Use of Lux_P_<i>tolC</i> to monitor survival of <i>Y. pestis</i> in the presence of antimicrobial compounds.

<p><i>Y. pestis</i> Lux_P<i>tolC</i> was incubated with increasing concentrations of antimicrobials (n = 9) in a 96-well format and bacterial survival was monitored by measuring bioluminescence. (A) Bioluminescence readings (RLU) from <i>Y. pestis</i> Lux_P<i>tolC</i> incubated with MicroChem-Plus for 14 mins. (B) At 6 mins during incubation with MicroChem-Plus, bacteria were harvested from a subset of wells, washed, serially diluted, and spot plated on agar to determine bacterial CFU. (C) Bioluminescence readings (RLU) from <i>Y. pestis</i> Lux_P<i>tolC</i> incubated with carbenicillin for 12 hrs. (D) At 4 (white), 8 (gray), and 12 (black) hrs during incubation with carbenicillin bacteria were harvested from a subset of wells to determine bacterial CFU. (E) Bioluminescence readings (RLU) from <i>Y. pestis</i> Lux_P<i>tolC</i> incubated with gentamicin for 12 hrs. (F) At 4 (white), 8 (gray), and 12 (black) hrs during incubation with gentamicin, bacteria were harvested from a subset of wells to determine bacterial CFU. For D and F, the dotted line represents the limit of detection.</p

Strains and plasmids used in this work.

<p>Strains and plasmids used in this work.</p

Dissemination of <i>Y. pestis</i> during bubonic infection.

<p>Mice were infected with ∼200 CFU of <i>Y. pestis</i> Lux_{P<i>cysZK</i>} subcutaneously at the base of the tail (A) or in the footpad (B) and imaged using an IVIS Spectrum. The lymph node drainage basin for each inoculation site is diagrammed above the images <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047123#pone.0047123-Harrell1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047123#pone.0047123-VandenBroeck1" target="_blank">[25]</a>. Location of the inoculation site is shown as a green circle, lymph nodes as blue circles, and the spleen as a red oval. For (A), the white arrow denotes the subiliac LN and the red arrow the axillary LN. For (B), the white arrow denotes the popliteal LN, the red arrow the sciatic LN, and the yellow arrow the renal LN. All images were adjusted to the radiance scale shown, except for the images in (B) marked with * in upper right corners. For these each image was adjusted to a different radiance to allow for visualization of specific tissues.</p

Extended imaging of animals intranasally infected with Δ<i>pla</i>.

<p>30% of animals infected intranasally with the Δ<i>pla</i> mutant in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047123#pone-0047123-g006" target="_blank">Figure 6</a> developed bioluminescence signal from regions corresponding to the head. A, B, and C represent three individual animals. Animal A also represents an example of a Δ<i>pla</i> infected animal that developed septicemic plague.</p

Sensitivities of chromosomal Lux reporters.

<p>The <i>luxCDABE</i> operon driven by different promoters was integrated into the <i>Y. pestis</i> chromosome using Tn7 transposition. Sensitivities of the Lux reporters were determined by making serial dilutions of the <i>Y. pestis</i> Lux strains (grown for 15 hrs) and determining the number of bacteria (CFU) and bioluminescence (RLU) in each dilution (n = 3). Linear regression analysis of the Log transformed data was used to calculate the trend line, R² values, and the limit of detection [LD = Log₁₀CFU (± standard deviation)]. (A) pGEN-<i>luxCDABE</i>, (B) Lux_PEM7, (C) Lux_P<i>tolC</i>, (D) Lux_{P<i>cysZK</i>}.</p

Survival of <i>Y. pestis</i> Lux reporters in macrophages.

<p>RAW264.7 macrophages were infected with <i>Y. pestis</i> Lux reporter strains, extracellular bacteria killed by gentamicin, and bacterial survival monitored by CFU determination (A–C) or bioluminescence (D–F). Data from WT <i>Y. pestis</i> is represented by black symbols and from an attenuated Δ<i>phoP</i> mutant by white symbols. (A and D) are strains with the Lux_PEM7 reporter (n = 3 for CFU, n = 24 for RLU), (B and E) are strains with the Lux_P<i>tolC</i> reporter (n = 3 for CFU, n = 12 for RLU), and (C and F) are strains with the Lux_{P<i>cysZK</i>} reporter (n = 3 for CFU, n = 12 for RLU).</p

Progression of pneumonic infection.

<p>Mice were infected with 5×10⁴–1×10⁵ CFU of <i>Y. pestis</i> Lux_{P<i>cysZK</i>} intranasally and imaged using an IVIS Spectrum. (A) Sequential images from representative animals. (B) For each animal, average bioluminescence was calculated for the thoracic cavity using the ROI tool in Living Image 3.2 software package. Black and white symbols represent animals infected with WT or Δ<i>pla Y. pestis</i>, respectively. Dotted line represents the limit of detection based on images from uninfected animals. ** = p<0.005, *** = p<0.001. At various time points, lungs were harvested from a subset of animals to determine bacterial loads (CFU) and compared to bioluminescence from the thoracic cavity (C) or from the lungs ex vivo (D).</p

Correlation between bioluminescence and bacterial number.

<p><i>Y. pestis</i> Lux_P<i>tolC</i> and Lux_{P<i>cysZK</i>} were diluted in BHI broth (n = 3) and grown at 26°C (A and B) or 37°C (C and D) for 12 hrs. Samples were harvested at multiple time points during growth to determine bioluminescence (RLU) and bacterial numbers (CFU). Linear regression analysis of the Log transformed data was used to calculate the trend line and R² values. (E) To determine if temperature impacted expression of the Lux_P<i>tolC</i> (white circles) or Lux_{P<i>cysZK</i>} (black circles) reporters, we calculated the RLU/CFU for each sample in A–D and compared the ratios. Black bars represent median values and statistical significance was determined using the Mann-Whitney <i>t</i> test with a two-tailed nonparametric analysis (**** = p<0.0001, ns = not significantly different).</p

Lux reporters do not impact fitness of <i>Y. pestis</i>.

<p>To determine if carriage of the Lux reporters impacted <i>Y. pestis</i> fitness, (A) growth of the <i>Y. pestis</i> Lux bioreporter strains (n = 3), and (B) survival in macrophages (n = 3) were compared to WT <i>Y. pestis</i> without a Lux reporter. WT (no reporter) = • or black bar; Lux_PEM7 = ○ or white bar; Lux_P<i>tolC</i> = □ or gray bar; Lux_{P<i>cysZK</i>} = ▪ or hatched bar.</p

Continued bioluminescence from inoculation site.

<p>Mice were infected with ∼200 CFU of WT (n = 5) or Δ<i>pla</i> (n = 5) <i>Y. pestis</i> Lux_{P<i>cysZK</i>} subcutaneously at the base of the tail and imaged using an IVIS Spectrum. (A) The average bioluminescence detected from the inoculation site was determined over the course of the infection. Black and white symbols represent animals infected with WT or Δ<i>pla Y. pestis</i>, respectively. (B) Sequential images from a representative animal infected with Δ<i>pla Y. pestis</i> Lux_{P<i>cysZK</i>}.</p