CspZ (TIGR open reading frame designation BBH06) is a part of

CspZ (TIGR open reading frame designation BBH06) is a part of a functionally related group of proteins that bind one or more members of the factor H (FH) protein family. that and CspZ orthologs possess a 64-amino-acid N-terminal domain name that is absent from CspZ. However binding analyses of recombinant proteins revealed that this domain name 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 Gefitinib of CspZ-ligand interactions and suggest that CspZ orthologs from diverse species can contribute to the host-pathogen conversation through their conversation with serum proteins. The binding of unfavorable regulators of the complement cascade such as factor H (FH) FH-like protein 1 (FHL-1) and C4b binding protein has been demonstrated to be an important virulence mechanism for numerous pathogens (reviewed in reference 19). A correlation between this phenotype and serum resistance has been exhibited for the Lyme disease spirochetes and several other microorganisms (6 19 The binding of FH and FHL-1 is certainly thought to donate to pathogenesis by facilitating evasion of go with and opsonophagocytosis (2 4 11 32 Many of the FH or FHL-1 binding proteins (FHBPs) made by spirochetes have already been determined (11 13 17 24 They consist of FhbA (13-15) FhbB (24) and CspZ (BBH06) CspA (BBA68) as well as the OspE paralogs (BBL39 BBN38 and BBP38) from the Lyme disease spirochetes (3 6 9 11 29 The CspZ proteins which may be the focus of the report may be the latest FHBP from the Lyme disease spirochetes to become determined. It has additionally been known as BbCRASP-2 in the books (9). Among the Lyme disease spirochetes shows the most complicated FH binding phenotype creating up to five different FHBPs owned by three different proteins households. and pathogenesis through multiple virulence systems. In B31MI is certainly continued a 28-kb linear plasmid specified lp28-3 (8). The distribution and conservation of among pathogenic Gefitinib types of the sensu lato complicated have not been assessed and little is known regarding the ligand binding abilities of CspZ proteins from diverse species. In addition the determinants required for ligand binding have not been fully identified. The goals of Gefitinib this study were to address voids in our current understanding of the molecular basis of the CspZ-ligand conversation and the contribution of CspZ to the pathogenesis of diverse species. MATERIALS AND METHODS Bacterial isolates and cultivation and contamination of mice. Table ?Table11 lists and describes the species/isolates that were Tmem34 analyzed as part of this report. All strains were cultivated in altered Barbour-Stoenner-Kelley (BSK-H) complete medium (Sigma) at 33°C in sealed bottles in Gefitinib CO2 incubators (3% CO2). Bacteria were harvested by centrifugation and gently washed with phosphate-buffered saline (PBS). To generate contamination serum mice (C3H/HeJ; Jackson Labs) were infected with B31MI by intradermal inoculation as previously described (7). Serum was harvested from the infected mice at week 6 postinoculation. Blots were screened using a 1:1 0 dilution of contamination serum in blocking buffer (1% PBS 0.2% Tween 5 Carnation nonfat dry milk) and antibody binding was detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG) (1:40 0 Pierce). TABLE 1. isolates used in this study SDS-PAGE immunoblotting and FH/FHL-1 ALBI analyses. cell lysates and recombinant proteins (r-proteins) were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane as previously described (23). FH and/or FHL-1 binding to membrane-immobilized Gefitinib proteins was assessed using the affinity ligand binding immunoblot (ALBI) assay approach (referred to as the FH ALBI assay). The membranes were incubated in blocking buffer for 1 h followed by incubation with purified human FH (5 ng μl?1; Calbiochem) or r-FHL-1 (2.5 ng μl?1;.