A Dual Luciferase Reporter System for B. burgdorferi Measures Transcriptional Activity during Tick-Pathogen Interactions

Abstract

Knowledge of the transcriptional responses of vector-borne pathogens at the vector-pathogen interface is critical for understanding disease transmission. Borrelia (Borreliella) burgdorferi, the causative agent of Lyme disease in the United States, is transmitted by the bite of infected Ixodes sp. ticks. It is known that B. burgdorferi has altered patterns of gene expression during tick acquisition, persistence and transmission. Recently, we and others have discovered in vitro expression of RNAs found internal, overlapping, and antisense to annotated open reading frames in the B. burgdorferi genome. However, there is a lack of molecular genetic tools for B. burgdorferi for quantitative, strand-specific, comparative analysis of these transcripts in distinct environments such as the arthropod vector. To address this need, we have developed a dual luciferase reporter system to quantify B. burgdorferi promoter activities in a strand-specific manner. We demonstrate that constitutive expression of a B. burgdorferi codon-optimized Renilla reniformis luciferase gene (rluc_Bb ) allows normalization of the activity of a promoter of interest when fused to the B. burgdorferi codon-optimized Photinus pyralis luciferase gene (fluc_Bb) on the same plasmid. Using the well characterized, differentially regulated, promoters for flagellin (flaBp), outer surface protein A (ospAp) and outer surface protein C (ospCp), we document the efficacy of the dual luciferase system for quantitation of promoter activities during in vitro growth and in infected ticks. Cumulatively, the dual luciferase method outlined herein is the first dual reporter system for B. burgdorferi, providing a novel and highly versatile approach for strand-specific molecular genetic analyses.

Keywords: Borrelia (Borreliella) burgdorferi; Lyme disease; Photinus pyralis luciferase; Photinus reniformis luciferase; bioluminescence reporter; tick-pathogen interactions.

Figure 1

B. burgdorferi luciferase plasmids. All of the B. burgdorferi luciferase shuttle vectors were derived from pJSB161, which contains a Rho-independent transcription terminator sequence (terminator); ORFs 1, 2, and 3 of the B. burgdorferi cp9 replication machinery (cp9 ori); E. coli origin of replication (ColE1 ori); and the spectinomycin/streptomycin resistance cassette (flg-aadA) (Blevins et al., 2007). (A) The B. burgdorferi shuttle vector pJSB161 features a promoterless, B. burgdorferi codon optimized Photinus pyralis luciferase (fluc_Bb), an upstream ribosome binding site (RBS) and a unique BlgII restriction site (Blevins et al., 2007). The plasmid pJSB175 was generated by addition of the flaBp promoter upstream of fluc_Bb in pJSB161 (Blevins et al., 2007). (B) The B. burgdorferi codon optimized Renilla reniformis luciferase (rluc_Bb) gene under the control of the flaB promoter (flaBp-rluc_Bb) was added to pJSB161, generating the B. burgdorferi dual luciferase shuttle vector, pCFA701. Plasmids, pCFA801, pCFA802, and pCFA803, harbor the flaB, ospA, and ospC promoters, respectively, upstream of fluc_Bb. (C) The density of B. burgdorferi clone A3-68Δbbe02 (wild type) alone or harboring various B. burgdorferi luciferase plasmids was assessed over a period of 144 h using a Petroff Hauser counting chamber and dark-field microscopy. The data are presented as the mean spirochete density (spirochetes/ml) ± standard deviation over time (hours).

Figure 2

Selectivity and sensitivity of the dual luciferase assay in B. burgdorferi. (A) B. burgdorferi clones were grown to mid-logarithmic phase, and the in vitro luciferase assay performed with 700 μM D-luciferin or 3.5 mM coelenterazine. Relative luciferase units were normalized to optical density at 600 nm (OD₆₀₀) and presented as the mean relative luciferase units/OD₆₀₀ ± standard deviation for biological triplicate samples. The data were square root transformed and analyzed with a one-way ANOVA followed by Dunnett's multiple comparison test compared to B. burgdorferi containing the promoterless fluc_Bb (+pJSB161) for each substrate. Unless indicated, means were not significantly different from the control. Significant differences are indicated with asterisks (^****p ≤ 0.0001). (B) Mid-logarithmic phase grown B. burgdorferi expressing both flaBp-rluc_Bb and flaBp-fluc_Bb (+pCFA801) were serial diluted from 2 × 10⁸ to 2 × 10⁰ spirochetes, and incubated with 3.5 mM coelenterazine. The limit of detection (LoD) was established as the mean relative luciferase units for PBS alone plus 3 standard deviations (gray dotted line). The limit of quantitation (LoQ) was established as the mean relative luciferase units for PBS alone plus 10 standard deviations (red dotted line). Data are presented as the mean relative luciferase units ± standard deviation for biological triplicate samples.

Figure 3

Dual luciferase assay with in vitro grown B. burgdorferi. Spirochetes were grown to either mid-logarithmic or stationary phase and the in vitro luciferase assay performed with 700 μM D-luciferin or 3.5 mM coelenterazine. All data are presented as mean normalized relative luciferase units ± standard deviation for biological triplicate samples. (A) Relative Rluc_Bb units normalized to OD₆₀₀. The data set for each growth condition was analyzed with a one-way ANOVA followed by Dunnett's multiple comparison test compared to B. burgdorferi expressing flaBp-rluc_Bb (+pCFA701) and Bonferroni's multiple comparison test to compare the same clone in the two growth phases. (B) Relative Fluc_Bb units normalized to OD₆₀₀ or 10⁸ relative Rluc_Bb units of the same sample. Each data set was square root transformed and analyzed with a one-way ANOVA followed by Dunnett's multiple comparison test compared to B. burgdorferi expressing flaBp-rluc_Bb (+pCFA701). Unless indicated, means were not significantly different from the control. Significant differences are indicated with asterisks (^*p ≤ 0.05; ^**p ≤ 0.01; ^***p ≤ 0.001; ^****p ≤ 0.0001).

Figure 4

The dual luciferase plasmids do not affect B. burgdorferi acquisition efficiency. Groups of ~200 naïve Ixodes scapularis larvae were fed to repletion on mice infected with B. burgdorferi containing distinct luciferase plasmids. Subsets of individual fed larvae per B. burgdorferi clone were surface sterilized, crushed, and plated in solid BSKII containing RPA cocktail and 50 μg/ml streptomycin. Data points represent the number of colony forming units (CFUs) per individual fed larva. Uninfected larvae (CFU = 0) are represented as data points on the X-axis. No significant differences were detected across the data as determined with a one-way ANOVA.

Figure 5

The dual luciferase assay quantitates known patterns of B. burgdorferi transcript expression in nymphs. (A) Groups of eight fed nymphs per B. burgdorferi clone were surface sterilized and crushed in 250 μl of PBS and the in vivo tick luciferase assay performed with 100 μl of tick extract and 700 μM D-luciferin or 3.5 mM coelenterazine. The data are presented as the mean relative Fluc_Bb units per 10⁸ relative Rluc_Bb units ± standard deviation (n = 3; B. burgdorferi carrying pCFA803 n = 2). (B) Relative Rluc_Bb luciferase activity is reflective of spirochete numbers in extracts from fed infected nymphs. 1 μl of fed nymph extract was plated for CFUs in solid BSKII containing RPA cocktail and 50 μg/ml streptomycin. The number of CFUs/100 μl of tick extract (CFUs) was plotted against the relative Rluc_Bb units for the same extract. The red dotted line indicates the established LoQ for relative Rluc_Bb units. Black symbols represent extracts with quantifiable relative Rluc_Bb units and red symbols represent extracts with non-quantifiable Rluc_Bb units. A nymph extract with no detectable CFU (CFU = 0) is represented as the data point on the Y-axis. A significant positive correlation was detected between CFU and relative Rluc_Bb units (Pearson coefficient, r = 0.8022, p = 0.0017).