Peter Kraiczy

Host association of Borrelia burgdorferi sensu lato – the key role of host complement

Borrelia burgdorferi sensu lato (s.l.), the tick-borne agent of Lyme borreliosis, is a bacterial species complex comprising 11 genospecies. Here, we discuss whether the delineation of genospecies is ecologically relevant. We provide evidence that B. burgdorferi s.l. is structured ecologically into distinct clusters that are host specific. An immunological model for niche adaptation is proposed that suggests the operation of complement-mediated selection in the midgut of the feeding tick. We conclude that vertebrate hosts rather than tick species are the key to Lyme borreliosis spirochaete diversity.

Immune evasion of Borrelia burgdorferi by acquisition of human complement regulators FHL-1/reconectin and Factor H.

To understand immune evasion mechanisms of Borrelia burgdorferi we compared serum‐resistant B. afzelii and serum‐sensitive B. garinii isolates for their capacity toacquire human complement regulators. Here we demonstrate that the two borrelial genospecies show different binding of the two important human complement regulators, FHL‐1/reconectin and Factor H. All serum‐resistant B. afzelii isolates bound FHL‐1/reconectin and also Factor H, and all analyzed serum‐sensitive B. garinii isolates showed no or a significantly lower binding activity. Using recombinant deletion mutants, the binding domains were localized to the C terminus of FHL‐1/reconectin to short consensus repeats 5–7. The borrelial binding proteins were located in the surface of the bacteria as demonstrated by immunofluorescence staining of intact, serum‐exposed bacteria and by enrichment of outer membrane proteins. The surface‐attached complement regulators maintained complement regulatory activity as demonstrated in a cofactor assay. By ligand blotting two different borrelial binding proteins were identified that were responsible for the surface attachment of FHL‐1/reconectin and Factor H. These borrelial complement regulators acquiring surface proteins (CRASP) were further characterized as either CRASP‐1, a 27.5‐kDa molecule which preferentially binds FHL‐1/reconectin and which was present in all serum‐resistant borreliae, or CRASP‐2, a 20/21‐kDa protein which interacts preferentially with Factor H and the expression of which was more restricted, being detected in four of the six isolates analyzed. In summary, we describe a new immune evasion mechanism of B. burgdorferi, as these bacteria acquire human complement regulators to control complement activation on their surface and to prevent formation of toxic activation products.

Further characterization of complement regulator-acquiring surface proteins of Borrelia burgdorferi

ABSTRACT The three genospecies Borrelia burgdorferi,Borrelia garinii, and Borrelia afzelii, all causative agents of Lyme disease, differ in their susceptibilities to human complement-mediated lysis. We recently reported that serum resistance of borrelias correlates largely with their ability to bind the human complement regulators FHL-1/reconectin and factor H. To date, two complement regulator-acquiring-proteins (CRASP-1 and CRASP-2) have been identified in serum-resistant B.afzelii isolates (P. Kraiczy, C. Skerka, M. Kirschfink, V. Brade, and P. F. Zipfel, Eur. J. Immunol. 31:1674–1684, 2001). Here, we present a comprehensive study of the CRASPs detectable in both serum-resistant and intermediate serum-sensitive B. afzelii and B. burgdorferi isolates. These CRASPs were designated according to the genospecies either as BaCRASPs, when derived fromB. afzelii, or as BbCRASPs, for proteins identified in B. burgdorferi isolates. Each borrelial isolate expresses distinct CRASPs that can be differentiated by their mobility and binding phenotypes. A detailed comparison reveals overlapping and even identical binding profiles for BaCRASP-1 (27.5 kDa), BbCRASP-1 (25.9 kDa), and BbCRASP-2 (23.2 kDa), which bind FHL-1/reconectin strongly and interact weakly with factor H. In contrast, two B. afzelii proteins (BaCRASP-4 [19.2 kDa] and BaCRASP-5 [22.5 kDa]) and three B. burgdorferi proteins (BbCRASP-3 [19.8 kDa], BbCRASP-4 [18.5 kDa], and BbCRASP-5 [17.7 kDa]) bind factor H but not FHL-1/reconectin. Most CRASPs bind both human immune regulators at their C-terminal ends. Temperature-dependent up-regulation of CRASPs (BaCRASP-1, BaCRASP-2, and BaCRASP-5) is detected in low-passage borrelias cultured at 33 or 37°C compared with those cultured at 20°C. The characterization of the individual CRASPs on the molecular level is expected to identify new virulence factors and potential vaccine candidates.

Complement resistance of Borrelia burgdorferi correlates with the expression of BbCRASP-1, a novel linear plasmid-encoded surface protein that interacts with human factor H and FHL-1 and is unrelated to Erp proteins.

The etiologic agent of Lyme disease, Borrelia burgdorferi, is capable of circumventing the immune defense of a variety of potential vertebrate hosts. Previous work has shown that interaction of host-derived complement regulators, factor H and factor H-like protein 1 (FHL-1), with up to five complement regulator-acquiring surface proteins (CRASPs) expressed by resistant B. burgdorferi sensu lato isolates conferred complement resistance. In addition expression of CRASP-1 is directly correlated with complement resistance of Borrelia species. This work describes the functional characterization of BbCRASP-1 as the dominant factor H and FHL-1-binding protein of B. burgdorferi. The corresponding gene, zs7.a68, is located on the linear plasmid lp54 and is different from factor H-binding Erp proteins that are encoded by genes localized on circular plasmids (cp32). Deletion mutants of BbCRASP-1 were generated, and a high affinity binding site for factor H and FHL-1 was mapped to the C terminus of BbCRASP-1. Similarly, the predominant binding site of factor H and FHL-1 was localized to the short consensus repeat 7. Factor H and FHL-1 maintain their cofactor activity for factor I-mediated C3b inactivation when bound to BbCRASP-1, and factor H is up to 6-fold more efficient in mediating C3b conversion than FHL-1. In conclusion, BbCRASP-1 (i) binds the host complement regulators factor H and FHL-1 with high affinity, (ii) is the key molecule of the complement resistance of spirochetes, and (iii) is distinct from the Erp protein family. Thus, BbCRASP-1 most likely contributes to persistence of B. burgdorferi and to pathogenesis of Lyme disease.

LfhA, a Novel Factor H-Binding Protein of Leptospira interrogans

ABSTRACT The early phase of leptospiral infection is characterized by the presence of live organisms in the blood. Pathogenic Leptospira interrogans is resistant to the alternative pathway of complement mediated-killing, while nonpathogenic members of the genus are not. Consistent with that observation, only pathogenic leptospires bound factor H, a host fluid-phase regulator of the alternative complement pathway. Ligand affinity blot analyses revealed that pathogenic L. interrogans produces at least two factor H-binding proteins. Through screening of a lambda phage expression library, we identified one of these as the novel membrane protein LfhA. Ligand affinity assays and surface plasmon resonance analyses of recombinant LfhA revealed specific binding of both factor H and factor H-related protein 1. Serological examination of infected humans and horses demonstrated that LfhA is expressed by L. interrogans during mammalian infection. LfhA may therefore contribute to the resistance of pathogenic leptospires to complement-mediated killing during leptospiremic phases of the disease.

Immune evasion of Borrelia burgdorferi: mapping of a complement-inhibitor factor H-binding site of BbCRASP-3, a novel member of the Erp protein family.

The causative agents of Lyme disease, Borrelia burgdorferi s.s., B. garinii, and B. afzelii, differ in their susceptibility to complement‐mediated lysis. This phenomenon apparently depends on the expression of proteins termed complement regulator‐acquiring surface proteins (CRASP) and their binding to the inhibitory plasma proteins factor H and FHL‐1. To characterize these bacterial proteins in more detail we have now isolated from a B. burgdorferi expression library a novel factor H‐binding protein. In accordance with our previous studies this protein was termed BbCRASP‐3 and represents a novel member of the polymorphic Erp (OspE/F‐related) protein family. On the basis of protease accessibility assays using intact spirochetes, BbCRASP‐3 isidentified as a surface‐exposed protein and binds the C‐terminal short consensus repeats of factor H. Applying deletion mutants of BbCRASP‐3, the factor H‐binding site was mapped to the nine‐amino‐acid motif LEVLKKNLK localized at the C‐terminal end of BbCRASP‐3. Factor H bound to BbCRASP‐3 maintains its cofactor activity in factor I‐mediated C3b inactivation. Binding of BbCRASP‐3 to factor H can be inhibited by heparin, a physiological ligand of the complement regulator factor H. Blocking of factor‐H‐binding by soluble BbCRASP‐3 leads to an increase of complement deposition on intermediate serum‐resistant strain ZS7. In conclusion, BbCRASP‐3 has been identified as a novel factor H‐binding protein on B. burgdorferi which by conferring complement resistance to the pathogen may contribute to its persistence in the mammalian host.

Functional characterization of BbCRASP-2, a distinct outer membrane protein of Borrelia burgdorferi that binds host complement regulators factor H and FHL-1.

Borrelia burgdorferi, the aetiological agent of Lyme disease, employs sophisticated means to survive in diverse mammalian hosts. Recent studies demonstrated that acquisition of complement regulators factor H and factor H‐like protein‐1 (FHL‐1) allows spirochetes to resist complement‐mediated killing. Serum‐resistant B. burgdorferi express up to five distinct complement regulator‐acquiring surface proteins (CRASPs) that bind factor H and/or FHL‐1. In this study we have identified and characterized one of those B. burgdorferi proteins, named BbCRASP‐2. BbCRASP‐2 is distinct from the four previously identified factor H/FHL‐1‐binding CRASPs of B. burgdorferi strains. The single copy of the gene encoding BbCRASP‐2, cspZ, is located on the linear plasmid lp28‐3. BbCRASP‐2 is highly divergent from the factor H/FHL‐1‐binding protein BbCRASP‐1 and from members of the factor H‐binding Erp (OspE/F‐related) protein family. Peptide mapping analysis revealed that the factor H/FHL‐1 binding site is discontinuous and it was found that C‐terminal truncations abrogate factor H and FHL‐1 binding. The predominant BbCRASP‐2 binding site of both host complement regulators was mapped to the short consensus repeat 7 (SCR 7). Factor H and FHL‐1 bound to BbCRASP‐2 maintain cofactor activity for factor I‐mediated C3b inactivation and accelerate the decay of the C3 convertase. Expression of BbCRASP‐2 in serum‐sensitive B. burgdorferi mutant B313 increased resistance to complement‐mediated lysis. The characterization of BbCRASP‐2 now provides a complete picture of the three diverse complement regulator‐binding protein families of B. burgdorferi yielding new insights into the pathogenesis of Lyme disease.

Identification and Functional Characterization of Complement Regulator-Acquiring Surface Protein 1 of the Lyme Disease Spirochetes Borrelia afzelii and Borrelia garinii

ABSTRACT Complement regulator-acquiring surface protein 1 (CRASP-1) is the dominant factor-H-like protein 1 (FHL-1)- and factor-H-binding protein of Borrelia burgdorferi and is suggested to contribute to persistence of the pathogen. The prototype CRASP-1 of B. burgdorferi sensu stricto (CRASP-1Bb) has been formerly characterized. As shown recently, serum-resistant Borrelia afzelii strains express a unique FHL-1 and factor H-binding protein, designated CRASP-1Ba. Here, we describe for the first time the isolation and functional characterization of the gene encoding the full-length CRASP-1Ba of 28 kDa, which, upon processing, is predicted to be 26.4 kDa. CPASP-1Ba of B. afzelii spirochetes is associated with a genetic locus encoding the orthologous gbb54 gene family that maps to the linear plasmid of approximately 54 kb. Ligand affinity blotting techniques demonstrate that both native and recombinant CRASP-1Ba molecules strongly bind to FHL-1 and much more weakly to factor H. The FHL-1 and factor-H-binding site in CRASP-1Ba is shown to be localized to a 12-amino-acid residue domain at the C terminus of the protein. For comparison, the corresponding cspA-like gene(s) of a serum-sensitive Borrelia garinii strain has also been cloned and characterized. Most notably, two CRASP-1-related B. garinii proteins were identified; however, both molecules bind only weakly to FHL-1 and not at all to factor H. The present identification of the binding site of CRASP-1Ba represents an important step forward in our understanding of the pathogenesis of Lyme disease and may be helpful to design therapeutic regimens to interfere with complement evasion strategies of human pathogenic Borrelia strains.

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

ABSTRACT 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 bacteriums 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, ε-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.

Binding of Human Factor H-Related Protein 1 to Serum-Resistant Borrelia burgdorferi Is Mediated by Borrelial Complement Regulator-Acquiring Surface Proteins

BACKGROUND Isolates of Borrelia burgdorferi, the causative agent of Lyme disease, express up to 5 distinct complement regulator-acquiring surface proteins (CRASP-1, -2, -3, -4, and -5). METHODS By use of ligand affinity blotting, enzyme-linked immunosorbent assay, surface plasmon resonance, and functional complement assays, we have identified factor H-related protein 1 (FHR-1) as a novel protein that binds to the bacterium via CRASP-3, -4, and -5. RESULTS When incubated in serum, serum-resistant Borrelia burgdorferi strain LW2 bind FHR-1, an additional member of the human factor H protein family, and, similarly, 2 mouse FHR proteins bind to the surface. Recombinant FHR-1 binds to 3 borrelial surface proteins (CRASP-3, -4, and -5) but not to CRASP-1 and -2. A comparative analysis of the individual CRASPs revealed common as well as distinct binding profiles for the 3 human regulators. FHR-1 binds to 3 CRASPs, and factor H binds to all 5 CRASPs. In addition, factor H-like protein 1 interacts with CRASP-1 and -2 but with no other borrelial proteins. CONCLUSIONS Thus, by expressing multiple surface proteins with different binding properties, the pathogen can attach a unique combination of host complement regulators to its surface. For the pathogen, this type of surface decoration and specific acquisition of different host plasma proteins allows fine-tuning of the host immune attack.