Decorin-binding protein of Borrelia burgdorferi is encoded within a two-gene operon and is protective in the murine model of Lyme borreliosis - PubMed (original) (raw)

Decorin-binding protein of Borrelia burgdorferi is encoded within a two-gene operon and is protective in the murine model of Lyme borreliosis

K E Hagman et al. Infect Immun. 1998 Jun.

Abstract

Isolated outer membranes of Borrelia burgdorferi were used in immunoblotting experiments with sera from immune mice to identify new putative Lyme disease vaccine candidates. One immunoreactive polypeptide migrated on polyacrylamide gels just proximal to outer surface protein C and comigrated with [3H]palmitate-labeled polypeptides. A degenerate oligonucleotide primer based upon internal amino acid sequence information was used to detect the corresponding gene within a B. burgdorferi total genomic library. The relevant open reading frame (ORF) encoded a polypeptide comprised of a 24-amino-acid putative signal peptide terminated by LLISC, a probable consensus sequence for lipoprotein modification, and a mature protein of 163 amino acids. Immunoblots of a recombinant fusion protein corresponding to this ORF supported the idea that the encoded protein was a previously reported decorin-binding protein (DBP) of B. burgdorferi N40 (B. P. Guo, S. J. Norris, L. C. Rosenberg, and M. Höök, Infect. Immun. 63:3467-3472, 1995). However, further DNA sequencing revealed the presence of a second ORF, designated ORF-1, whose termination codon was 119 bp upstream of the dbp gene. ORF-1 also encoded a putative lipoprotein with a mature length of 167 amino acids. Northern blots, Southern blots, and primer extension analyses indicated that ORF-1 and dbp comprised a two-gene operon located on the 49-kb linear plasmid. Both proteins, which were 40% identical and 56% similar, partitioned into Triton X-114 detergent extracts of B. burgdorferi isolated outer membranes. Mice infected with B. burgdorferi produced high titers of antibodies against the ORF-1-encoded protein and DBP during both early and later stages of chronic infection. Both DBP and the ORF-1-encoded protein were sensitive to proteinase K treatment of intact borreliae, suggesting that they were surface exposed. In active immunization experiments, 78% of mice immunized with recombinant DBP were immune to challenge. While it is not clear whether the two lipoproteins encoded by the ORF-1-dbp operon have analogous decorin-binding functions in vivo, the combined studies implicate DBP as a new candidate for a human Lyme disease vaccine.

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Figures

FIG. 1

Comparison of passive immunization (▪) and growth inhibition (○) titers in sera of mice infected with B. burgdorferi. Values are reciprocal titers (means ± standard errors of the means).

FIG. 2

SDS-PAGE analysis of isolated outer membranes (OM) of B. burgdorferi. Spirochetes were labeled with radioactive palmitate, OM were isolated, and a portion of the OM material was phase partitioned with Triton X-114. Lane 1 (OM) and lane 3 (detergent-phase [D] OM proteins) were immunoblotted with pooled sera from mice 8 weeks postinfection. Other lanes: 5, immunoblot of OM material partitioning into the aqueous phase after Triton X-114 extraction (note the presence of p66); 2 and 4, OM or D OM proteins, respectively, subjected to autoradiography to assess the incorporation of radioactive palmitate. Protein designations to the right of lane 4 are according to convention. The asterisk denotes the region of the gel harvested for amino acid sequence procedures. Numbers at left correspond to apparent molecular weights (in thousands).

FIG. 3

Schematic diagram (A) and Northern blot (B) of the ORF-1-dbp operon of B. burgdorferi 297. (A) The putative −10 and −35 sites and ribosome-binding site (RBS) are indicated. The arrow denotes a transcriptional initiation site determined from primer extension analysis of B. burgdorferi RNA. Predicted leader peptides culminating in signal peptidase II cleavage sites are denoted by shaded boxes. The black box within dbp corresponds to the amino acid (aa) sequence obtained from internal sequencing of trypsin fragments. The intergenic region is 119 bp long. A putative stem-loop structure for termination is present downstream of dbp. (B) The hybridization probes used to detect transcripts for ORF-1 and dbp were generated by PCR with the primer pairs listed in Table 1. Molecular size markers (kilobases) are shown at left.

FIG. 3

FIG. 4

Deduced amino acid sequences of DBPs from B. burgdorferi 297 and N40. The arrow indicates the predicted site of cleavage by signal peptidase II. Dashes indicate sequence gaps used to align the two DBPs. Black boxes on the lower line indicate identity to the upper line.

FIG. 5

Immunoblots of native and recombinant DBP and ORF-1-encoded protein. Lanes: 1, whole-cell lysates of B. burgdorferi 297; 2, Triton X-114-extracted outer membranes; 3, recombinant DBP (cleaved from its six-His fusion partner) (A and B) or recombinant ORF-1-encoded protein (cleaved) (D); 4 (and panel C), recombinant OspC (cleaved from GST) as a control. Antibody probes are indicated at the top of each panel; rab, rabbit. Numbers at left denote apparent molecular weights (in thousands).

FIG. 6

Appearance of antibodies against ORF-1-encoded protein and DBP during the course of experimental B. burgdorferi infection of C3H/HeJ mice. Recombinant ORF-1-encoded protein (top panel) and recombinant DBP (bottom panel) were subjected to SDS-PAGE. Nitrocellulose strips (containing 750 ng of protein) were immunoblotted with sera harvested from mice at various weeks (wk) (denoted at tops of strips) postinfection with B. burgdorferi. Lanes 7 contain strips probed with rat antiserum directed against either protein. Numbers at left denote apparent molecular weights (in thousands).

FIG. 7

Assessment of the immunological cross-reactivity between DBPs of B. burgdorferi 297 and N40. Recombinant DBPs from strain 297 (lanes 1, 3, 5, and 7) or strain N40 (lanes 2, 4, 6, and 8) were subjected to SDS-PAGE. Individual nitrocellulose strips (containing 750 ng of protein) were then immunoblotted with sera from either 297-infected (lanes 1 and 2) or N40-infected (lanes 3 and 4) mice. Note that during experimental infection, mice did not produce cross-reactive antibodies. For lanes 5 to 8, proteins were probed with either rat anti-297 DBP antibody (lanes 5 and 6) or rat anti-N40 DBP antibody (lanes 7 and 8); artificial immunization of rats with recombinant proteins produced low-level cross-reactive antibodies. Numbers at left denote apparent molecular weights (in thousands).

FIG. 8

Proteinase K accessibility of DBP and ORF-1-encoded protein in B. burgdorferi 297. Growing spirochetes were divided, and one-half of the population was treated with proteinase K. Borrelial proteins were separated by SDS-PAGE. Proteins on nitrocellulose strips from either untreated (lanes 1, 3, 5, 7, and 9) or proteinase K-treated (lanes 2, 4, 6, 8, and 10) borreliae were then immunoblotted with rat antisera as follows: lanes 1 and 2, anti-ORF-1; lanes 3 and 4, anti-DBP; lanes 5 and 6, anti-Fla; lanes 7 and 8, anti-p66; and lanes 9 and 10, anti-OspA. Numbers at left denote apparent molecular weights (in thousands).

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Decorin-binding protein of Borrelia burgdorferi is encoded within a two-gene operon and is protective in the murine model of Lyme borreliosis - PubMed (original) (raw)