Delineation of Species-Specific Binding Properties of the CspZ Protein (BBH06) of Lyme Disease Spirochetes: Evidence for New Contributions to the Pathogenesis of Borrelia spp

Abstract

Borrelia burgdorferiCspZ (TIGR open reading frame designation, BBH06) is part of a functionally related group of proteins that bind one or more members of the factor H (FH) protein family. In this report we assess the conservation, distribution, properties, and ligand binding abilities of CspZ from the three main Borrelia species associated with Lyme disease infections in humans. CspZ (also referred to as BbCRASP-2 in the literature) was found to be highly conserved at the intraspecies level but divergent at the interspecies level. All CspZ orthologs that originated from B. burgdorferi isolates bound FH from a diverse group of mammals. In contrast, CspZ derived from B. garinii and B. afzelii did not. Regardless of the Borrelia species of origin, all CspZ proteins tested bound to unknown ϳ60-kDa serum proteins produced by different mammals. To further define the molecular basis for the differential binding of CspZ orthologs to host proteins, DNA sequence, truncation, and site-directed mutagenesis analyses were performed. DNA sequence analyses revealed that B. garinii and B. afzelii CspZ orthologs possess a 64-amino-acid N-terminal domain that is absent from B. burgdorferi CspZ. However, binding analyses of recombinant proteins revealed that this domain does not in and of itself influence ligand binding properties. Truncation and mutagenesis analyses further revealed that the key determinants required for ligand binding are discontinuous and that the presentation of the ligand binding pocket is dependent on alpha helices with high coiled-coil formation probability. The data presented here provide insight into the molecular basis of CspZ-ligand interactions and suggest that CspZ orthologs from diverse Borrelia species can contribute to the host-pathogen interaction through their interaction with serum proteins.

Comparative Analysis of the Properties and Ligand Binding Characteristics of CspZ, a Factor H Binding Protein, Derived fromBorrelia burgdorferiIsolates of Human Origin

“…While cspZ has been demonstrated for both B. burgdorferi and B. garinii, it is not clear if this gene is widely distributed among B. afzelii isolates (37,39). In addition, although B. garinii produces CspZ, the protein lacks FH binding ability (39). However, CspZ appears to have other roles during infection, as suggested by its ability to bind to other, unidentified serum proteins (39).…”

“…CspZ is the most recent of these proteins to be identified. While cspZ has been demonstrated for both B. burgdorferi and B. garinii, it is not clear if this gene is widely distributed among B. afzelii isolates (37,39). In addition, although B. garinii produces CspZ, the protein lacks FH binding ability (39).…”

Identification of an Antiparallel Coiled-Coil/Loop Domain Required for Ligand Binding by theBorrelia hermsiiFhbA Protein: Additional Evidence for the Role of FhbA in the Host-Pathogen Interaction

“…Coiled-coil domains have been implicated as being key determinants in the formation of the FH-binding pocket of multiple spirochetal FH-binding proteins (38,42,51). In addition, these structural domains are also involved in the formation of conformational or discontinuous epitopes that are presented at the cell surface during infection.…”

“…It is important to note that this upregulation of fhbA occurred while the number of spirochetes were decreasing. It has been demonstrated for some spirochetal FH-binding proteins that the formation of the FH-and/or FHL-1-binding pocket or antibody recognition domains is dependent on hydrophobic interactions between alpha helices that have a significant predictive probability of coiled-coil formation (28,38,39,45,51). Two C-terminal, highly conserved, putative coiledcoil elements are present in both FhbA1 and FhbA2 (28).…”

“…We have demonstrated that the binding interaction between FH and the spirochetal FH-binding proteins OspE, CspA (BbCRASP-1), and CspZ (BbCRASP-2) involves conformational determinants (38,42,45,51). Independent N-or C-terminal truncations of these proteins or site-directed mutagenesis of specific alpha helices abolished ligand binding ability.…”