Constitutive expression of outer surface protein C diminishes the ability of Borrelia burgdorferi to evade specific humoral immunity

Abstract

The Lyme disease spirochete Borrelia burgdorferi reduces the expression of outer surface protein C (OspC) in response to the development of an anti-OspC humoral response, leading to the hypothesis that the ability to repress OspC expression is critical for the pathogen to proceed to chronic infection. B. burgdorferi was genetically modified to constitutively express OspC by introducing an extra ospC copy fused with the borrelial flagellar gene (flaB) promoter. Such a genetic modification did not reduce infectivity or pathogenicity in severe combined immunodeficiency mice but resulted in clearance of infection by passively transferred OspC antibody. Spirochetes with constitutive ospC expression were unable to establish chronic infections in immunocompetent mice unless they had undergone very destructive mutations in the introduced ospC copy. Two escape mutants were identified; one had all 7 bp deleted between the putative ribosome-binding site and the start codon, ATG, causing a failure in translational initiation, and the other mutant had an insertion of 2 bp between nucleotides 315 and 316, resulting in a nonsense mutation at codon 108. Thus, the ability of B. burgdorferi to repress ospC expression during mammalian infection allows the pathogen to avoid clearance and to preserve the integrity of the important gene for subsequent utilization during its enzootic life cycle.

FIG. 1.

Fused flaB promoter leads to constitutive ospC expression in cultured B. burgdorferi. (A) Constitutive ospC expression at the transcriptional level. Transformants 5A13/pBBE22 and 5A13/pBBE22-ospC′ were grown to late exponential phase at 23°C and 37°C. RNA samples were analyzed by RT-qPCR for flaB and ospC mRNA copy numbers. Each datum point was calculated from a triplicate experiment and is presented as the mean ospC mRNA copy number per 1,000 flaB transcripts. (B) Constitutive ospC expression at the translational level. Spirochetes were prepared as described for panel A, and lysates were subjected to immunoblotting with a mixture of FlaB and OspC MAbs.

FIG. 2.

Infectivity and ospC expression of transformants in SCID mice. (A) Groups of 10 BALB/c SCID mice were infected with either transformant 5A13/pBBE22 or 5A13/pBBE22-ospC′ for 1 month. DNA samples were prepared from heart, joint, and skin specimens and analyzed for spirochete flaB and murine actin DNA copies by qPCR. The data are expressed as spirochete numbers per 10⁶ host cells. (B) RNA samples were prepared from the heart, joint, and skin tissues and quantified for flaB and ospC expression by RT-qPCR. The data are presented as ospC mRNA copy numbers per 1,000 flaB transcripts.

FIG. 3.

5A13/pBBE22-ospC′ spirochetes evade the immune system less effectively than 5A13/pBBE22 spirochetes during early infection. (A) Groups of 10 BALB/c mice were inoculated with either transformant 5A13/pBBE22 or 5A13/pBBE22-ospC′ and sacrificed on day 19 postinoculation. The anti-OspC humoral response was titrated by an end-point ELISA, using sera collected on days 10 and 19 postinoculation (p.i.). (B) RNA samples were prepared from heart, joint, and skin specimens from mice sacrificed on day 19 postinoculation and quantified for flaB and ospC expression by RT-qPCR. The data are presented as ospC mRNA copy numbers per 1,000 flaB transcripts. (C) DNA samples were prepared from heart, joint, and skin specimens and analyzed for spirochete flaB and actin DNA copies by qPCR. The data are expressed as spirochete numbers per 10⁶ host cells.

FIG. 4.

Recovered spirochetes no longer produce an antigen reactive with OspC MAb from the introduced ospC copy. Spirochetes were isolated from ear biopsies of mice BC13 and BC18, which had been infected with transformant 5A13/pBBE22-ospC′ for 17 weeks, designated BC13E and BC18E, respectively, and grown to late exponential phase at either 37°C (A) or 23°C (B and C). The initial inoculum, 5A13/pBBE22-ospC′, was used as a control. Lysates were subjected to immunoblot analysis with either a mixture of FlaB and OspC MAbs (A and B) or a mixture of FlaB MAb and mouse antisera raised against recombinant OspC (C). tOspC, truncated OspC antigen.

FIG. 5.

Escape mutants have destructive mutations in the introduced ospC copy. Spirochetes were isolated from ear biopsies of mice BC13 and BC18 at 17 weeks postinoculation. The two mice were sacrificed after 5 months of infection; spirochetes were recovered from the heart, joint, and skin specimens. DNAs were extracted from these eight isolates and used to transform E. coli. Three to five kanamycin-resistant clones transformed with each DNA preparation were randomly selected and sequenced. In the four panels of the left column, the sequences represent isolates recovered from the ear biopsy (A) and the heart (C), joint (E), and skin (G) specimens of mouse BC13. All isolates obtained from this mouse had a 2-bp insertion (in bold) between nucleotides 315 and 316, producing a nonsense mutation, TAA (shaded), at codon 108. In the four panels of the right column, the sequences represent isolates from the ear biopsy (B) and the heart (D), joint (F), and skin (H) specimens of mouse BC18. All isolates recovered from this animal had 7 bp deleted between the putative ribosome-binding site (RBS) and the initial start codon, ATG (underlined).