Real-Time Bioluminescence Imaging of Mixed Mycobacterial Infections

Molecular analysis of infectious processes in bacteria normally involves construction of isogenic mutants that can then be compared to wild type in an animal model. Pathogenesis and antimicrobial studies are complicated by variability between animals and the need to sacrifice individual animals at specific time points. Live animal imaging allows real-time analysis of infections without the need to sacrifice animals, allowing quantitative data to be collected at multiple time points in all organs simultaneously. However, imaging has not previously allowed simultaneous imaging of both mutant and wild type strains of mycobacteria in the same animal. We address this problem by using both firefly (Photinus pyralis) and click beetle (Pyrophorus plagiophthalamus) red luciferases, which emit distinct bioluminescent spectra, allowing simultaneous imaging of two different mycobacterial strains during infection. We also demonstrate that these same bioluminescence reporters can be used to evaluate therapeutic efficacy in real-time, greatly facilitating our ability to screen novel antibiotics as they are developed. Due to the slow growth rate of mycobacteria, novel imaging technologies are a pressing need, since they can they can impact the rate of development of new therapeutics as well as improving our understanding of virulence mechanisms and the evaluation of novel vaccine candidates

Luminescence of mycobacteria during growth in bacteriological media.

<p>Luminescent (FFlux and CBRlux) and non-luminescent (pJDC89) BCG carrying the vector backbone display similar growth rates in M-ADC-TW plus hygromycin (80 µg/ml) laboratory medium (A). Luminescence for both firefly (FFlux) and click beetle red (CBRlux) luciferase expressing BCG (BCG16 and BCG26) increases steadily over time until approximately 12 days post-inoculation (B). The correlation between luminescence and optical density of BCG cultures expressing FFlux, CBRlux or the vector (pJDC89) alone (C). Data and error bars represent the means and standard deviations, respectively, of triplicate samples. Error bars are often too small to be visible around the marker for the mean. RLU  =  relative light units. * indicate data points with P<0.05 for FFlux vs. CBRlux.</p

Strains and Plasmids.

a<p>Hyg  =  hygromycin, Tet  =  tetracycline, oriM  =  mycobacterial pAL5000 origin of replication <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108341#pone.0108341-Stolt1" target="_blank">[47]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108341#pone.0108341-Ranes1" target="_blank">[62]</a>, P_hsp60  =  promoter from 60 kDa heat shock protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108341#pone.0108341-Stover1" target="_blank">[63]</a>, P_L5  =  promoter from the L5 mycobacteriophage <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108341#pone.0108341-Barletta1" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108341#pone.0108341-Lee2" target="_blank">[64]</a>, CBRlux  =  click beetle red luciferase, FFlux  =  firefly luciferase, optCBRlux  =  click beetle red luciferase with all amino acid codons modified to the optimal codons for mycobacteria, optFFlux  =  firefly luciferase with all amino acid codons modified to be optimal for mycobacteria.</p><p>Strains and Plasmids.</p

Luminescence from mycobacteria within macrophages.

<p>Luminescence detected from firefly (FFlux) and click beetle red (CBRlux) luciferase expressing BCG (BCG16 and BCG26) or BCG carrying the vector (pJDC89) alone within J774A.1 macrophages. Infections in macrophages were carried out with various multiplicities of infection (MOI) from 0.1 to 100 bacteria per cell for 30 min and washed to remove extracellular mycobacteria. Luciferin was added immediately after infection (day 0) and on days 2 and 7 post-infection ∼15 min before luminescence measurements. Data and error bars represent the means and standard deviations, respectively, of triplicate samples. RLU  =  relative light units. * indicate data points with P<0.05 for luciferase vs. pJDC89 expressing BCG at the same time point and same MOI.</p

Codon optimized click beetle red luciferase (CBRlux) allows rapid therapeutic evaluation.

<p>Percent inhibition of light production (A) and bacterial killing (B) for 10⁴ of BCG expressing codon optimized CBRlux (optCBRlux) in the presence of different concentrations of isoniazid (INH) plus rifampin (RIF) during culture in bacterial media after 24 (day 1) or 48 h (day 2) as compared to the absence of antimicrobials. Whole body imaging during pulmonary infection in BALB/C mice (4/group) with 4×10⁶ cfu of BCG expressing optCBRlux after 24 and 48 hr treatment with 10 mg/kg INH+RIF results in a reduction in luminescence (C). Quantitation of the percentage of the initial luminescence as compared to each time point out to six days post-treatment confirms the reduced luminescence in treated animals (D). Similarly, ex-vivo imaging of lungs at 2 days to confirm luminescence observed in whole body images is derived from the lungs and is reduced after antibiotic treatment (E). Untreated and treated panels for lung images are indicated in panel C. Correlation of luminescence with cfu present was confirmed by plating homogenates from the same animals at each time point (F). Data and error bars represent the means and standard deviations, respectively, of four mice. * indicates p<0.05 and ** indicates p<0.01 as compared to treated group at the same time point.</p

Codon optimized luciferases produce more light than non-optimized luciferases.

<p>BCG (10⁴ cfu) expressing wild type click beetle red (CBRlux) or firefly (FFlux) luciferases or the same luciferases synthetically optimized for mycobacterial codon usage (opt) were examined in liquid culture for light production over time in the presence of 2 mM luciferin (A, B). Results shown are for the individual strains of each type selected for maximal luminescence as described in the methods. BCG containing the vector alone (pJDC89) was used as a control. Both the L5 and hsp60 promoters were examined with the codon optimized luciferases to evaluate which promoter results in the greatest light production. Whole body images of BALB/C mice (2/group) subcutaneously infected with 10⁶ cfu of the same BCG strains show similar results to those found in liquid culture where codon optimized luciferases produce greater luminescence (C). A map of subcutaneous inoculation sites on mice is shown in the right panel. Quantitation of each inoculation site for total Flux photons per second (p/sec) indicates that the codon optimized CBRlux expressed from hsp60 emits the most light (D). Data and error bars represent the means and standard deviations, respectively, of two mice. ** indicates p<0.01 as compared to FFlux in the same construct.</p

Spectral characteristics of firefly and click beetle red luciferase.

<p>Collection of whole body images of BALB/C mice (2/group) infected subcutaneously with 10⁶ cfu of BCG expressing click beetle red (CBRlux) or firefly (FFlux) luciferase at defined wavelengths between 540 nm and 720 nm demonstrates that very little luminescence is obtained from CBRlux at short wavelengths and, similarly, less is obtained for FFlux at longer wavelengths (A). Template map for subcutaneous inoculation of BCG strains and PBS control (B). Quantitation of total Flux (p/sec) at different wavelengths for areas with CBRlux of FFlux indicates that luminescence from each reporter can be spectrally separated (C). Comparing luminescence kinetics obtained with mixed cultures of 10⁴ total cfu CBRlux and FFlux expressing BCG at ratios from 1∶1 to 1∶100 shows that the kinetic curve of a mixed culture resembles that of the luciferase that is at higher numbers in the culture. Furthermore, at the similar numbers (1∶1) the kinetic curve appears to be a mix between the two curves for the luciferases. Since the kinetic curves are directly relates to the numbers of each strain present, it is likely that these luciferases compete similarly for available substrate (D). Luminescence was measured using a plate reader after injecting 50 µl of 2 mM luciferin. Measurements began 10 s after adding luciferin and were taken every 10 s up to 5 min.</p

Real-Time Bioluminescence Imaging of Mixed Mycobacterial Infections

Molecular analysis of infectious processes in bacteria normally involves construction of isogenic mutants that can then be compared to wild type in an animal model. Pathogenesis and antimicrobial studies are complicated by variability between animals and the need to sacrifice individual animals at specific time points. Live animal imaging allows real-time analysis of infections without the need to sacrifice animals, allowing quantitative data to be collected at multiple time points in all organs simultaneously. However, imaging has not previously allowed simultaneous imaging of both mutant and wild type strains of mycobacteria in the same animal. We address this problem by using both firefly (Photinus pyralis) and click beetle (Pyrophorus plagiophthalamus) red luciferases, which emit distinct bioluminescent spectra, allowing simultaneous imaging of two different mycobacterial strains during infection. We also demonstrate that these same bioluminescence reporters can be used to evaluate therapeutic efficacy in real-time, greatly facilitating our ability to screen novel antibiotics as they are developed. Due to the slow growth rate of mycobacteria, novel imaging technologies are a pressing need, since they can they can impact the rate of development of new therapeutics as well as improving our understanding of virulence mechanisms and the evaluation of novel vaccine candidates

Evidence of a causal effect of genetic tendency to gain muscle mass on uterine leiomyomata

Uterine leiomyomata (UL) are the most common tumours of the female genital tract and the primary cause of surgical removal of the uterus. Genetic factors contribute to UL susceptibility. To add understanding to the heritable genetic risk factors, we conduct a genome-wide association study (GWAS) of UL in up to 426,558 European women from FinnGen and a previous UL meta-GWAS. In addition to the 50 known UL loci, we identify 22 loci that have not been associated with UL in prior studies. UL-associated loci harbour genes enriched for development, growth, and cellular senescence. Of particular interest are the smooth muscle cell differentiation and proliferation-regulating genes functioning on the myocardin-cyclin dependent kinase inhibitor 1A pathway. Our results further suggest that genetic predisposition to increased fat-free mass may be causally related to higher UL risk, underscoring the involvement of altered muscle tissue biology in UL pathophysiology. Overall, our findings add to the understanding of the genetic pathways underlying UL, which may aid in developing novel therapeutics.Peer reviewe