AEM Accepted Manuscript Posted Online 29 May 2015 Appl. Environ. Microbiol. doi:10.1128/AEM.01042-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Title: Dual color bioluminescence imaging for simultaneous monitoring of intestinal
2
persistence of Lactobacillus plantarum and Lactococcus lactis in living mice
3
Catherine Daniela, b, c #, Sabine Poireta, b, c, Véronique Dennina, b, c, Denise Boutilliera, b, c, Delphine
4
Armelle Lacorreb, c, d, e, Benoit Folignéa, b, c and Bruno Pota, b, c
5
Lactic acid Bacteria and Mucosal Immunity team, Institut Pasteur de Lille, Center for Infection
6
and Immunity of Lille, F-59019 Lille, Francea; Université de Lille, F-59000 Lille, Franceb;
7
CNRS, UMR 8204, F-59021 Lille, Francec; BioImaging Center Lille-Nord de France, F-59800
8
Lille, Franced; Cellular Microbiology and Physics of Infection, Institut Pasteur de Lille, Center
9
for Infection and Immunity of Lille, F-59019 Lille, France e
10 11
# Corresponding author: Daniel, C. ([email protected])
12 13
Running title: Dual color bioluminescent lactic acid bacteria
14
Journal section: Methods
15
1
16
ABSTRACT:
17
Lactic acid bacteria are found in the gastrointestinal tract of mammals and have received
18
tremendous attention due to their health-promoting properties. We report the development of two
19
dual color luciferase-producing Lactobacillus plantarum and Lactococcus lactis for noninvasive
20
simultaneous tracking in the mouse gastrointestinal tract. We previously described the functional
21
expression of the red luciferase mutant (CBRluc) from Pyrophorus plagiophtalamus in L.
22
plantarum NCIMB8826 and L. lactis MG1363. In this study, we determined that CBRluc is a
23
better performing luciferase for in vivo localization of both lactic acid bacteria after oral
24
administration, as compared with the green click beetle luciferase mutant construct developed in
25
this study. We furthermore established the possibility to simultaneously detect red and green
26
emitting lactic acid bacteria by dual wavelength bioluminescence imaging in combination with
27
spectral unmixing. The difference in spectra of light emission by the red and green click beetle
28
luciferase mutants and dual bioluminescence detection allowed in vitro and in vivo quantification
29
of the red and green emitted signals and thus monitoring the dynamics and fate of the two
30
bacterial populations simultaneously. Persistence and viability of both strains simultaneously
31
administered to mice in different ratios was studied in vivo in anesthetized mice and ex vivo in
32
mouse feces. The application of dual-luciferase-labeled bacteria has considerable potential to
33
simultaneous study the interactions and potential competitions of different targeted bacteria and
34
their host.
35
2
36
INTRODUCTION
37
Lactococci and lactobacilli are lactic acid bacteria (LAB) that have been used for thousands of
38
years for the production and preservation of fermented food such as milk, vegetables and meat.
39
Some specific strains are commercialized as probiotics and claimed to have health-promoting
40
properties (1). The gastrointestinal tract (GIT) is the most important field of activity of LAB
41
although distal effects outside the gut have been described (2). It is thus important to understand
42
the interactions of the administered bacteria with their host GIT system. LAB are able to survive
43
and adapt to the GIT conditions, as shown by comparative and functional genomic
44
characterization of various human isolates, essentially lactobacilli (for a review, see references (2,
45
3)). Some LAB, when present in the GIT of mice or humans, express a number of common
46
characteristics that may relate to their intestinal niche adaptation (3, 4). However, direct in vivo
47
tracking of these actions in terms of both spatial and temporal evolution would allow a better
48
understanding of the survival and metabolic activities of these LAB in the gut.
49
In the past decade, bioluminescence imaging (BLI) has become essential for in vivo
50
noninvasive monitoring of biological processes (4). The technique relies on the detection of
51
photons emitted from cells or tissues in a living organism. Bioluminescence is a biological
52
process that requires an enzyme known as luciferase, an enzyme-specific substrate (e.g.
53
luciferin), oxygen, magnesium and / or ATP, depending on the luciferase. Luciferases encompass
54
a wide range of enzymes that catalyze light-producing chemical reactions in living organisms.
55
The most used luciferase for in vivo bioluminescence imaging is represented by the Photinus
56
pyralis (firefly) luciferase (Fluc) and the click beetle luciferase from Pyrophorus
57
plagiophtalamus (CBluc). Both use D-luciferin as the substrate, depend on ATP, Mg and O2 and
58
result in the production of green light. Red click beetle (CBRluc) and firefly luciferase variants
3
59
with higher emission wavelengths have also been developed and greatly enhance the penetration
60
and sensitivity of BLI in deep tissues (4). We previously showed that CBRluc produced in
61
Lactobacillus plantarum NCIMB8826, one of most studied strains in LAB research, and
62
Lactococcus lactis MG1363, a dairy starter derivative, can be successfully detected in the GIT of
63
anesthetized mice by noninvasive BLI after oral administration (5).
64
It has been reported that dual color BLI can be applied in vitro and in vivo using appropriate
65
emission filters for the separation of bioluminescent signals and mathematical corrections for
66
their deconvolution using spectral unmixing algorithms (6-8). Dual color bioluminescence has
67
been described with red and green luciferases expressed by mammalian cells that can be
68
separated in vivo (9). Very recently, Chang et al. showed that Fluc and CBRluc, which emit
69
distinct light wavelengths, allow simultaneous imaging of two mycobacterial strains during
70
infection (10).
71
As LAB are studied and given to humans and animals mostly in mixtures with multiple strains
72
(11), we describe the development and application of two dual color luciferase-expressing L.
73
plantarum and L. lactis for noninvasive simultaneous monitoring in the GIT of small laboratory
74
animals. We demonstrate that CBRluc is a better performing luciferase in vivo, as compared with
75
the green click beetle luciferase (CBGluc) mutant also described in this study. We also explore
76
the possibility to simultaneously detect red and green emitting LAB by dual wavelength
77
bioluminescence imaging in combination with spectral unmixing.
78
MATERIALS AND METHODS
79
Bacterial strains, plasmids and growth conditions. Bacterial strains and plasmids are listed in
80
Table 1. The L. plantarum codon-optimized cbgluc gene under the control of Pldh and the L.
81
lactis codon-optimized cbgluc gene under the control of Pusp45 (the two DNA fragments were
4
82
synthetized by Eurogentec, Belgium) were cloned into pNZ8148 as BglII-XbaI fragments,
83
respectively. The two resulting constructs were subsequently introduced into L. lactis MG1363
84
and L. plantarum NCIMB8826 by electrotransformation, as described elsewhere (12) and named
85
L. lactis-CBGluc (Ll-CBGluc) and L. plantarum-CBGluc (Lp-CBGluc), respectively. L. lactis
86
MG1363 and L. plantarum NCIMB8826 containing the empty vector pNZ8148 (named Ll-
87
pNZ8148 and Lp-pNZ8148, respectively) described previously (5) served as controls in all the in
88
vitro and in vivo experiments. Lp-CBRluc and Ll-CBRluc were described previously (5). Strain
89
stability was tested as described previously (13).
90
Escherichia coli was cultured in Luria–Bertani broth at 37°C. L. lactis was grown at 30°C in M17
91
medium (Difco, Becton Dickinson, Sparks, MD) supplemented with 0.5% of glucose. L.
92
plantarum was grown at 37°C in MRS medium (Difco, Becton Dickinson). Chloramphenicol
93
(Sigma-Aldrich, St Quentin Fallavier, France) was added to culture media for bacterial selection
94
when necessary, at a final concentration of 20µg/mL for E. coli and 10µg/mL for LAB strains.
95
In vitro bioluminescence measurement. The bioluminescence of each recombinant luciferase
96
was quantified as described previously (5). Briefly, recombinant bacteria were grown overnight,
97
harvested by centrifugation, and washed with phosphate-buffered saline (PBS). 50 µl of each
98
culture adjusted to approximately 2.5 × 1011 CFU/ml was distributed in black microplates (Nunc,
99
Thermo Fisher, NY, USA) and imaged after addition of 50µl of the Bright-Glo Luciferase
100
(Promega, Madison, WI, USA). Luminescence was measured at room temperature on the IVIS
101
Lumina XR imaging system (Perkin Elmer, Alameda, CA, USA) using the Living Image
102
software (Perkin Elmer). Each individual well was manually selected as a region of interest
103
(ROI). Strains were compared on the basis of total flux of emitted light expressed as photons per
104
second (p/s) measured per 1.2 × 1010 CFU of each bacterial culture. L. lactis MG1363 and L.
5
105
plantarum NCIMB8826 containing the empty vector pNZ8148 were used to measure the
106
background luminescence.
107
Preparation of bacteria and administration to mice. Bacterial strains were grown
108
overnight, harvested by centrifugation, and washed with PBS. Mice received 5 × 1010 CFU in
109
200µl gavage buffer (0.2 M NaHCO3 buffer containing 1% glucose, pH 8). Eight-week-old
110
female BALB/c mice were purchased from Charles River (St Germain sur l’Arbresle, France).
111
Experiments were performed in an accredited establishment (no. A59107; Institut Pasteur de
112
Lille) according to European guidelines (number 86/609/CEE), and animal protocols were
113
approved by the local ethics committee.
114
In vivo persistence of LAB in the GIT of mice. Groups of five mice received a unique, daily
115
dose of 5 × 1010 CFU of live recombinant strains (Lp-CBRluc, Ll-CBRluc, Lp-CBGluc, or Ll-
116
CBGluc) or different doses of the two different recombinant strains (one expressing CBRluc and
117
the other CBGluc) administered for four consecutive days by oral gavage. Control mice received
118
Lp-pNZ8148 and Ll-pNZ8148 in the different experiments. Fecal samples were individually
119
collected at different time-points and mechanically homogenized in PBS. No chloramphenicol-
120
resistant bacteria were detected in non-inoculated mice. Mice were anesthetized with 2%
121
isoflurane and sacrificed by cervical dislocation and mouse digestive tracts were immediately
122
excised before ex vivo bioluminescence imaging. As described previously, the intestines were
123
injected with air to enhance the bioluminescent signal (5). Experiences were repeated twice.
124
In vivo bioluminescence imaging. Bioluminescence imaging was performed using a
125
multimodal IVIS Lumina XR imaging system (Perkin Elmer) as described previously (5). Mice
126
were anesthetized with 2% isoflurane and D-luciferin potassium salt (Perkin Elmer) was
127
administered to animals, intragastrically 15 minutes before the oral administration of the bacteria.
128
Two hours later, mice were placed into the specimen chamber of the IVIS imaging system where 6
129
a controlled flow of 1.5% isoflurane was administered through a nose cone via a gas anesthesia
130
system (Tem Sega, Lormont, France). A grayscale reference photography under low illumination
131
was taken as an overlay prior to detection of emitted photons over 1s to 5 min, depending on
132
signal intensity and using the Living Image software (Perkin Elmer). A pseudocolour image
133
representing detected light intensity was generated using the Living Image software and
134
superimposed over the grayscale reference photography. For each individual mouse, the ROI
135
corresponding to the mouse digestive tract was manually selected.
136
In vitro and in vivo spectral unmixing of emission wavelengths. To separate the red
137
bioluminescent signal (CBRluc with an emission peak at 620 nm) from the green signal (CBGluc
138
with an emission peak at 540 nm) in mixed cultures of L. lactis and L. plantarum, either (i) in
139
vitro in microplates, (ii) in vivo in mice after co-administration of both strains by oral gavage,
140
(iii) ex vivo on dissected GITs, or (iv) directly in feces, images were taken using a set of 20 nm
141
step emission filters (500 Series high spectral resolution emission filters, Perkin Elmer) from 500
142
nm to 620 nm. The Living Image software was used for generating spectrally unmixed images
143
and quantification of each signal.
144
RESULTS
145
In vitro comparison of the production of CBGluc and CBRluc in L. lactis and L. plantarum.
146
The bioluminescent signals produced by the different recombinant L. lactis and L. plantarum was
147
quantified and compared in vitro on bacterial cultures adjusted to 2.5 × 1011 CFU/ml (Fig. 1).
148
Results show that maximum bioluminescence is obtained with Lp-CBRluc and Lp-CBGluc with
149
mean values of 5.1 × 1010 and 4.1 × 1010 p/s (per 1.2 × 1010 CFU, equivalent to 50µl of bacterial
150
culture). Approximately 36-fold lower bioluminescent signals were obtained with L. lactis strains
151
producing CBGluc and CBRluc. No statistical difference was found between the production of 7
152
CBGluc and CBRluc for the respective strains. In L. lactis and L. plantarum, plasmids 271 and
153
269, respectively, were remarkably stable with 100% of bioluminescent colonies even after
154
approximately 100 generations (10 subcultures) in non-selective medium.
155
As observed earlier for CBRluc-producing strains (5), there was an excellent correlation
156
between CFU counts and in vitro bioluminescent signals from CBGluc-expressing strains (data
157
not shown). The bioluminescence system allowed the detection of bacterial amounts as low as 5
158
× 104 CFU/ml for Lp-CBGluc [similar to Lp-CBRluc (5)] and 5 × 105 CFU/ml for Ll-CBGluc
159
[similar to Ll-CBGluc (5)].
160
Comparison of the persistence of CBGluc- and CBRluc-producing L. lactis and L.
161
plantarum in the digestive tract of mice after 4 daily oral administrations. As we had
162
previously shown that differences in bacterial persistence in mice are more pronounced after
163
multiple daily oral administrations (5), groups of mice were given a daily dose of 5 × 1010 CFU
164
of each recombinant strain for four consecutive days. The bioluminescent signal was quantified
165
each day by direct imaging in 5 anesthetized mice (Fig. 2). The same mice were used during the
166
whole experiment. The signal was very strong for the four strains on day 1 and remained at a
167
similar level till the last day of oral administration (day 4). Results show that from day 1 to day 4,
168
Lp-CBRluc emitted the highest bioluminescent signal (mean value of 1.2 × 108 p/s), 6-fold higher
169
than Lp-CBGluc, 14-fold higher than Ll-CBRluc and 111-fold higher than Ll-CBGluc. Signals of
170
both L. lactis strains decreased to background level at day 5, whereas signals of both L.
171
plantarum strains decreased to background level only at day 7.
172
Simultaneous monitoring of red and green bioluminescent signals by dual color in vivo
173
imaging. In order to test the CBRluc/CBGluc pair of luciferases for in vivo applications, groups
174
of mice were orally given a dose of Lp-CBRluc, Lp-CBGluc, or a mixture (50/50) of L. 8
175
plantarum expressing CBRluc and expressing CBGluc for four days. The bioluminescent signal
176
was measured each day in mice and a series of images at different wavelengths ranging from 500
177
to 620 nm were acquired (Fig. 3A) Representative images of a spectral unmixing of the red and
178
green signals are shown in Figure 3. The respective red and green signals are separated and
179
quantified after applying the spectral unmixing algorithm to the acquired images series. The
180
resulting Unmix1 image displays the green signal from Lp-CBGluc while the resulting Unmix 2
181
image displays the red signal from Lp-CBRluc (Fig. 3B). The red signal was not detected in the
182
green emission light image and the green signal was not detected in the red emission light image
183
which confirmed the good spectral unmixing of both signals. Moreover, both signals were
184
detected in mice administered with a mixture of Lp-CBRluc and Lp-CBGluc.
185
Simultaneous monitoring of red and green bioluminescent signals after co-administration
186
of L. plantarum-CBRluc and L. lactis-CBGluc by dual color in vivo imaging. In order to
187
simultaneously study the persistence of L. plantarum and L. lactis in mice, groups of mice were
188
orally given a daily dose of Lp-CBRluc, Ll-CBGluc, or different mixtures (20/80, 50/50 and
189
80/20) of Lp-CBRluc/Ll-CBGluc for four days. We found that red signals in mice administered
190
with a mixture of 50/50 or 80/20 of Lp-CBRluc/Ll-CBGluc, are similar to red signals of mice
191
administered with 100% Lp-CBRluc (no statistical difference) from day 1 to day 6 (Fig. 4A). The
192
same observation was made for green signals in mice administered only with Ll-CBGluc (Fig.
193
4B). However, red signals in mice administered with L. plantarum only are statistically higher
194
than red signals in mice administered with a mixture of 20% of L. plantarum/80% of L. lactis
195
from day 1 to day 6. Similarly, green signals in mice administered with 100% L. lactis are
196
statistically higher than green signals in mice administered with a mixture of 20% of L.
197
lactis/80% of L. plantarum. The values of red signals from L. plantarum given alone (mean value
9
198
of 1.4 × 108 p/s from day 1 to day 4) are statistically higher than green signals from L. lactis
199
given alone (p
1
Title: Dual color bioluminescence imaging for simultaneous monitoring of intestinal
2
persistence of Lactobacillus plantarum and Lactococcus lactis in living mice
3
Catherine Daniela, b, c #, Sabine Poireta, b, c, Véronique Dennina, b, c, Denise Boutilliera, b, c, Delphine
4
Armelle Lacorreb, c, d, e, Benoit Folignéa, b, c and Bruno Pota, b, c
5
Lactic acid Bacteria and Mucosal Immunity team, Institut Pasteur de Lille, Center for Infection
6
and Immunity of Lille, F-59019 Lille, Francea; Université de Lille, F-59000 Lille, Franceb;
7
CNRS, UMR 8204, F-59021 Lille, Francec; BioImaging Center Lille-Nord de France, F-59800
8
Lille, Franced; Cellular Microbiology and Physics of Infection, Institut Pasteur de Lille, Center
9
for Infection and Immunity of Lille, F-59019 Lille, France e
10 11
# Corresponding author: Daniel, C. ([email protected])
12 13
Running title: Dual color bioluminescent lactic acid bacteria
14
Journal section: Methods
15
1
16
ABSTRACT:
17
Lactic acid bacteria are found in the gastrointestinal tract of mammals and have received
18
tremendous attention due to their health-promoting properties. We report the development of two
19
dual color luciferase-producing Lactobacillus plantarum and Lactococcus lactis for noninvasive
20
simultaneous tracking in the mouse gastrointestinal tract. We previously described the functional
21
expression of the red luciferase mutant (CBRluc) from Pyrophorus plagiophtalamus in L.
22
plantarum NCIMB8826 and L. lactis MG1363. In this study, we determined that CBRluc is a
23
better performing luciferase for in vivo localization of both lactic acid bacteria after oral
24
administration, as compared with the green click beetle luciferase mutant construct developed in
25
this study. We furthermore established the possibility to simultaneously detect red and green
26
emitting lactic acid bacteria by dual wavelength bioluminescence imaging in combination with
27
spectral unmixing. The difference in spectra of light emission by the red and green click beetle
28
luciferase mutants and dual bioluminescence detection allowed in vitro and in vivo quantification
29
of the red and green emitted signals and thus monitoring the dynamics and fate of the two
30
bacterial populations simultaneously. Persistence and viability of both strains simultaneously
31
administered to mice in different ratios was studied in vivo in anesthetized mice and ex vivo in
32
mouse feces. The application of dual-luciferase-labeled bacteria has considerable potential to
33
simultaneous study the interactions and potential competitions of different targeted bacteria and
34
their host.
35
2
36
INTRODUCTION
37
Lactococci and lactobacilli are lactic acid bacteria (LAB) that have been used for thousands of
38
years for the production and preservation of fermented food such as milk, vegetables and meat.
39
Some specific strains are commercialized as probiotics and claimed to have health-promoting
40
properties (1). The gastrointestinal tract (GIT) is the most important field of activity of LAB
41
although distal effects outside the gut have been described (2). It is thus important to understand
42
the interactions of the administered bacteria with their host GIT system. LAB are able to survive
43
and adapt to the GIT conditions, as shown by comparative and functional genomic
44
characterization of various human isolates, essentially lactobacilli (for a review, see references (2,
45
3)). Some LAB, when present in the GIT of mice or humans, express a number of common
46
characteristics that may relate to their intestinal niche adaptation (3, 4). However, direct in vivo
47
tracking of these actions in terms of both spatial and temporal evolution would allow a better
48
understanding of the survival and metabolic activities of these LAB in the gut.
49
In the past decade, bioluminescence imaging (BLI) has become essential for in vivo
50
noninvasive monitoring of biological processes (4). The technique relies on the detection of
51
photons emitted from cells or tissues in a living organism. Bioluminescence is a biological
52
process that requires an enzyme known as luciferase, an enzyme-specific substrate (e.g.
53
luciferin), oxygen, magnesium and / or ATP, depending on the luciferase. Luciferases encompass
54
a wide range of enzymes that catalyze light-producing chemical reactions in living organisms.
55
The most used luciferase for in vivo bioluminescence imaging is represented by the Photinus
56
pyralis (firefly) luciferase (Fluc) and the click beetle luciferase from Pyrophorus
57
plagiophtalamus (CBluc). Both use D-luciferin as the substrate, depend on ATP, Mg and O2 and
58
result in the production of green light. Red click beetle (CBRluc) and firefly luciferase variants
3
59
with higher emission wavelengths have also been developed and greatly enhance the penetration
60
and sensitivity of BLI in deep tissues (4). We previously showed that CBRluc produced in
61
Lactobacillus plantarum NCIMB8826, one of most studied strains in LAB research, and
62
Lactococcus lactis MG1363, a dairy starter derivative, can be successfully detected in the GIT of
63
anesthetized mice by noninvasive BLI after oral administration (5).
64
It has been reported that dual color BLI can be applied in vitro and in vivo using appropriate
65
emission filters for the separation of bioluminescent signals and mathematical corrections for
66
their deconvolution using spectral unmixing algorithms (6-8). Dual color bioluminescence has
67
been described with red and green luciferases expressed by mammalian cells that can be
68
separated in vivo (9). Very recently, Chang et al. showed that Fluc and CBRluc, which emit
69
distinct light wavelengths, allow simultaneous imaging of two mycobacterial strains during
70
infection (10).
71
As LAB are studied and given to humans and animals mostly in mixtures with multiple strains
72
(11), we describe the development and application of two dual color luciferase-expressing L.
73
plantarum and L. lactis for noninvasive simultaneous monitoring in the GIT of small laboratory
74
animals. We demonstrate that CBRluc is a better performing luciferase in vivo, as compared with
75
the green click beetle luciferase (CBGluc) mutant also described in this study. We also explore
76
the possibility to simultaneously detect red and green emitting LAB by dual wavelength
77
bioluminescence imaging in combination with spectral unmixing.
78
MATERIALS AND METHODS
79
Bacterial strains, plasmids and growth conditions. Bacterial strains and plasmids are listed in
80
Table 1. The L. plantarum codon-optimized cbgluc gene under the control of Pldh and the L.
81
lactis codon-optimized cbgluc gene under the control of Pusp45 (the two DNA fragments were
4
82
synthetized by Eurogentec, Belgium) were cloned into pNZ8148 as BglII-XbaI fragments,
83
respectively. The two resulting constructs were subsequently introduced into L. lactis MG1363
84
and L. plantarum NCIMB8826 by electrotransformation, as described elsewhere (12) and named
85
L. lactis-CBGluc (Ll-CBGluc) and L. plantarum-CBGluc (Lp-CBGluc), respectively. L. lactis
86
MG1363 and L. plantarum NCIMB8826 containing the empty vector pNZ8148 (named Ll-
87
pNZ8148 and Lp-pNZ8148, respectively) described previously (5) served as controls in all the in
88
vitro and in vivo experiments. Lp-CBRluc and Ll-CBRluc were described previously (5). Strain
89
stability was tested as described previously (13).
90
Escherichia coli was cultured in Luria–Bertani broth at 37°C. L. lactis was grown at 30°C in M17
91
medium (Difco, Becton Dickinson, Sparks, MD) supplemented with 0.5% of glucose. L.
92
plantarum was grown at 37°C in MRS medium (Difco, Becton Dickinson). Chloramphenicol
93
(Sigma-Aldrich, St Quentin Fallavier, France) was added to culture media for bacterial selection
94
when necessary, at a final concentration of 20µg/mL for E. coli and 10µg/mL for LAB strains.
95
In vitro bioluminescence measurement. The bioluminescence of each recombinant luciferase
96
was quantified as described previously (5). Briefly, recombinant bacteria were grown overnight,
97
harvested by centrifugation, and washed with phosphate-buffered saline (PBS). 50 µl of each
98
culture adjusted to approximately 2.5 × 1011 CFU/ml was distributed in black microplates (Nunc,
99
Thermo Fisher, NY, USA) and imaged after addition of 50µl of the Bright-Glo Luciferase
100
(Promega, Madison, WI, USA). Luminescence was measured at room temperature on the IVIS
101
Lumina XR imaging system (Perkin Elmer, Alameda, CA, USA) using the Living Image
102
software (Perkin Elmer). Each individual well was manually selected as a region of interest
103
(ROI). Strains were compared on the basis of total flux of emitted light expressed as photons per
104
second (p/s) measured per 1.2 × 1010 CFU of each bacterial culture. L. lactis MG1363 and L.
5
105
plantarum NCIMB8826 containing the empty vector pNZ8148 were used to measure the
106
background luminescence.
107
Preparation of bacteria and administration to mice. Bacterial strains were grown
108
overnight, harvested by centrifugation, and washed with PBS. Mice received 5 × 1010 CFU in
109
200µl gavage buffer (0.2 M NaHCO3 buffer containing 1% glucose, pH 8). Eight-week-old
110
female BALB/c mice were purchased from Charles River (St Germain sur l’Arbresle, France).
111
Experiments were performed in an accredited establishment (no. A59107; Institut Pasteur de
112
Lille) according to European guidelines (number 86/609/CEE), and animal protocols were
113
approved by the local ethics committee.
114
In vivo persistence of LAB in the GIT of mice. Groups of five mice received a unique, daily
115
dose of 5 × 1010 CFU of live recombinant strains (Lp-CBRluc, Ll-CBRluc, Lp-CBGluc, or Ll-
116
CBGluc) or different doses of the two different recombinant strains (one expressing CBRluc and
117
the other CBGluc) administered for four consecutive days by oral gavage. Control mice received
118
Lp-pNZ8148 and Ll-pNZ8148 in the different experiments. Fecal samples were individually
119
collected at different time-points and mechanically homogenized in PBS. No chloramphenicol-
120
resistant bacteria were detected in non-inoculated mice. Mice were anesthetized with 2%
121
isoflurane and sacrificed by cervical dislocation and mouse digestive tracts were immediately
122
excised before ex vivo bioluminescence imaging. As described previously, the intestines were
123
injected with air to enhance the bioluminescent signal (5). Experiences were repeated twice.
124
In vivo bioluminescence imaging. Bioluminescence imaging was performed using a
125
multimodal IVIS Lumina XR imaging system (Perkin Elmer) as described previously (5). Mice
126
were anesthetized with 2% isoflurane and D-luciferin potassium salt (Perkin Elmer) was
127
administered to animals, intragastrically 15 minutes before the oral administration of the bacteria.
128
Two hours later, mice were placed into the specimen chamber of the IVIS imaging system where 6
129
a controlled flow of 1.5% isoflurane was administered through a nose cone via a gas anesthesia
130
system (Tem Sega, Lormont, France). A grayscale reference photography under low illumination
131
was taken as an overlay prior to detection of emitted photons over 1s to 5 min, depending on
132
signal intensity and using the Living Image software (Perkin Elmer). A pseudocolour image
133
representing detected light intensity was generated using the Living Image software and
134
superimposed over the grayscale reference photography. For each individual mouse, the ROI
135
corresponding to the mouse digestive tract was manually selected.
136
In vitro and in vivo spectral unmixing of emission wavelengths. To separate the red
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bioluminescent signal (CBRluc with an emission peak at 620 nm) from the green signal (CBGluc
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with an emission peak at 540 nm) in mixed cultures of L. lactis and L. plantarum, either (i) in
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vitro in microplates, (ii) in vivo in mice after co-administration of both strains by oral gavage,
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(iii) ex vivo on dissected GITs, or (iv) directly in feces, images were taken using a set of 20 nm
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step emission filters (500 Series high spectral resolution emission filters, Perkin Elmer) from 500
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nm to 620 nm. The Living Image software was used for generating spectrally unmixed images
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and quantification of each signal.
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RESULTS
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In vitro comparison of the production of CBGluc and CBRluc in L. lactis and L. plantarum.
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The bioluminescent signals produced by the different recombinant L. lactis and L. plantarum was
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quantified and compared in vitro on bacterial cultures adjusted to 2.5 × 1011 CFU/ml (Fig. 1).
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Results show that maximum bioluminescence is obtained with Lp-CBRluc and Lp-CBGluc with
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mean values of 5.1 × 1010 and 4.1 × 1010 p/s (per 1.2 × 1010 CFU, equivalent to 50µl of bacterial
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culture). Approximately 36-fold lower bioluminescent signals were obtained with L. lactis strains
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producing CBGluc and CBRluc. No statistical difference was found between the production of 7
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CBGluc and CBRluc for the respective strains. In L. lactis and L. plantarum, plasmids 271 and
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269, respectively, were remarkably stable with 100% of bioluminescent colonies even after
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approximately 100 generations (10 subcultures) in non-selective medium.
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As observed earlier for CBRluc-producing strains (5), there was an excellent correlation
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between CFU counts and in vitro bioluminescent signals from CBGluc-expressing strains (data
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not shown). The bioluminescence system allowed the detection of bacterial amounts as low as 5
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× 104 CFU/ml for Lp-CBGluc [similar to Lp-CBRluc (5)] and 5 × 105 CFU/ml for Ll-CBGluc
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[similar to Ll-CBGluc (5)].
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Comparison of the persistence of CBGluc- and CBRluc-producing L. lactis and L.
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plantarum in the digestive tract of mice after 4 daily oral administrations. As we had
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previously shown that differences in bacterial persistence in mice are more pronounced after
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multiple daily oral administrations (5), groups of mice were given a daily dose of 5 × 1010 CFU
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of each recombinant strain for four consecutive days. The bioluminescent signal was quantified
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each day by direct imaging in 5 anesthetized mice (Fig. 2). The same mice were used during the
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whole experiment. The signal was very strong for the four strains on day 1 and remained at a
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similar level till the last day of oral administration (day 4). Results show that from day 1 to day 4,
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Lp-CBRluc emitted the highest bioluminescent signal (mean value of 1.2 × 108 p/s), 6-fold higher
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than Lp-CBGluc, 14-fold higher than Ll-CBRluc and 111-fold higher than Ll-CBGluc. Signals of
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both L. lactis strains decreased to background level at day 5, whereas signals of both L.
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plantarum strains decreased to background level only at day 7.
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Simultaneous monitoring of red and green bioluminescent signals by dual color in vivo
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imaging. In order to test the CBRluc/CBGluc pair of luciferases for in vivo applications, groups
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of mice were orally given a dose of Lp-CBRluc, Lp-CBGluc, or a mixture (50/50) of L. 8
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plantarum expressing CBRluc and expressing CBGluc for four days. The bioluminescent signal
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was measured each day in mice and a series of images at different wavelengths ranging from 500
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to 620 nm were acquired (Fig. 3A) Representative images of a spectral unmixing of the red and
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green signals are shown in Figure 3. The respective red and green signals are separated and
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quantified after applying the spectral unmixing algorithm to the acquired images series. The
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resulting Unmix1 image displays the green signal from Lp-CBGluc while the resulting Unmix 2
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image displays the red signal from Lp-CBRluc (Fig. 3B). The red signal was not detected in the
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green emission light image and the green signal was not detected in the red emission light image
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which confirmed the good spectral unmixing of both signals. Moreover, both signals were
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detected in mice administered with a mixture of Lp-CBRluc and Lp-CBGluc.
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Simultaneous monitoring of red and green bioluminescent signals after co-administration
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of L. plantarum-CBRluc and L. lactis-CBGluc by dual color in vivo imaging. In order to
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simultaneously study the persistence of L. plantarum and L. lactis in mice, groups of mice were
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orally given a daily dose of Lp-CBRluc, Ll-CBGluc, or different mixtures (20/80, 50/50 and
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80/20) of Lp-CBRluc/Ll-CBGluc for four days. We found that red signals in mice administered
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with a mixture of 50/50 or 80/20 of Lp-CBRluc/Ll-CBGluc, are similar to red signals of mice
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administered with 100% Lp-CBRluc (no statistical difference) from day 1 to day 6 (Fig. 4A). The
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same observation was made for green signals in mice administered only with Ll-CBGluc (Fig.
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4B). However, red signals in mice administered with L. plantarum only are statistically higher
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than red signals in mice administered with a mixture of 20% of L. plantarum/80% of L. lactis
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from day 1 to day 6. Similarly, green signals in mice administered with 100% L. lactis are
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statistically higher than green signals in mice administered with a mixture of 20% of L.
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lactis/80% of L. plantarum. The values of red signals from L. plantarum given alone (mean value
9
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of 1.4 × 108 p/s from day 1 to day 4) are statistically higher than green signals from L. lactis
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given alone (p
