Table 1.
Strains and Plasmids used in this study.
Table 2.
Primers used in this study.
Fig 1.
Characterization of a bioluminescent B. burgdorferi PospC reporter strain.
The response of borrelial ospC reporter strain to pH was assessed to determine the validity of PospC-luc relative to native OspC production. PflaB-luc and PospC-luc B. burgdorferi strains were grown at pH 7 and pH 8 to mid-log phase to assess in vitro luminescence assay and protein production via Western blot analysis. (A) PflaB-luc and PospC-luc cultures were serial diluted from 106 to 10 cells, treated with D-luciferin, and luminescence was measured in photons/sec. PflaB-luc pH 7 (red squares) and PflaB-luc pH 8 (black circles) did not differ in bioluminescence. PospC-luc pH 7 (blue squares) induces greater luminescence relative to PospC-luc pH 8 (green circles). Values represent three independent cultures that were normalized to background and averaged. Error bars represent standard error. (B) Differential protein production of Luc in PospC-luc reporter strain in response to pH reflects changes observed for the native OspC in PflaB-luc and PospC-luc. Cell lysates of PflaB-luc and PospC-luc at pH 7 or pH 8 were immunoblotted and probed with anti-sera against OspC, FFluc and FlaB that served as a loading control.
Fig 2.
Temporal monitoring of PflaB-luc and PospC-luc expressing B. burgdorferi during experimental infection.
Balb/c mice were infected with PflaB-luc (A) or PospC-luc (B) reporter strains at 105 by ventral intradermal infection, treated with D-luciferin and imaged by IVIS at 0, 4, 7, 10, 14 and 21 days post-infection. A background control mouse was included in each group that was infected with luminescent B. burgdorferi but not treated with D-luciferin; such mouse is shown in the far left position of each image. D-luciferin treatment or the lack thereof is designated by a + or -, respectively. A 10 minute exposure was utilized to obtain images. Normalization to subtract background was performed per strain for all time points displayed in the color spectrum position under the images. PflaB-luc and PospC-luc images are set on individual scales to display the full spectrum of bioluminescence. (A) PflaB-luc images were normalized to radiance range of 6.95x103-1.83x105 p/sec/cm2/sr. One-way ANOVA followed by Tukey’s Multiple Comparison test was performed to determine significant difference resulting in a p-value of 0.0132. A p-value < 0.05 is considered significant. (B) PospC-luc images were normalized to radiance range of 2.38x103-1.1x105 p/sec/cm2/sr. One-way ANOVA followed by Tukey’s Multiple Comparison test was performed to determine significant difference resulting in a p-value of 0.0009. A p-value < 0.05 is considered significant.
Fig 3.
Quantitation of PflaB-luc and PospC-luc expressing B. burgdorferi using bioluminescent readout.
Five Balb/c mice were infected with 105 PflaB-luc or PospC-luc containing B. burgdorferi and 4 mice treated with D-luciferin for imaging 0, 4, 7, 10, 14, and 21 days post-inoculation for bioluminescent imaging. (A) Quantitation of 1 minute exposures was performed. At all time points the whole mouse was measured to obtain a measurement in photons/sec, representing total flux. Bioluminescence from the 4 mice treated with D-luciferin was normalized by subtracting the measurement from the no D-luciferin control and averaged. Blue circles represent PflaB-luc and red squares PospC-luc. Error bars represent standard error. One-way ANOVA analysis resulted in statistical significance for PflaB-luc and PospC-luc with a p-value of 0.0132 and 0.0262, respectively. (B) To assess the expression of ospC independent of changes in borrelial load, the ratio differential of PospC-luc and PflaB-luc was calculated and represented on the y-axis. Permutation analyses comparing time points to each other found statistically significant differences (p < 0.05) between all comparisons, except between day 4 and day 21.
Fig 4.
Validation of equivalent bacterial load of PflaB-luc and PospC-luc infected Balb/c mice.
Skin samples from adjacent to the inoculation site of Balb/c mice infected with 105 PflaB-luc or PospC-luc on day 10 (A) or day 21 (B) following inoculation were harvested for qPCR analysis of borrelial genomes (recA) per copies of 106 β-actin. Horizontal bars denotes average copies of recA per 106 β-actin and error bars represent standard error. Statistical analysis using the Mann-Whitney test indicated a lack of significance between the PflaB-luc and PospC-luc at day 10 and 21 post-infection with p-values of 0.2222 and 0.9444, respectively.
Fig 5.
Temporal and spatial expression of B. burgdorferi containing PflaB-luc and PospC-luc in murine tissues.
Skin, inguinal lymph node, heart, bladder, and tibiotarsal joint, from PflaB-luc or PospC-luc infected Balb/c mice were quantitatively assessed for bioluminescent emission at 4, 7, 10, 14 and 21 days post-infection. Four of the five mice were treated with a double bolus of D-luciferin and the remaining mouse served as a background control for normalization of ex vivo tissues. The—represents the no luciferin control and + designates the tissues treated with D-luciferin. Tissues were evaluated for bacterial load or ospC expression as represented by PflaB-luc and PospC-luc, respectively. Normalization to subtract background was performed per strain for all time points displayed in the color spectrum position under the images. PflaB-luc and PospC-luc images are set on individual scales to display the full spectrum of bioluminescence over the experimental time course. Measurable radiance above background is detectable on all days evaluated in all tissues with the exception of PospC-luc infected inguinal lymph node that emits minimal luminescence. Graphs for each tissue display the ratio of PospC-luc/PflaB-luc to depict the expression of ospC as measured by PospC-luc relative to bacterial load (scored by PflaB-luc) for a given time point. The PospC-luc/PflaB-luc ratio underwent permutation analyses comparing all time points to determine statistical significance. (A) The radiance range of skin at the site of inoculation for both strains is 4.9e3-1.83e5 p/sec/cm2/sr. All comparisons have a p-value < 0.05. (B) Heart radiance for PflaB-luc is 4.3e2-1.1e4 and PospC-luc is 3.92e2-3.9e3 p/sec/cm2/sr. Two comparisons, day 4 versus day 21 and day 7 versus day 14, were not statistically significant. Remaining comparisons were significantly different (p-value<0.05). (C) Inguinal lymph node radiance for PflaB-luc is 1.1e3-1.1e4 and PospC-luc is 8.32e2-1.5e3 p/sec/cm2/sr. Comparisons were statistically significant with p-values of 0.0160 or less, except day 10 versus day 21. (D) Bladder radiance for PflaB-luc is 1.1e3-5e4 and PospC-luc is 3.78e2-1.1e4 p/sec/cm2/sr. There is statistical difference between early time points (day 4, 7, & 10) and late time points (day 14 & 21) with p-values no greater than 0.0305. (E) Tibiotarsal joint radiance for PflaB-luc is 1.15e3-1.15e5 and PospC-luc is 4.95e2-1.1e4 p/sec/cm2/sr. All comparisons were statistically different (p-value < 0.05), except for day 4 versus day 21.
Fig 6.
Correlation of ospC radiance and quantitation measure of ospC transcript in mammalian tissues.
PospC-luc infected tissues from day 10 and 21 post-infection were evaluated for radiance (p/sec/cm2/sr) relative to total ospC transcript for the whole tissue sample. Skin (A), heart (B), bladder (C), and tibiotarsal joint (D) had r values of 0.944, 0.961, 0.854, and 0.812, respectively.
Fig 7.
Evaluation of ospC expression as represented by PospC-luc expressing B. burgdorferi during early infection.
Balb/c mice infected by ventral intradermal injection with 105 PospC-luc and PflaB-luc B. burgdorferi for the quantitation of ospC relative to bacterial load during the 96 hours following needle inoculation. (A) Mice were treated with D-luciferin and imaged 2, 8, 24, 48, 72, and 96 hours with the exception of one no D-luciferin background control at each time point. Ten minute exposures were used to obtain images and were normalized to radiance range of 6.95x103-1.83x105 p/sec/cm2/sr. (B) Quantification of bioluminescence was determined from 1 minute exposure images. Values represent flux (photon/sec) normalized to background and the averaged value from four mice treated with D-luciferin. PospC-luc is represented by red squares and PflaB-luc is represented by blue circles. Error bars represent standard error. An asterisk represents p < 0.05, indicating significant differences in bioluminescence between PospC-luc and PflaB-luc containing B. burgdorferi. There was no significant difference in PospC-luc radiance during the first 96 hours. One-way ANOVA of PflaB-luc radiance had a of p-value < 0.0001 indicating a statistically significant change in luminescence.