Taru Meri

The complement regulator factor H binds to the surface protein OspE of Borrelia burgdorferi.

Spirochete bacteria of the Borrelia burgdorferi sensu lato complex cause Lyme borreliosis. The three pathogenic subspecies Borrelia garinii, Borrelia afzelii, and Borrelia burgdorferi sensu stricto differ in their disease profiles and susceptibility to complement lysis. We investigated whether complement resistance ofBorreliae could be due to acquisition of the main soluble inhibitors of the alternative complement pathway, factor H and the factor H-like protein 1. When exposed to nonimmune EDTA-plasma, the serum-resistant B. afzelii and B. burgdorferisensu stricto strains bound factor H/factor H-like protein 1 to their surfaces. Assays with radiolabeled proteins showed that factor H bound strongly to the B. burgdorferi sensu stricto strain. To identify factor H ligands on the borrelial surface, we analyzed a panel of outer surface proteins of B. burgdorferi sensu stricto with the surface plasmon resonance technique. The outer surface lipoprotein OspE was identified as a specific ligand for factor H. Using recombinant constructs of factor H, the binding site for OspE was localized to the C-terminal short consensus repeat domains 15–20. Specific binding of factor H to B. burgdorferi sensu stricto OspE may help the pathogen to evade complement attack and phagocytosis.

Complement Inhibitor Factor H Binding to Lyme Disease Spirochetes Is Mediated by Inducible Expression of Multiple Plasmid-Encoded Outer Surface Protein E Paralogs

Borrelia burgdorferi spirochetes can circumvent the vertebrate host’s immune system for long periods of time. B. burgdorferi sensu stricto and B. afzelii, but not B. garinii, bind the complement inhibitor factor H to protect themselves against complement-mediated opsonophagocytosis and killing. We found that factor H binding and complement resistance are due to inducible expression of a wide repertoire of outer surface protein E (OspE) lipoproteins variably called OspE, p21, ErpA, and ErpP. Individual Borrelia strains carry multiple plasmid-encoded OspE paralogs. Together the OspE homologs were found to constitute an array of proteins that bind factor H via multiple C-terminal domains that are exposed outwards from the Borrelial surface. Charged residue substitutions in the key binding regions account for variations between OspE family members in the optimal binding pH, temperature, and ionic strength. This may help the spirochetes to adapt into various host environments. Our finding that multiple plasmid-encoded OspE proteins act as virulence factors of Borrelia can provide new tools for the prevention and treatment of borreliosis.

Lysine-Dependent Multipoint Binding of the Borrelia burgdorferi Virulence Factor Outer Surface Protein E to the C Terminus of Factor H

Serum resistance, an important virulence determinant of Borrelia burgdorferi sensu lato strains belonging to the Borrelia afzelii and B. burgdorferi sensu stricto genotypes, is related to binding of the complement inhibitor factor H to the spirochete surface protein outer surface protein E (OspE) and its homologues. In this study, we show that the C-terminal short consensus repeats 18–20 of both human and mouse factor H bind to OspE. Analogously, factor H-related protein 1, a distinct plasma protein with three short consensus repeat domains homologous to those in factor H, bound to OspE. Deleting 15-aa residues (region V) from the C terminus of the OspE paralog P21 (a 20.7-kDa OspE-paralogous surface lipoprotein in the B. burgdorferi sensu stricto 297 strain) abolished factor H binding. However, C-terminal peptides from OspE, P21, or OspEF-related protein P alone and the C-terminal deletion mutants of P21 inhibited factor H binding to OspE only partially when compared with full-length P21 or its N-terminal mutant. Alanine substitution of amino acids in peptides from the key binding regions of the OspE family indicated that several lysine residues are required for factor H binding. Thus, the borrelial OspE family proteins bind the C inhibitor factor H via multiple sites in a lysine-dependent manner. The C-terminal site V (Ala151-Lys166) is necessary, but not sufficient, for factor H binding in both rodents and humans. Identification of the necessary binding sites forms a basis for the development of vaccines that block the factor H-OspE interaction and thereby promote the killing of Borreliae.

Onchocerca volvulus microfilariae avoid complement attack by direct binding of factor H

The filarial parasite Onchocerca volvulus is the causative agent of river blindness. The adult worms produce microfilariae (mf), which are responsible for the disease pathogenesis; mf activate the complement system, but the activation stops before the formation of terminal complement complexes. Because of the arrest of complement activation, this study analyzed binding of the main alternative pathway regulator, factor H (fH), to the mf. The mf bound fH after incubation in nonimmune human serum or with purified radiolabeled fH. In the presence of factor I, mf-bound fH promoted the cleavage of complement 3 molecule b (C3b) to iC3b. An analysis with recombinant constructs of fH showed that the C-terminal short consensus repeats (SCRs) 8-20 of fH bound to mf, whereas the N-terminal SCRs 1-7 containing the complement-regulatory domains in SCRs 1-5 did not. Thus, mf of the nematode O. volvulus may evade human complement by binding fH and by promoting inactivation of C3b into iC3b.

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 ∼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.

Distribution of exogenous complement factor H in mice in vivo

Factor H is an important regulator of complement activation in plasma and on cell surfaces in both humans and mice. If FH function is compromised, inappropriate complement activation on self‐surfaces can have disastrous effects as seen in the kidney diseases atypical haemolytic uremic syndrome (aHUS) and C3 glomerulopathy. As FH constructs have been proposed to be used in treatment for these diseases, we studied the distribution of exogenous FH fragments in mice. Full‐length mFH, mFH1‐5 and mFH18‐20 fragments were radiolabelled, and their distribution was examined in WT, FH−/− and FH−/−C3−/− mice in vivo. Whole body scintigraphy revealed accumulation of radioactivity in the abdominal part of the mice, but also to the thyroid gland and urinary bladder. At organ level in WT mice, some full‐length FH accumulated in internal organs, but most of it remained in the circulation. Both of the mFH fragments accumulated in the kidneys and were excreted in urine. For mFH1‐5, urinary secretion is the likely cause for the accumulation. Concentration of mFH18‐20 to kidneys was slower, and at tissue level, mFH18‐20 was localized at the proximal tubuli in WT and FH−/−C3−/− mice. No C3‐independent binding to glomeruli was detected. In conclusion, these results show that glomerular glycosaminoglycans and sialic acids alone do not collect FH in kidneys. Deposition of C3 fragments is also needed, which implies that in aHUS, the problem is in simultaneous recognition of C3 fragments and glycosaminoglycans or sialic acids by FH, not just the inability of FH to recognize glomerular endothelium as such.

Lysine-Dependent Binding of OspE to the C-terminus of Factor H Mediates Complement Resistance in Borrelia burgdorferi

Serum resistance of Borrelia burgdorferi strains belonging to the B. afzelii and B. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor H. We recently reported that factor H binding by B. burgdorferi is due to inducible expression of several approximately 20u2003kDa plasmid‐encoded, surface‐exposed lipoproteins related to OspE (e.g. ErpA, ErpP and P21). In addition, a second class of factor H‐binding proteins of approximately 27–35u2003kDa has been described. The OspE‐related lipoproteins are dramatically induced by B. burgdorferi during transmission from its tick vector into the mammalian host. The induction of OspE‐related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the hosts immune system. The goal of the present study was to define the factor H‐binding regions of OspE‐related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (Biacore). The combined studies revealed that the C‐terminal regions of both human and mouse factor H (SCRs 18–20) specifically bind to OspE‐related lipoproteins. We also found FHR‐1, whose C‐terminal SCRs 3–5 are homologous to SCRs 18–20 of factor H, to bind to OspE. Peptide mapping revealed five putative regions (designated I‐V) in OspE that could directly interact with factor H. Deleting the C‐terminal 15 amino acid residues from region V of P21 abolished its ability to bind factor H. At the same time, however, synthetic peptides corresponding to the C‐termini of OspE, P21 and ErpP did not inhibit factor H binding to OspE. Thus, the C‐terminal‐binding region V appears to be necessary but not sufficient for factor H binding. When a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor H‐binding regions were mutated to alanines, we observed that lysines in the factor H‐binding regions of OspE were required for factor H binding. The combined data have revealed that key lysine residues in OspE‐related lipoproteins and ionic interactions are crucial for factor H interactions. Furthermore, binding of OspE to the C‐termini of both mouse and human factor H suggests that Borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. In Borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the OspE sequences as well as in the expression of factor H‐binding proteins may account for their susceptibility to serum lysis.