Let there be bioluminescence: development of a biophotonic imaging platform for in situ analyses of oral biofilms in animal models

Abstract

Summary In the current study, we describe a novel biophotonic imaging-based reporter system that is particularly useful for the study of virulence in polymicrobial infections and interspecies interactions within animal models. A suite of luciferase enzymes was compared using three early colonizing species of the human oral flora (Streptococcus mutans, Streptococcus gordonii, and Streptococcus sanguinis) to determine the utility of the different reporters for multiplexed imaging studies in vivo. Using the multiplex approach, we were able to track individual species within a dual species oral infection model in mice with both temporal and spatial resolution. We also demonstrate how biophotonic imaging of multiplexed luciferase reporters could be adapted for real-time quantification of bacterial gene expression in situ. By creating an inducible dual-luciferase expressing reporter strain of S. mutans, we were able to exogenously control and measure expression of nlmAB (encoding the bacteriocin mutacin IV) within mice to assess its importance for the persistence ability of S. mutans in the oral cavity. The imaging system described in the current study circumvents many of the inherent limitations of current animal model systems, which should now make it feasible to test hypotheses that were previously impractical to model.

Enhanced Probiotic Potential of Lactobacillus reuteri When Delivered as a Biofilm on Dextranomer Microspheres That Contain Beneficial Cargo

“…The resulting DNA fragment was inserted into pJC156 using the XhoI/SalI restriction sites. The click beetle luciferase (CBluc) gene was amplified from the Streptococcus mutans strain ldhCBGSm (Merritt et al, 2016) using oligos oSG1067-1068 and inserted downstream of the gtfW promoter region in pJC156 using SalI/NotI restriction sites. The resulting reporter plasmid pWAR501 was transformed into L. reuteri 23272 as described above to create the reporter strain LMW501.…”

Options and Limitations in Clinical Investigation of Bacterial Biofilms

“…Bioluminescence imaging of localized infections not only is well suited for monitoring the effectiveness of experimental antimicrobial therapeutics but also has a major role in the study of microbial virulence and pathogenicity. A virulence-specific study of an oral mouse infection model that employed a multiplexed biophotonic imaging-based inducible luciferase reporter was used to track individual species temporally and spatially in polymicrobial biofilms (377). Bioluminescence technology has expanded to include imaging in three dimensions for biofilm infections that would otherwise be challenging to monitor and treat (378)(379)(380)(381)(382).…”

LytTR Regulatory Systems: A potential new class of prokaryotic sensory system

“…Briefly, the luciferase open reading frame (ORF) containing the S . mutans ldh (lactate dehydrogenase) ribosome binding site was amplified from the strain ldhRenGSm [ 55 ] using the primer pair RenG-F/RenG-R. The ermAM erythromycin resistance cassette was PCR amplified from the plasmid pJY4164 [ 56 ] using the primer pair (RenG) erm-F/erm-R. Primers used to amplify the respective upstream and downstream homologous fragments for each reporter construct are as follows: wild-type SMU_294/295 LRS [SMU294-LF/SMU295(RenG)-R and (erm)SMU295-RF/SMU295-RR], SMU_294/Δ295 LRS [SMU294-LF/SMU294(RenG)-R and (erm)SMU294-RF/SMU295-RR], wild-type SMU_1070c/1069c LRS [SMU1070c-LF/SMU1069c(RenG)-R and (erm)SMU1069c-RF/ SMU1070c-RR], SMU_1070c/Δ1069c LRS [SMU1070c-LF/SMU1070c(RenG)-R and (erm)SMU1070c-RF/SMU1070c-RR], wild-type SMU_1854/1855 ( hdrRM ) LRS [hdrRM159-LF/hdrM(RenG)-R and (erm)hdrM-RF/hdrRM159-RR-2], SMU_1854/Δ1855 ( hdrR Δ M ) LRS [hdrRM159-LF/hdrR(RenG)-R and (erm)hdrR-RF/hdrRM159-RR-2], SMU_2080/2081 ( brsRM ) LRS [brsM-LF/brsM(RenG)-R and (erm)brsM-RF/brsM-RR], SMU_2080/Δ2081 ( brsR Δ M ) LRS [brsM-LF/brsR(RenG)-R and (erm)brsR-RF/brsM-RR].…”

“…Assays of firefly and green renilla luciferase activity were performed using a previously described methodology [ 55 ] with mid-log phase cultures. Reporter data were normalized by dividing luciferase values by their corresponding optical density (OD 600 ) values.…”