4A). shown that tick-borne pathogens, and and was shown to bind PLG via a multitude of lipoproteins, such as CRASP-3 (ErpP), CRASP-4 (ErpC), CRASP-5 (ErpA), OspA and as yet to be defined molecules [17], [18]. have been proposed [21], but so far these proteins have not been isolated. Here we provide evidence that communicate a novel multifunctional surface lipoprotein, which by exploiting sponsor proteins confers resistance to both, match assault and opsonization and simultaneously acquires an SDZ 205-557 HCl increased potential to invade sponsor cells. Materials and Methods Bacterial strains and growth conditions Relapsing fever spirochetes strain A1 and A17, (ATCC35209) strain HS1 and the Lyme disease spirochete strain B313, which is a clonal mutant of B31 lacking all linear and circular plasmids with the exception of cp32-1, cp32-2, cp32-4, cp26 and lp17, were cultivated in BSK-H total medium (PAN Biotech, Aidenbach, Germany) supplemented with 5% rabbit serum (Cell Concept, Freiburg, Germany) at 30C [22]. Bacteria were harvested by centrifugation and washed with phosphate-buffered saline. The denseness of spirochetes was identified using dark-field microscopy and a Kova counting chamber (Hycor Biomedical, Garden Grove, CA). JM109 SDZ 205-557 HCl were cultivated at 37C in LB medium. Isolation and cloning of HcpA, building of manifestation plasmids and production of recombinant proteins Isolation of the CFH binding protein of was carried out by co-immunoprecipitation. Protein G sepharose beads (Amersham Bioscience, Freiburg, Germany) were loaded with polyclonal anti-factor H antibody (Calbiochem, Schwalbach, Germany) and purified human being element H (Calbiochem). Subsequently, beads were incubated over night at 4C with whole cell lysates of A1. Immunoprecipitates were separated by SDS-PAGE and visualized by staining with colloidal Coomassie (Pierce/Thermofisher, Bonn, Germany). The selected protein band of approximately 17 kDa was cored from your gel and subjected to mass spectrometric analysis. The recognized peptides matched an open reading framework of 525 bp of the A1 genome, designated strain JM109 and recombinant proteins were purified as recommended by the manufacturer (Qiagen). Table 1 Oligonucleotides used in this study. geneBrR geneBrBam geneBrR1 geneBrSal Sf9 insect cells infected having a recombinant baculovirus. The cloning, manifestation and purification have been explained previously [14], [23]. The CFH deletion mutant CFHSCR19-20 was amplified by PCR, ligated in framework into pQE-30Xa vector and indicated as fusion protein with an N-terminal His6-tag. Manifestation and purification was carried Rabbit Polyclonal to Tyrosinase out as recommended by the manufacturer (Qiagen). SDS-PAGE, Ligand affinity blot and ELISA Borrelial whole cell lysates (15 g) or purified recombinant HcpA proteins (200 ng) were subjected to Tris/Tricine-SDS-PAGE under reducing conditions and transferred to nitrocellulose as previously explained [24]. For ELISA using SDZ 205-557 HCl non-denatured recombinant proteins, microtiter plates (MaxiSorp, Nunc) were coated with HcpA or the deletion mutants (100 l; 1 g/ml) for 2 h at space temp. The wells were washed with PBS/0.05%Tween, blocked with PBS/5% BSA and incubated with CFH (10 g/ml), 50% normal human serum (NHS) or PLG (20 g/ml) for 1 h at RT. After washing, HcpA bound proteins were recognized by goat anti-CFH (Calbiochem) or goat anti-PLG (Acris) antibodies or CFHR-1 specific mouse mAb JHD8 followed by peroxidase-conjugated rabbit anti-goat IgG (Dianova) or sheep SDZ 205-557 HCl anti-mouse secondary antibody (GE Healthcare), respectively. Substrate reaction was performed with o-phenyldiamine dihydrochloride (Sigma-Aldrich) and absorbance was measured at 492 nm. For competition inhibition assay, HcpA (2 g/ml) was coated on microtiter plates. To analyze the ability of PLG to inhibit the binding of CFH to HcpA, plates were incubated simultaneously with constant amounts of CFH (3 g/ml) and different amounts of PLG (0.001C100 g/ml). The ability of CFH to inhibit the binding of PLG to HcpA was determined by adding constant amounts of PLG (10 g/ml) with different amounts of SDZ 205-557 HCl CFH (0.001C100 g/ml). HcpA bound CFH and PLG were recognized as explained above. For detection of purified recombinant HcpA full-length protein and deletion mutants, the anti His6-tag monoclonal mouse antibody (Calbiochem) was used. Immunofluorescence analysis Spirochetes (1107) were washed with Tris buffer (30 mM Tris, 60 mM NaCl, pH 7.4) and incubated with mAb directed against HcpA (BR-1) for 1 h at RT. For detection of CFH-binding cells were treated with purified human being CFH for 1 h at RT followed by.
