binding region from OspE sequences of resistant strains. Recombinant B. garinii BITS OspE protein was found to have a considerably lower FH-binding activity than the B. burgdorferi sensu stricto 297 OspE protein P21 (P21-297). Unlike bacteria that had been kept in culture for a long time, neurovirulent B. garinii strains from neuroborreliosis patients were found to express approximately 27-kDa FH-binding proteins. These were not recognized by polyclonal anti-OspE or anti-BbA68 antibodies. We conclude that B. garinii strains carry ospE genes but have a decreased expression of OspE proteins and a reduced ability to bind FH, especially when grown for prolonged periods in vitro. Recently isolated neuroinvasive B. garinii strains, however, can express FH-binding proteins, which may contribute to the virulence of neuroborreliosis-causing B. garinii strains.     

Borrelia burgdorferi Infection-Associated Surface Proteins ErpP,ErpA, and ErpC Bind Human Plasminogen

Host-derived plasmin plays a critical role in mammalian infection by Borrelia burgdorferi. The Lyme disease spirochete expresses several plasminogen-binding proteins. Bound plasminogen is converted to the serine protease plasmin and thereby may facilitate the bacterium''s dissemination throughout the host by degrading extracellular matrix. In this work, we demonstrate plasminogen binding by three highly similar borrelial outer surface proteins, ErpP, ErpA, and ErpC, all of which are expressed during mammalian infection. Extensive characterization of ErpP demonstrated that this protein bound in a dose-dependent manner to lysine binding site I of plasminogen. Removal of three lysine residues from the carboxy terminus of ErpP significantly reduced binding of plasminogen, and the presence of a lysine analog, ɛ-aminocaproic acid, inhibited the ErpP-plasminogen interaction, thus strongly pointing to a primary role for lysine residues in plasminogen binding. Ionic interactions are not required in ErpP binding of plasminogen, as addition of excess NaCl or the polyanion heparin did not have any significant effect on binding. Plasminogen bound to ErpP could be converted to the active enzyme, plasmin. The three plasminogen-binding Erp proteins can also bind the host complement regulator factor H. Plasminogen and factor H bound simultaneously and did not compete for binding to ErpP, indicating separate binding sites for both host ligands and the ability of the borrelial surface proteins to bind both host proteins.Lyme disease is the most commonly reported arthropod-borne disease in the United States (8). Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to its hosts through the bites of infected Ixodes ticks. In the earliest stage of Lyme disease, a bull''s-eye-shaped rash, erythema migrans, occurs as the spirochete spreads outward from the site of the tick bite. If left untreated, serious clinical outcomes can occur, including arthritis, neuropathies, and carditis (48).The bacterium disseminates from the bite site to other host tissues. B. burgdorferi can traverse the epithelium and invade vascular walls but is rarely abundant in blood (1). In addition, B. burgdorferi can pass through the blood-brain barrier to enter the central nervous system (58). The spirochete, unlike many invasive pathogens, lacks surface protease activities (12, 26). Therefore, binding of host proteases to the surface of the bacterium may aid in the spirochete''s dissemination. Indeed, B. burgdorferi binds plasminogen, a component of the host''s fibrinolytic system (12, 19). Plasminogen circulates in the plasma as an inactive proenzyme and is activated by tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA) to plasmin (55). Plasminogen binding is an important virulence factor for invasive pathogens such as group A streptococci and Staphylococcus, as well as Borrelia species (10, 43, 55). The binding of plasminogen to bacteria and its subsequent activation allow bacteria to degrade the host''s extracellular matrix and basement membranes either through the direct protease activity of plasmin or by plasmin''s activation of host matrix metalloproteases (MMPs). B. burgdorferi has previously been shown to bind plasminogen, which is rapidly converted to active plasmin in the presence of host plasminogen activator (11). In vitro, plasmin-coated B. burgdorferi is able to penetrate endothelial cell monolayers (12). Surface-associated plasmin on B. burgdorferi can directly degrade fibronectin, a major component of the extracellular matrix, as well as laminin and vitronectin (11, 19). B. burgdorferi induces the release of MMP-9 (gelatinase) and MMP-1 (collagenase) from human cells, and plasmin-coated B. burgdorferi activates pro-MMP-9 (20), initiating a cascade that leads to degradation of basement membranes. Plasminogen has previously been shown to be important in B. burgdorferi pathogenesis. Although not strictly required for infection, plasminogen was required for efficient dissemination in ticks, and its absence decreased spirochetemia in plasminogen-deficient mice (10).Plasminogen-binding proteins of B. burgdorferi have previously been identified, including the outer-surface lipoprotein OspA (19). A role for OspC in plasminogen binding has also been suggested (31). However, OspA is generally not expressed during human infection, and OspC production ceases within the first few days of mammalian infection (13, 24, 25, 34, 42). Other, unidentified plasminogen-binding proteins have been observed in B. burgdorferi, including a protein(s) with an approximate molecular mass of 20 kDa, which is close to the size of several Erp proteins (12, 19). The members of the Erp family of outer-surface lipoproteins are expressed at high levels during mammalian infection (15, 23, 38-41).Lyme disease spirochetes contain numerous DNA elements, including the main chromosome as well as linear and circular plasmids (6). Infectious isolates carry several distinct yet homologous elements called cp32s, circular prophages of approximately 32 kb (54). All cp32 elements encode one or two Erp proteins, which can vary widely in amino acid sequence (50). However, all erp loci are preceded by nearly identical promoter regions (36, 53). Hence, most of the erp genes analyzed follow the same pattern of expression, being repressed in the tick vector but synthesized during mammalian infection (15, 21, 23, 35, 37-41). Roles for most of the Erp proteins have yet to be defined. ErpX has been demonstrated to bind host laminin (our unpublished results and reference 3). Three Erp proteins bind the host complement regulator factor H and factor H-related protein 1: ErpP, ErpC, and ErpA (22, 28, 29). Some factor H binding proteins of other human pathogens have been demonstrated to bind multiple ligands, including plasminogen (30, 47). These data, and the presence of unidentified plasminogen-binding proteins in B. burgdorferi, prompted us to examine if Erp proteins are able to bind plasminogen.     

Heterogeneity in the complement-dependent bacteriolysis within the species of Borrelia burgdorferi

Sixteen Borrelia burgdorferi strains, including all three species, were compared in a colorimetric bactericidal assay for their ability to escape the complement-dependent bacteriolysis on incubation in normal human serum free of specific antibodies (NHS). The species B. afzelii was found to be serum resistant (EB1, EB3, FEM1, FEM2, Pko), whereas strains of the species B. garinii were found to be serum sensitive (1/B29, G1, G2, PSth, PBr, PTrob). Six strains, mainly B. burgdorferi sensu stricto, were only partially sensitive (Z25, 297, B31, PKa-I, PBi). All strains activated the complement cascade in NHS, whereas only four strains (G1, G2, PBr, PSth) could activate complement in the presence of EGTA-Mg. After complement activation, covalently bound C3 fragments (C3b, iC3b) were detected on serum-sensitive as well as serum-resistant borrelial strains. Heterogeneity, however, was observed between serum-resistant and serum-sensitive strains with respect to deposition of C6 and C9. Whereas serum-sensitive strains were strongly positive for C6 and C9 and were, therefore, killed by the terminal complement complex (TCC), serum-resistant strains were devoid of C6 and C9 on their cell surface. The serum resistance may, therefore, be due to an absent or only transient formation of TCC on the bacterial surface. Received: 17 September 1996     

Identification and characterization of the factor H and FHL-1 binding complement regulator-acquiring surface protein 1 of the Lyme disease spirochete Borrelia spielmanii sp. nov

Borrelia spielmanii, one of the etiological agents of Lyme disease found in Europe, evades host complement-mediated killing by recruitment of the immune regulators factor H and FHL-1 from human serum. Serum-resistant and intermediate serum-resistant isolates express up to 3 distinct complement regulator-acquiring surface proteins (CRASPs) that bind factor H and/or FHL-1. The present study describes identification and functional characterization of BsCRASP-1 as the dominant factor H and FHL-1 binding protein of B. spielmanii. BsCRASP-1 is a 27.7kDa outer surface lipoprotein, which after processing has a predicted mass of 24.9kDa. BsCRASP-1 is encoded by a single copy gene, cspA, that maps to a linear plasmid of approximately 55kb. Ligand affinity blot techniques revealed that both native and recombinant BsCRASP-1 from different isolates can strongly bind FHL-1, but only weakly factor H. Deletion mutants of recombinant BsCRASP-1 were generated and a high-affinity binding site for factor H and FHL-1 was mapped to its carboxy-terminal 10-amino-acid residue domain. Similarly, the dominant binding site of factor H and FHL-1 was localized to short consensus repeats (SCRs) 5-7. Factor H and FHL-1 maintained cofactor activity for factor I-mediated C3b inactivation when bound to full-length BsCRASP-1 but not to a deletion mutant lacking the carboxy-terminal 10-amino-acid residue domain. In conclusion, BsCRASP-1 binds the host immune regulators factor H and FHL-1, and is suggested to represent a key molecule of B. spielmanii for complement resistance. Thus, BsCRASP-1 most likely contributes to persistence of B. spielmanii and to pathogenesis of Lyme disease.     

The opportunistic human pathogenic fungus Aspergillus fumigatus evades the host complement system

The opportunistic human pathogenic fungus Aspergillus fumigatus causes severe systemic infections and is a major cause of fungal infections in immunocompromised patients. A. fumigatus conidia activate the alternative pathway of the complement system. In order to assess the mechanisms by which A. fumigatus evades the activated complement system, we analyzed the binding of host complement regulators to A. fumigatus. The binding of factor H and factor H-like protein 1 (FHL-1) from human sera to A. fumigatus conidia was shown by adsorption assays and immunostaining. In addition, factor H-related protein 1 (FHR-1) bound to conidia. Adsorption assays with recombinant factor H mutants were used to localize the binding domains. One binding region was identified within N-terminal short consensus repeats (SCRs) 1 to 7 and a second one within C-terminal SCR 20. Plasminogen was identified as the fourth host regulatory molecule that binds to A. fumigatus conidia. In contrast to conidia, other developmental stages of A. fumigatus, like swollen conidia or hyphae, did not bind to factor H, FHR-1, FHL-1, and plasminogen, thus indicating the developmentally regulated expression of A. fumigatus surface ligands. Both factor H and plasminogen maintained regulating activity when they were bound to the conidial surface. Bound factor H acted as a cofactor to the factor I-mediated cleavage of C3b. Plasminogen showed proteolytic activity when activated to plasmin by urokinase-type plasminogen activator. These data show that A. fumigatus conidia bind to complement regulators, and these bound host regulators may contribute to evasion of a host complement attack.     

The human complement regulator factor H binds pneumococcal surface protein PspC via short consensus repeats 13 to 15

The innate ability of Streptococcus pneumoniae to resist complement activation and complement-mediated phagocytosis may be a direct consequence of the ability of the bacteria to bind components of the complement regulatory system. One such component, factor H (fH), is a crucial fluid-phase negative regulator of the alternative pathway of complement and is utilized by a number of pathogenic organisms to resist complement attack. The pneumococcal surface protein C (PspC [also known as CbpA] and SpsA) has been shown to bind fH, although the exact binding site within one or more of the 20 short consensus repeats (SCRs) of the molecule is not known. The purpose of the current study was to map specific SCRs on fH responsible for this binding. Initial experiments utilizing type 2 pneumococcal strain D39 and its isogenic PspC-negative derivative (D39/pspC mutant) showed that fH binding was PspC dependent. A purified recombinant protein derivative of PspC that lacked the proline-rich region (PspCDeltaPro) had a reduced binding efficiency for fH, thereby directly showing the importance of this region for the fH interaction. We have specifically shown by inhibition experiments that SCRs responsible for heparin and C3b binding of fH are not involved in binding PspC and the interaction between fH and PspC is largely hydrophobic, since no inhibition was observed in the presence of high concentrations of NaCl. Construction of SCR proteins encompassing the whole fH molecule showed that SCRs 8 to 15 (SCR 8-15) mediated binding to PspC. Further localization experiments revealed that SCR 13 and SCR 15 were required for full binding, although partial binding was retained when either SCR was removed.     

Binding of Endotoxin to Macrophages: Distinct Effects of Serum Constituents

The respective roles of serum lipoproteins, and of the complement component C3, in the binding of endotoxin (LPS) to macrophages were analyzed by an in vitro assay using [³H]LPS. The addition of an anti-C3 serum in the medium induced an apparent abolishment of the specific binding of LPS to mouse macrophages, but this effect appeared to be due to an actual increase of nonspecific binding. Isolated complexes of LPS with lipoproteins of high density (HDL3) and of very high density (VHDL) did not bind to macrophages. Furthermore, addition of HDL3 and VHDL in the incubation medium inhibited the specific binding of LPS to macrophages. These results suggest that C3 reduces nonspecific interactions between LPS and macrophages whereas associations between LPS and HDL3 or VHDL inhibit specific LPS-macrophage interactions.     

Decreased membrane complement regulators in the retinal pigmented epithelium contributes to age‐related macular degeneration

Dysregulated complement is thought to play a central role in age‐related macular degeneration (AMD) pathogenesis, but the specific mechanisms have yet to be determined. In maculae of AMD specimens, we found that the complement regulatory protein, CD59, was increased in regions of uninvolved retinal pigmented epithelium (RPE) of early AMD, but decreased in the RPE overlying drusen and in geographic atrophy, an advanced form of AMD. While CD46 immunostaining was basolaterally distributed in the RPE of unaffected controls, it was decreased in diseased areas of early AMD samples. Since oxidized low‐density lipoproteins (oxLDL) collect in drusen of AMD and are a known complement trigger, we treated ARPE‐19 cells with oxLDL and found that cellular CD46 and CD59 proteins were decreased by 2.9‐ and nine‐fold (p < 0.01), respectively. OxLDLs increased complement factor B mRNA and Bb protein, but not factor D, I or H. OxLDLs increased C3b, but not C3a, C5 or C5b‐9. C5b‐9 was increased by 27% (p < 0.01) when the medium was supplemented with human serum, which was sufficient to induce poly(ADP‐ribose) polymerase cleavage, a marker of apoptosis. The decreased levels of CD46 and CD59 were in part explained by their release in exosomal and apoptotic membranous particles. In addition, CD59 was partially degraded through activation of IRE1α. Collectively, these results suggest that a combination of impaired complement regulators results in inadequately controlled complement by the RPE in AMD that induces RPE damage. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd .     

Expression and sequence diversity of the complement regulating outer surface protein E in Borrelia afzelii vs. Borrelia garinii in patients with erythema migrans or neuroborreliosis

Outer surface protein E (OspE) is a complement factor H-binding virulence factor of borrelial subspecies. It is usually absent from in vitro grown Borrelia garinii, although in vivo B. garinii causes neuroborreliosis (NB). We analyzed the presence and sequence spectrum of the ospE genes in vivo in Borrelia spirochetes. DNA samples from the skin, serum and cerebrospinal fluid (CSF) of patients with infections caused by Borrelia afzelii or B. garinii were studied, and anti-OspE antibodies in the corresponding patient sera were detected by IgG ELISA using recombinant OspE as an antigen. ospE genes were found in 20 of 23 erythema migrans (EM) skin biopsies with B. afzelii, in 2 EM skin biopsies with unknown underlying subspecies, in 5 of 9 EM biopsies with B. garinii, and in 1 of 4 CSF samples of NB patients with B. garinii infection. All OspE sequences from B. garinii samples were identical. In contrast, OspE of B. afzelii origin showed more variation. Anti-OspE antibodies were found in 8/21 (38.0%) sera from patients with B. afzelii-associated EM. In conclusion, our results indicate that all borrelial subspecies, but not necessarily all strains, causing human infections can carry ospE genes to protect themselves against complement attack in vivo.     

Demonstration of factor H-like protein 1 binding to Treponema denticola, a pathogen associated with periodontal disease in humans

Treponema denticola is an important contributor to periodontal disease. In this study we investigated the ability of T. denticola to bind the complement regulatory proteins factor H and factor H-like protein 1 (FHL-1). The binding of these proteins has been demonstrated to facilitate evasion of the alternative complement cascade and/or to play a role in adherence and invasion. Here we demonstrate that T. denticola specifically binds FHL-1 via a 14-kDa, surface-exposed protein that we designated FhbB. Consistent with its FHL-1 binding specificity, FhbB binds only to factor H recombinant fragments spanning short consensus repeats (SCRs) 1 to 7 (H7 construct) and not to SCR constructs spanning SCRs 8 to 15 and 16 to 20. Binding of H7 to FhbB was inhibited by heparin. The specific involvement of SCR 7 in the interaction was demonstrated using an H7 mutant (H7AB) in which specific charged residues in SCR 7 were replaced by alanine. This construct lost FhbB binding ability. Analyses of the ability of FHL-1 bound to the surface of T. denticola to serve as a cofactor for factor I-mediated cleavage of C3b revealed that C3b is cleaved in an FHL-1/factor I-independent manner, perhaps by an unidentified protease. Based on the data presented here, we hypothesize that the primary function of FHL-1 binding by T. denticola might be to facilitate adherence to FHL-1 present on anchorage-dependent cells and in the extracellular matrix.