Regulation of the virulence determinant OspC by bbd18 on linear plasmid lp17 of Borrelia burgdorferi

Abstract

Persistent infection of a mammalian host by Borrelia burgdorferi, the spirochete that causes Lyme disease, requires specific downregulation of an immunogenic outer surface protein, OspC. Although OspC is an essential virulence factor needed by the spirochete to establish infection in the mammal, it represents a potent target for the host acquired immune response, and constitutive expression of OspC results in spirochete clearance. In this study, we demonstrate that a factor encoded on a linear plasmid of B. burgdorferi, lp17, can negatively regulate ospC transcription from the endogenous gene on the circular plasmid cp26 and from an ospC promoter-lacZ fusion on a shuttle vector. Furthermore, we have identified bbd18 as the gene on lp17 that is responsible for this effect. These data identify a novel component of ospC regulation and provide the basis for determining the molecular mechanisms of ospC repression in vivo.

Fig. 1.

Negative regulation of ospC by lp17 in B. burgdorferi B312. (A) Total B. burgdorferi protein lysates analyzed by immunoblotting with OspC and FlaB antisera, as indicated. Lane 1, wild-type clone B31-A3; lane 2, B312; lane 3, B312_lp17.8; lane 4, mutant ospC K1; lane 5, ospC K1 + ospC. (B) ospC transcript levels in total RNAs from B312 and B312_lp17.8 (with lp17), as measured by quantitative RT-PCR and normalized to the chromosomal flaB gene transcript. ***, P < 0.002 based on Student's two-tailed t test. (C) ospC promoter activity in B. burgdorferi B312 (no lp17) and B312_lp17.8 (with lp17), monitored by the lacZ reporter gene. Beta-galactosidase activity in B. burgdorferi transformed with a shuttle vector carrying a lacZ reporter gene fused to the ospC promoter (pBHospCp-lacZBb*) was detected by the addition of X-Gal to solid BSK II medium without phenol red, resulting in blue B312 colonies (left), whereas B312_lp17.8 colonies remained white (right).

Fig. 2.

Introduction of truncated and cloned segments of lp17 into B. burgdorferi B312 and the resulting OspC phenotype. (A) Schematic diagram of truncated forms of lp17 created by insertion of replicated telomeres (3). lp17 sequences were deleted to the left of the insertion point for pGCB413 (red arrowhead above diagram) and to the right of the insertion points for pGCB409, pGCB426, and pGCB473 (yellow arrowheads beneath diagram). Light shading indicates lp17 sequences that were not present in the respective lp17 truncated variants. Gene designations are as previously annotated (13, 23). The presence (+) or absence (−) of OspC in protein lysates of B312 transformants carrying different truncated forms of lp17 is indicated to the right of each plasmid diagram. (B) OspC immunoblots of B312 and transformants carrying truncated forms of lp17, as well as the ospC K1 mutant. Whole-cell lysates of B. burgdorferi strains, as identified at the top of each lane, were separated by SDS-PAGE and analyzed by immunoblotting with anti-OspC antiserum. (C) Schematic diagram of lp17 sequences introduced on the shuttle vector pBSV2*. The presence (+) or absence (−) of OspC in protein lysates of B312 transformants carrying the shuttle vectors is indicated to the right of each plasmid diagram. The number in parentheses to the left of each diagram corresponds with the lane designation in the immunoblot panels below. NP, native promoter; flaB_p, constitutive promoter. (D) OspC, BBD18, and FlaB immunoblots of B. burgdorferi. Whole-cell lysates of B. burgdorferi wild-type clone B31-A3 (lane 1), high-passage clone B312 (lane 2), B312/pBSV2*-7′ (lane 3), B312/pBSV2*-NP-bbd18 (lane 4), B312/pBSV2*-flaB-bbd18 (lane 5), and B312/pBSV2*-bbd18 (lane 6) were analyzed by immunoblotting with antisera recognizing OspC, BBD18, and FlaB, as indicated. The estimated sizes of the proteins recognized in each immunoblot, relative to the mobilities of standards, are indicated to the right of each panel.

Fig. 3.

Assessing BBD18-mediated repression of the ospC promoter in E. coli. (A) ospC promoter-lacZBb expression in E. coli as monitored by colony color. Reporter constructs carrying lacZBb, with or without the ospC promoter (as identified at the left side of the figure), were introduced into E. coli that harbored or lacked a second plasmid with a constitutively expressed bbd18 gene (as designated at the top of the figure) and streaked on plates containing X-Gal. Expression of lacZBb from the ospC promoter in the presence or absence of BBD18 resulted in blue bacterial colonies (bottom panels), whereas bacteria carrying the promoterless lacZBb gene formed white colonies under both conditions (top panels). The number of each panel corresponds with the lane designation in the following immunoblot. (B) BBD18 immunoblot of E. coli strains harboring plasmids with the following genes: constitutively expressed flaBp::bbd18 (lane 1), promoterless lacZBb (lane 2), ospC promoter-lacZBb (lane 3), promoterless lacZBb plus constitutively expressed flaBp::bbd18 (lane 4), and ospC promoter-lacZBb plus constitutively expressed flaBp::bbd18 (lane 5). The estimated size of BBD18 relative to the mobilities of standards is indicated to the right of the immunoblot.

Fig. 4.

Proposed model of ospC regulation in wild-type and B312 strains of B. burgdorferi. Activation of the response regulator Rrp2 in wild-type B. burgdorferi by environmental signals present during tick feeding leads to rpoS transcription through the alternative sigma factor RpoN (σ⁵⁴), which interacts with RNA polymerase (RNAP) (29, 59). RpoS in turn positively regulates ospC transcription, resulting in OspC on the spirochete's outer membrane during the critical initial stage of mammalian infection (26, 29). Persistent infection of mammals by wild-type B. burgdorferi requires specific repression of ospC to evade host acquired immunity (57). Clone B312 constitutively synthesizes OspC in the absence of stimuli typically required for induction in wild-type B. burgdorferi. Likewise, bbd18 is constitutively expressed when lp17 is restored to B312, resulting in ospC repression. Inward-facing arrowheads indicate a putative operator site upstream of ospC. Parts of this figure are modeled closely after a figure by Burtnick et al. (9).