Regulation of OspE-related, OspF-related, and Elp lipoproteins of Borrelia burgdorferi strain 297 by mammalian host-specific signals

Abstract

In previous studies we have characterized the cp32/18 loci in Borrelia burgdorferi 297 which encode OspE and OspF orthologs and a third group of lipoproteins which possess OspE/F-like leader peptides (Elps). To further these studies, we have comprehensively analyzed their patterns of expression throughout the borrelial enzootic cycle. Serial dilution reverse transcription-PCR analysis indicated that although a shift in temperature from 23 to 37 degrees C induced transcription for all nine genes analyzed, this effect was often markedly enhanced in mammalian host-adapted organisms cultivated within dialysis membrane chambers (DMCs) implanted within the peritoneal cavities of rats. Indirect immunofluorescence assays performed on temperature-shifted, in vitro-cultivated spirochetes and organisms in the midguts of unfed and fed ticks revealed distinct expression profiles for many of the OspE-related, OspF-related, and Elp proteins. Other than BbK2.10 and ElpA1, all were expressed by temperature-shifted organisms, while only OspE, ElpB1, OspF, and BbK2.11 were expressed in the midguts of fed ticks. Additionally, although mRNA was detected for all nine lipoprotein-encoding genes, two of these proteins (BbK2.10 and ElpA1) were not expressed by spirochetes cultivated in vitro, within DMCs, or by spirochetes within tick midguts. However, the observation that B. burgdorferi-infected mice generated specific antibodies against BbK2.10 and ElpA1 indicated that these antigens are expressed only in the mammalian host and that a form of posttranscriptional regulation is involved. Analysis of the upstream regions of these genes revealed several differences between their promoter regions, the majority of which were found in the -10 and -35 hexamers and the spacer regions between them. Also, rather than undergoing simultaneous upregulation during tick feeding, these genes and the corresponding lipoproteins appear to be subject to progressive recruitment or enhancement of expression as B. burgdorferi is transmitted from its tick vector to the mammalian host. These findings underscore the potential relevance of these molecules to the pathogenic events of early Lyme disease.

FIG. 1

Schematic representation of the ospE-related, ospF-related, and elp genes characterized in B. burgdorferi strain 297. Evolutionarily related genes are shaded similarly. Below each gene, the primers used for the serial dilution RT-PCR and transcriptional linkage analyses are indicated. The cp32 or cp18 plasmid which harbors each locus is indicated at left, and the direction of transcription for each gene or operon is indicated by an arrow.

FIG. 2

Serial dilution RT-PCR and immunoblot analyses of the ospE-related, ospF-related, and elp genes and proteins. (A) Total RNA was isolated and processed as indicated in Materials and Methods from spirochetes cultivated in vitro at 23°C, shifted from 23 to 37°C, or cultivated within rat peritoneal DMCs. Twofold serial dilutions of cDNA were amplified for 45 cycles by PCR with the specific primers indicated in Table 1 to detect mRNA abundance under the various conditions. The final amounts of cDNA used were from 5 pg to 39 fg for flaB, 313 to 2.4 pg for ospA and ospC, and 20 ng to 156 pg for the various ospE-related, ospF-related, and elp genes. −RT indicates reaction mixtures lacking reverse transcriptase; + and − indicate control PCRs using 10 ng of total B. burgdorferi strain 297 genomic DNA and water, respectively. (B) B. burgdorferi whole-cell lysates probed with 1:100 dilutions of affinity-purified rat anti-OspC, -OspE, -ElpB1, -p21, -ElpOs2, -BbK2.10, -ElpA1, and -ElpA2 or MAb 10D7-42 for OspF/BbK2.11, 1H6-33 for FlaB, and 1402-27 for OspA. Lysates were generated from organisms cultivated in vitro at 23°C, shifted from 23 to 37°C, or cultivated within rat peritoneal DMCs. Protein expression was detected using the Enhanced Chemiluminescence Plus Western blotting system as described in Materials and Methods.

FIG. 3

Ontogeny of the antibody response against the OspE-related, OspF-related, and Elp proteins following syringe inoculation or tick infestation of C3H/HeJ mice. Pooled sera collected over an 8-week period from five C3H/HeJ mice either syringe inoculated or tick infested were analyzed for reactivity against the cleaved, recombinant OspE-related, OspF-related, and Elp proteins by ELISA. Additionally, antibody reactivity to the constitutively expressed FlaB protein, temperature-induced OspC lipoprotein, and OspA (which is downregulated or repressed during infection) was included in the analysis. To ensure monospecificity for the highly homologous p21/OspE and OspF/BbK2.11 proteins, sera from all time points were preadsorbed overnight at 4°C with the potentially cross-reactive paralog prior to ELISA analysis.

FIG. 4

Primer extension, multiple sequence alignment, and phylogenetic analysis of the ospE/ospF/elp promoter regions. (A) Primer extension analysis showing the product of the bbk2.10 mRNA generated with reverse transcriptase (lane RT) and the sequencing ladder of the homologous region of the bbk2.10 gene. Lanes loaded with individual dideoxy sequencing reaction mixtures are labeled A, T, C, and G. The arrow indicates the position of the primer extension product in relation to the sequencing ladder. RBS indicates the putative ribosomal binding site. Also shown are ClustalW alignment (B) and phenogram analysis (C) of the upstream 85 bp of the putative promoter regions for the ospE-related, ospF-related, and elp loci (left); for comparison, a phenogram of the full-length lipoproteins also is shown (right).