A stable luciferase reporter plasmid for in vivo imaging in murine models of Staphylococcus aureus infections

Abstract

In vivo imaging of bioluminescent bacteria permits their visualization in infected mice, allowing spatial and temporal evaluation of infection progression. Most available bioluminescent strains were obtained by integration of the luciferase genes into the bacterial chromosome, a challenging and time-consuming approach. Recently, episomal plasmids were used, which were introduced in bacteria and expressed all genes required for bioluminescence emission. However, the plasmid was progressively lost in vitro and in vivo, if bacteria were not maintained under antibiotic selective pressure. Increased stability could be obtained inserting into the plasmid backbone sequences that assured plasmid partition between daughter bacterial cells, or caused death of bacteria that had lost the plasmid. So far, no detailed analysis was performed of either plasmid stability in vivo or contribution of different stabilizing sequence types. Here we report the construction of a plasmid, which includes the Photorhabdus luminescens lux cassette expressed under the control of a Staphylococcus aureus specific gene promoter, and toxin/antitoxin (T/A) and partition sequences (Par) conferring stability and transmissibility of the plasmid. Following infection of mice with S. aureus carrying this plasmid, we demonstrated that the promoter-lux fusion was functional in vivo, that the plasmid was retained by 70-100% of bacterial cells 7 days post-infection, and that both stabilizing sequence types were required to maximize plasmid retention. These data suggest that the plasmid can be a valuable tool to study gene expression and bacterial spread in small laboratory animals infected with S. aureus or possibly other Gram-positive human pathogens.

In Vivo Analysis of Staphylococcus aureus-Infected Mice Reveals Differential Temporal and Spatial Expression Patterns of fhuD2

“…With the purpose of evaluating fhuD2 promoter activity in vitro and in vivo, we used a reporter plasmid, pMABA-Par/TA-P fhuD2 -lux, with the lux operon of Photorhabdus luminescens under the control of the 213-bp upstream region of the gene containing the fhuD2 promoter from the S. aureus Newman strain. The plasmid had been molecularly engineered to be stably maintained in vitro and in vivo in the absence of selective antibiotic pressure (21).…”

A Bioluminescent Francisella tularensis SCHU S4 Strain Enables Noninvasive Tracking of Bacterial Dissemination and the Evaluation of Antibiotics in an Inhalational Mouse Model of Tularemia

“…This plasmid is derived from the pFN10 family of Francisella shuttle vectors carrying orf4 and orf5 (31). Based on the homology of orf5 with the axe (32) and phd (33) antitoxins, orf4 and orf5 are thought to encode components of a plasmid addiction system which likely improve plasmid retention in vivo and in vitro (13). In this study, the concentration of bacteria required to generate a detectable signal in vivo was relatively high, which may have limited the detection of bacteria very early in infection.…”

“…Pivotal experiments by Contag et al have demonstrated the usefulness of BLI in the noninvasive study of bacterial pathogenesis and the evaluation of antibiotic treatments for Salmonella enterica serovar Typhimurium (11). Subsequently, bioluminescent reporters have been introduced into other bacteria, including Mycobacterium tuberculosis (10,12), Staphylococcus aureus (13,14), Burkholderia mallei (15), and Yersinia pestis (16,17). Further, BLI has been used to evaluate vaccines (18), antibiotics (19)(20)(21), and supportive therapies (22) for a range of pathogens.…”

Reconstructing promoter activity from Lux bioluminescent reporters

“…Gene expression can be measured by cloning a promoter of interest upstream of the lux operon, and interpreting the bioluminescence from bacteria containing such constructs as a measure of transcription [ 3 , 4 ]. This provides a reporter that can measure gene expression at high frequency and with less background noise than other reporters, such as GFP [ 4 , 5 ], and has found great value in both bacteria [ 6 ] and eukaryotes [ 7 ], with important recent applications in whole cell biosensors [ 8 ], live animal infection models [ 9 , 10 ] and live tumour infection models [ 11 ].…”