Adaptation of a luciferase gene reporter and lac expression system to Borrelia burgdorferi

Abstract

The development of new genetic systems for studying the complex regulatory events that occur within Borrelia burgdorferi is an important goal of contemporary Lyme disease research. Although recent advancements have been made in the genetic manipulation of B. burgdorferi, there still remains a paucity of basic molecular systems for assessing differential gene expression in this pathogen. Herein, we describe the adaptation of two powerful genetic tools for use in B. burgdorferi. The first is a Photinus pyralis firefly luciferase gene reporter that was codon optimized to enhance translation in B. burgdorferi. Using this modified reporter, we demonstrated an increase in luciferase expression when B. burgdorferi transformed with a shuttle vector encoding the outer surface protein C (OspC) promoter fused to the luciferase reporter was cultivated in the presence of fresh rabbit blood. The second is a lac operator/repressor system that was optimized to achieve the tightest degree of regulation. Using the aforementioned luciferase reporter, we assessed the kinetics and maximal level of isopropyl-beta-D-thiogalactopyranoside (IPTG)-dependent gene expression. This lac-inducible expression system also was used to express the gene carried on lp25 required for borrelial persistence in ticks (bptA). These advancements should be generally applicable for assessing further the regulation of other genes potentially involved in virulence expression by B. burgdorferi.

FIG. 1.

Diagram illustrating the two B. burgdorferi shuttle plasmids used in this study (A) and the relevant regions and restriction sites of derivative constructs generated and transformed into B. burgdorferi (B). pJSB66 and pJSB82 were used to assess the impact of codon optimization on luciferase expression. pJSB161, pJSB165, and pJSB175 were created for the luc_Bb⁺ reporter (indicated as Bbluc+on the figure) validation studies. pJSB56, pJSB70, pJSB104, and pJSB252 were generated for the development and evaluation of a lac repressor/operator system optimized for use in B. burgdorferi. pJSB194 was used to express the BptA protein under the control of the lac-inducible expression system. SPluc+, luc_Sp⁺.

FIG. 2.

The impact of codon optimization on the expression of firefly luciferase. The PflaB promoter was fused to either the luc_Sp⁺ (pJSB66) or luc_Bb⁺ (codon-optimized, pJSB82) ORFs and the resulting clones were transformed into B. burgdorferi. After cultures had grown to a cell density of approximately 0.5 × 10⁶ to 5 × 10⁶ spirochetes/ml, four 1-ml aliquots of each culture were collected for luciferase assays. RLU readings were normalized according to cell density and are presented as the RLU/1 × 10⁵ bacteria ± standard deviation. The results from two independent experiments (trials 1 and 2) are presented.

FIG. 3.

Influence of blood supplementation (in BSK medium) on luciferase expression driven by various B. burgdorferi promoters. Cultures of BbJSB161 (promoterless luc_Bb⁺), BbJSB165 (PospC-Bbluc+), and BbJSB175 (PflaB-Bbluc+) were treated with either 6% fresh heparinized rabbit blood (+6% blood) or with 6% heparin-supplemented BSK medium (−6% blood). Two days posttreatment, samples were removed and processed for luciferase assays. (A) Luciferase activities (RLU) from quadruplicate samples of each culture were standardized according to a cell density of 1 × 10⁵ spirochetes, and results are presented as the mean RLU/1 × 10⁵ bacteria ± standard deviation. (B) qRT-PCR analysis was performed on RNA extracted from the culture of BbJSB165 (PospC-Bbluc+) used for luciferase assays (above). SYBR Green one-step qRT-PCR was used to determine the relative levels of ospC, luc_Bb⁺, and flaB transcripts; flaB was included in the analyses for the purpose of signal standardization. The results from six replicate reactions are presented as the mean fold change (fold [relative to heparin-treated culture]) ± standard deviation. Two independent experiments were performed, and representative results from one trial are provided in the figure.

FIG. 4.

Kinetics of luciferase induction from the Borrelia-adapted lac repressor/operator expression construct (pJSB104). Cultures of BbJSB56 (promoterless luc_Bb⁺), BbJSB70 (PpQE30-Bbluc+), and BbJSB104 (PpQE30-Bbluc+/PflaB-BblacI) were induced with various concentrations of IPTG. (A) A culture of BbJSB104 was untreated (0 mM IPTG) or induced with 0.1, 1, and 10 mM IPTG; samples were collected at the designated times postinduction (h). Luciferase activities (RLU) from quadruplicate samples of each culture were standardized according to a cell density of 1 × 10⁵ spirochetes; results are presented as the mean RLU/1 × 10⁵ bacteria ± standard deviation. Four independent induction studies were performed with equivalent results; data from a representative study is shown. (B) SDS-PAGE/immunoblot analysis of Luc, LacI, and FlaB using specific antibodies. Cultures of BbJSB56 (−PpQE30/−lacI_Bb), BbJSB70 (+PpQE30/−lacI_Bb), and BbJSB104 (+PpQE30/+lacI_Bb) were untreated or induced with 1 mM IPTG. Cells were collected at 0, 6, and 24 h postinduction. Total protein from 2 × 10⁶ spirochetes was loaded in each gel lane for the FlaB immunoblot. FlaB detection was included to confirm that equivalent concentrations of lysates were loaded per gel lane. For gels involving Luc and LacI immunoblots, a volume of whole-cell lysate equivalent to 1 × 10⁷ spirochetes was loaded per gel lane. Values at left denote relevant molecular masses (kDa) of Bio-Rad All Blue Precision Plus standard (MW).

FIG. 5.

Kinetics of luciferase expression from the modified lac repressor/operator expression construct (pJSB252). A culture of BbJSB252 (analogous to pJSB104, but with PflaB-BblacI reoriented upstream and divergent from luc_Bb⁺) was untreated (0 mM IPTG) or induced with 1 mM IPTG. Samples were collected at the designated times (h), and luciferase assays were performed. Luciferase activities (RLU) from quadruplicate samples of each culture were standardized according to a cell density of 1 × 10⁵ spirochetes; results are presented as the mean RLU/1 × 10⁵ bacteria ± standard deviation. Two independent induction studies were performed with equivalent results; only data from one experiment are shown.

FIG. 6.

Control of BptA expression using the Borrelia-adapted lac repressor/operator expression system. Cultures of BbDTR596 (bptA-) and BbJSB194 (bptA-/PpQE30-bptA) were untreated or induced with 1 mM IPTG. Six hours postinduction, cells were collected and prepared for SDS-PAGE/immunoblot analysis; total protein from 1 × 10⁷ spirochetes was loaded in each gel lane. Antibody against recombinant BptA was used to assess induction. Equivalent protein loading per gel lane was verified by probing for FlaB. Values at left denote relevant molecular masses (kDa) of Bio-Rad All Blue Precision Plus standard (MW). Two independent induction studies were performed with equivalent results; data from one representative experiment are shown.