Joint Institute for
Biological Sciences
Tracking Human Cell Exposure to E. coli O157:H7 Using an
Autobioluminescent Cell Line Expressing the Bacterial Luciferase Gene
Cassette
Dan Close
The Joint Institute for Biological Sciences
The University of Tennessee, Knoxville/
Oak Ridge National Laboratory
865-974-8080
[email protected]
D.M. Close1,2, J.D. Webb3, S.A. Ripp2,4, and G.S. Sayler1,2,3,4
ORNL
1Joint
Institute for Biological Sciences, Oak Ridge National Laboratory
2490 BioTech, Inc., Knoxville, TN
3Department of Ecology and Evolutionary Biology, The University of Tennessee, Knoxville
4Center for Environmental Biotechnology, University of Tennessee, Knoxville
Abstract
Unlike substrate-requiring bioluminescent or fluorescent-based reporters, the bacterial
luciferase (lux) reporter system is capable of synthesizing and scavenging its substrates from within
its host cell environment, allowing for an autobioluminescent phenotype when expressed
exogenously. Here we leverage this phenotype to determine if changes in the autobioluminescent
production of cultured human cells can serve as a near real-time bioreporter for bacterial
contamination.
Autobioluminescent human cells (HEK293) stably expressing luxCDABEfrp genes were
plated into 24 well plates at 1 × 106 cells/ml in 1 ml of DMEM supplemented with 10% FBS and
lacking all antibiotic components. Cells were imaged over 24 h with a 10 min photon counting
integration every 15 min. After the first 3 h, cells were treated with doses of either virulent or nonvirulent E. coli O157:H7 ranging from 1 to 6 × 106 in a volume of 100 μl LB media. As a vehicle
control, autobioluminescent cells were similarly inoculated with 100 μl LB without bacteria.
The dynamics of autobioluminescent production were similar between groups of cells
treated with virulent or non-virulent E. coli strains. This pattern remained across all inoculation
dose ranges, but underwent shifts in the time to peak output and time to diminishment of signal
compared to control. A shift in the autobioluminescent pattern was also observed among cells
treated with equivalent inoculation doses of the virulent and non-virulent E. coli strains, with
virulent strain treatment leading to diminishment below positive control output levels (p < 0.05) and
complete extinction of bioluminescent production (p > 0.05 compared to cell free controls) 0.5 h
prior to cells treated with non-virulent strains. The time until autobioluminescent production
diminished below unexposed controls correlated strongly with the initial inoculation dose of toxic
E. coli strains (R2 > 0.999) across all doses tested. Because of their constitutive autobioluminescent phenotype, human cells expressing lux
genes present a means to accurately and inexpensively determine the magnitude of an E. coli
contamination event in cell culture. Virulent and non-virulent strains can be distinguished by the
length of time required for cessation of the autobioluminescent signal, but cannot provide
information about virulence on a cellular level. Comparison with Luciferin-Based Assay Testing
Validating the Relationship Between Light Production and Cellular Metabolism
Firefly luciferasebased metabolic
assessment
•  Equal numbers of autobioluminescent cells were plated per well
•  Triplicate
samples
were
treated
with
100
μg/ml,
200
μg/ml,
300
μg/ml,
or
400
μg/ml
of
the
antibiotic
Zeocin
(Life
Technologies)
•  Positive
and
negative
controls
were
included
for
reference
•  10
min
image
acquisitions
were
acquired
at
15
min
intervals
over
a 24 h period
Bacterial
luciferase-based
metabolic
assessment
•  The substrate-free bioluminescent production of the bacterial luciferase gene cassette
allowed for increased data collection and therefore more detailed elucidation of
metabolic dynamics
•  Despite the differences in assay design, the two approaches revealed similar patterns of
metabolic disruption upon Zeocin treatment
Correlation Between Cell Number and Bioluminescent Output
Infection Dose Correlates with Bioluminescent
Cessation
R2 > 0.999
Differences in Treatment with
Virulent vs. Non-Virulent
E. Coli Strains
2 X 105
1 x 105
1.5 X 105
!
8 x 104
Cells expressing the engineered lux cassette genes are capable of
continuously producing a bioluminescent phenotype without
application of a chemical or fluorescent trigger. This allows them to be
constantly and inexpensively monitored. As few as 15,000 cells can be
detected using standard equipment, and the resulting bioluminescent
signal correlates strongly (R2 = 0.9527) with the total bioluminescent
cell population size, allowing for rapid and continuous tracking of
cellular growth dynamics.
Acknowledgments
This work was supported by the National Science Foundation Division of Chemical, Bioengineering, Environmental, and
Transport Systems (CBET) under award number CBET-0853780, the National Institutes of Health, National Cancer Institute,
Cancer Imaging Program, award number CA127745-01, and the Army Defense University Research Instrumentation Program.
Funding for J.D. Webb was provided by National Science Foundation Division of Chemical, Bioengineering, Environmental, and
Transport Systems (CBET) under award number CBET-0853780 and the University of Tennessee Office for Undergraduate
Research.
Produced for presentation at:
112th General Meeting of the American Society for Microbiology
June 16th – 19th, 2012 San Francisco, California, USA
1 X 105
6 x 104
4 x 104
2 x 104
Control
1 x 100
1 x 101
1 x 102
1 x 103
1 x 104
5 X 104
1 x 105
Time (hours)
•  Autobioluminescent cells were imaged for 3 h prior to E. coli treatment to determine baseline
light production
•  Cells were then treated with varying numbers of E. coli
•  Bioluminescent readings were performed every 15 min for 18 h
•  Regardless of infection dose, bioluminescent dynamics remained similar •  The time until bioluminescent cessation correlated strongly with the initial infection dose
across all ranges surveyed
•  Equal numbers of autobioluminescent cells were plated in 24
well tissue culture plates
•  Cells were treated with equal numbers of either virulent, or
non-virulent E. coli O157:H7
•  Cells treated with virulent E. coli O157:H7 displayed greater
bioluminescent dynamics than cells treated with non-virulent
strains