Abstract
Measurement of bacterial burden in animal infection models is a key component for both bacterial pathogenesis studies and therapeutic agent research. The traditional quantification means for in vivo bacterial burden requires frequent animal sacrifice and enumerating colony forming units (CFU) recovered from infection loci. To address these issues, researchers have developed a variety of luciferase-expressing bacterial reporter strains to enable bacterial detection in living animals. To date, all such luciferase-based bacterial reporters are in cell-associated form. Production of luciferase-secreting recombinant bacteria could provide the advantage of reporting CFU from both infection loci themselves and remote sampling (eg. body fluid and plasma). Toward this end, we have genetically manipulated a pathogenic Escherichia coli (E. coli) strain, ATCC25922, to secrete the marine copepod Gaussia princeps luciferase (Gluc), and assessed the use of Gluc as both an in situ and ex situ reporter for bacterial burden in mouse tissue cage infections. The E. coli expressing Gluc demonstrates in vivo imaging of bacteria in a tissue cage model of infection. Furthermore, secreted Gluc activity and bacterial CFUs recovered from tissue cage fluid (TCF) are correlated along 18 days of infection. Importantly, secreted Gluc can also be detected in plasma samples and serve as an ex situ indicator for the established tissue cage infection, once high bacterial burdens are achieved. We have demonstrated that Gluc from marine eukaryotes can be stably expressed and secreted by pathogenic E. coli in vivo to enable a facile tool for longitudinal evaluation of persistent bacterial infection.
Highlights
The detection and quantification of bacterial colony-forming units (CFU) within animal infection models is critical for both basic research of host-pathogen interactions and pre-clinical evaluation of antibacterial agents and vaccines
It is documented that human codonoptimized Gaussia princeps luciferase (Gluc) is 1000-fold more sensitive than humanized firefly luciferase (FFLuc) or humanized Renilla reniformis luciferase (RLuc, 34 kDa) [12]; Second, Gluc is naturally secreted in active form from Gaussia princeps using its native secretion signal (SS), enabling reporting from both the cells themselves and their extracelluar environment; Third, Gluc exhibits good stability under adverse conditions including low pH, hydrogen peroxide, high temperature and even b-mercaptoethanol [13], making it well suited for reporting from stress-associated in vivo environments that are expected within sites of infection
Two further fusion proteins were designed from the latter construct, Gluc appended to the 21 N-terminal amino acid secretion signal from pectate lyase B (PelB), and a construct with the 60 amino acid secretion signal of Hemolysin A (HlyA) appended at the C-terminus of the Gluc protein
Summary
The detection and quantification of bacterial colony-forming units (CFU) within animal infection models is critical for both basic research of host-pathogen interactions and pre-clinical evaluation of antibacterial agents and vaccines. Luciferase from luminous marine eukaryotes such as Gaussia princeps has not been successfully applied for in vivo bacterial studies. It is documented that human codonoptimized Gluc is 1000-fold more sensitive than humanized firefly luciferase (FFLuc) or humanized Renilla reniformis luciferase (RLuc, 34 kDa) [12]; Second, Gluc is naturally secreted in active form from Gaussia princeps using its native secretion signal (SS), enabling reporting from both the cells themselves and their extracelluar environment; Third, Gluc exhibits good stability under adverse conditions including low pH, hydrogen peroxide, high temperature and even b-mercaptoethanol [13], making it well suited for reporting from stress-associated in vivo environments that are expected within sites of infection. The independence of Gluc activity from these metabolites avoids declines in detection sensitivity associated with the decreased concentration of bacterial metabolite, occuring upon stationary phase [13,18,19]
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