Food Sci. Biotechnol. 24(2): 595-599 (2015) DOI 10.1007/s10068-015-0077-0
RESEARCH ARTICLE
Construction of Bioluminescent Lactobacillus casei CJNU 0588 for Murine Whole Body Imaging Ji-Eun Eom, Won-Gyeong Ahn, Song Her, and Gi-Seong Moon
Received August 27, 2014; revised October 29, 2014; accepted November 11, 2014; published online April 30, 2015 © KoSFoST and Springer 2015
Abstract Recombinant DNA pLuc2 which contains a firefly luciferase cDNA from the commercial pGL3-Basic plasmid vector was constructed for bioluminescent Escherichia coli and lactobacilli. The bioluminescent signal strengths of E. coli DH5α and L. casei CJNU 0588 transformed with pLuc2 were 2.78×109 and 1.19×106 p/s, respectively. Approximately 1010 cells of bioluminescent L. casei CJNU 0588 (pLuc2) were given orally to 6-week-old female Balb/c mice. The bioluminescent signal was detected from all three mice at 1 h and one mouse at 3 h but was not detected thereafter. The signal was also confirmed from an opened abdomen and the extracted GI tract of a mouse at 1 h. From the results, we have known that L. casei CJNU 0588 pass through the murine stomach and reach the colon and cecum 1 h after oral administration. Keywords: Lactobacillus casei, bioluminescence, luciferase, whole body imaging, probiotics
Introduction Lactic acid bacteria (LAB) have been attracted, as they have many beneficial effects on human health. Thus, various studies on the bacteria have been performed, and the relationship between intestinal bacteria and human physiology has been revealed (1,2), resulting in an emphasis on intestinal beneficial bacteria such as LAB and Ji-Eun Eom, Gi-Seong Moon () Department of Biotechnology, Korea National University of Transportation, Jeungpyeong, Chungbuk 368-701, Korea Tel: +82-43-820-5251; Fax: +82-43-820-5272 E-mail:
[email protected] Won-Gyeong Ahn, Song Her Division of Bio-Imaging, Chuncheon Center, Korea Basic Science Institute, Chuncheon, Gangwon 200-701, Korea
bifidobacteria. Such good bacteria play key roles in the human gastrointestinal (GI) tract, i.e., i) inhibition of pathogenic bacteria (3), ii) stimulation of the immune response (4), iii) anti-cancer or anti-tumor effects (5), iv) prevention of diarrhea (6), v) improved constipation (7), and vi) production of useful metabolites such as vitamins (8). Probiotics and prebiotics have been used to boost the beneficial bacterial populations in the human GI tract (9). Probiotics are living bacteria that help improve GI homeostasis (10) and prebiotics are non-digestible agents for stimulating the growth of intestinal beneficial bacteria such as bifidobacteria and lactobacilli (11). Based on in vitro selection criteria, probiotic strains should be acidresistant, bile-tolerant, and colonize host epithelial tissue (12). Colonization is somewhat controversial, as probiotics can also play roles during passage in the GI tract. Nevertheless, it is clear that viable probiotics should be delivered to the host intestines for healthy function. In particular, they or their metabolites should contact immune cells that are linked to gut-associated lymphoid tissue to stimulate regional or systemic immune responses (13). However, it is difficult to monitor bacteria in the GI tract of living animals without sacrifice. To solve this problem, researchers have applied luciferasebased labeling systems and molecular imaging devices for in vivo whole body imaging. Cronin et al. (14) developed a labeling system to monitor Bifidobacterium breve persistence in mice. They monitored colonization and persistence of the strain in real time via ex vivo imaging of the mice intestinal tracts. However, they could not obtain the bioluminescent signal from whole body imaging. Loessner et al. (15) reported drug-inducible remote control gene expression by the probiotic Escherichia coli Nissle 1917 in the murine intestine, tumor, and gall bladder. In the study, they successfully detected bioluminescent signal
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from the recombinant strain labeled with a luciferase gene via in vivo whole body imaging. In our previous report (16), with the similar technique, Lactococcus lactis as well as E. coli could be detected from murine intestines via whole body imaging. The study was the first case for detection of LAB from the murine intestine via noninvasive whole body imaging. Similarly, Daniel et al. (17) reported a bioluminescent imaging study of Lactobacillus plantarum and L. lactis in living mice. Those authors developed a functional in vitro expression system using the click beetle, Gaussia, and bacterial luciferases in combination with vectors, promoters, and codon-optimized genes. They confirmed the precise gut compartments in which both strains located via in vivo and ex vivo imaging. L. casei CJNU 0588 strain was previously isolated from a natural cheese and confirmed to present bifidogenic growth stimulation (BGS) activity (18). As mentioned above, bifidobacteria are gut beneficial bacteria, so it might be possible for L. casei CJNU 0588 to positively modulate human gut microbiota. With the reason, L. casei CJNU 0588 will be further analyzed for the influence on the microbiota in animal model or human. Before then, we wanted to know whether the strain can survive in murine stomach and reach intestine. For the purpose, we constructed a luciferase expression vector for labeling E. coli and lactobacilli, and the bioluminescent L. casei CJNU 0588 was monitored in the murine GI tract after oral administration via in vivo and ex vivo imaging in this study.
Materials and Methods Bacterial strains and culture conditions E. coli DH5α was cultured in Luria-Bertani broth (10 g/L NaCl, 10 g/L tryptone, and 5 g/L yeast extract, pH 7.0) at 37oC with vigorous shaking (250 rpm), and L. casei CJNU 0588 was cultured in De Man, Rogosa, and Sharpe (MRS) broth (Difco, Sparks, MD, USA) at 37oC without shaking. Two hundred µg/mL erythromycin (Sigma-Aldrich Co., St.
Louis, MO, USA) was used for antibiotic selection of recombinant E. coli, and 5 µg/mL was used for L. casei CJNU 0588. Bacterial strains and plasmids used in this study are summarized in Table 1. Construction of recombinant plasmids and transformation The recombinant plasmid pLuc2 was constructed to label E. coli and lactobacilli with a luciferase. A luciferase (luc+) gene was liberated by restriction with EcoRI and XbaI endonuclease (Takara Bio Co., Shiga, Japan) from a previously constructed pMG36e_luc+ (16) and ligated with pUC18 restricted with the same enzymes. The resulting plasmid was named pLuc1 (pUC18::luc+). For replication and antibiotic selection in lactobacilli, pC7_Em from pCW4 (20) was liberated by EcoRI endonuclease and ligated with pUC18_luc+ restricted with the same enzyme. The resulting plasmid was named pLuc2 (pUC18::luc+::pC7_Em). pLuc3 (pUC18::pC7_Em) was also constructed as a control vector. The recombinant plasmids were transferred into electrocompetent E. coli DH5α and L. casei CJNU 0588 strains. The electrocompetent cells were prepared by a method reported previously (21). In vitro bioluminescent imaging E. coli and L. casei transformants were cultured until the mid-exponential phase (OD600: ~0.5) and 100 µL aliquots of cells were added to 96 black-well microplates supplemented with 150 µg/mL L-luciferin (sodium salt; GoldBio Co., St. Louis, MO, USA). Subsequently, in vitro bioluminescent imaging was performed using an IVIS-200 system (Xenogen Co., Alameda, CA, USA), and the bioluminescent signals were quantified as photon flux (p/s) using Living Image® software (Xenogen Co.). To achieve linearity between the viable cell concentration of the recombinant L. casei CJNU 0588 and the bioluminescent signal, cells cultured in MRS broth at 37oC for 24 h were washed with 1× phosphate buffered saline (PBS) and serially diluted two-fold with the same solution. Bioluminescent imaging and quantification were performed as described above. The initial cell count
Table 1. Bacterial strains and plasmids used in this study Strain or Plasmid Strains Escherichia coli DH5α Lactobacillus casei CJNU 0588 Plasmids pMG36e::luc+ pUC18 pCW4 pLuc1 pLuc2 pLuc3
Description
Reference
supE44 ∆lacU169 (Φ 80 lacZ ∆M 15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 Originally isolated from a natural cheese and present bifidogenic growth stimulation activity
(19)
pMG36e carrying a luciferase gene (luc+) Cloning vector for E. coli, Apr E. coli and lactobacilli shuttle vector, Emr pUC18 carrying luc+ pLuc1 carrying pC7 and Emr from pCW4 pUC18 carrying pC7 and Emr from pCW4
(18) (16) (19) (20) This work This work This work
Bioluminescent L. casei CJNU 0588
Fig. 1. Bioluminescence of Escherichia coli DH5α and Lactobacillus casei CJNU 0588. Both strains were transformed with pLuc3 as the control vector and pLuc2 harboring a luciferase gene, respectively.
was achieved with 10-fold serial dilution on MRS agar plate via spread-plate method. Each experiment was confirmed by a repeated test. In vivo bioluminescent imaging Six-week-old female Balb/c mice (Samtako Co., Osan, Korea) were used for in vivo whole body imaging. The mice were housed in portable plastic cages which were under optimized temperatures (22-24oC) and given normal feed for 1 week before intervention. All animal procedures were in accordance with the Guide for the Care and Use of Laboratory Animals issued by the Laboratory Animal Resources Commission of the Korea Basic Science Institute (KBSI). Approximately 1010 bioluminescent bacterial cells of L. casei CJNU 0588 (pLuc2) in 0.2 mL PBS were given orally to the mice by gavage. Then, D-luciferin (150 mg/kg of weight) was injected intraperitoneally (IP) according to the manufacturer’s guide. At times of 1, 3, 6, 12 h, 1, 3, 5, 7, and 9 day after oral administration, in vivo whole body imaging of isofluorane-anesthetized mice was performed using the IVIS-200 system, and the bioluminescent signals were quantified as photon flux (p/s) using Living Image® software (Xenogen Co.). In a repeat experiment, the time points for whole body imaging were 1, 3, and 6 h after oral administration. After whole body imaging at 1 h, mice were sacrificed and its abdomen was opened. Ex vivo bioluminescent images were taken before and after extracting the GI tracts.
Results and Discussion In vitro bioluminescent imaging The pLuc2 and pLuc3 recombinant plasmids were transferred to E. coli DH5α and L. casei CJNU 0588, respectively. The transformants
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were cultured until the mid-log phase and 100 µL of each culture was used to quantify the bioluminescent signal. The signal strengths of E. coli DH5α and L. casei CJNU 0588 transformed with the control vector pLuc3 were 2.25×104 p/s and 1.32×104 p/s, respectively, whereas those of E. coli DH5α and L. casei CJNU 0588 transformed with pLuc2 were 2.78×109 p/s and 1.19×106 p/s, respectively (Fig. 1). In our previous report (16), the bioluminescent signal of E. coli (pMG36e::luc+) was 100 times stronger than that of Lactococcus lactis (pMG36e::luc+). Loessner et al. (15) also easily detected a strong bioluminescent signal from a recombinant E. coli strain even in the murine intestine. The linearity (R2) between bioluminescent L. casei CJNU 0588 cell density and signal strength was measured (Fig. 2), and the R2 value was 0.999, indicating that the bioluminescent signal of L. casei CJNU 0588 (pLuc2) was well correlated with bacterial cell density. Theoretically the signal can be detected above 108 cells in vitro test. From these results, pLuc2 was considered suitable for labeling L. casei CJNU 0588 with bioluminescence. In vivo bioluminescent imaging To investigate survivability of L. casei CJNU 0588 in murine stomach and monitor its behavior, approximately 1010 bioluminescent bacterial cells of L. casei CJNU 0588 (pLuc2) in 0.2 mL PBS were given orally to 6-week-old female Balb/c mice, and whole body bioluminescent imaging for isofluorane-anesthetized mice was performed using the IVIS-200 system. As a result, the bioluminescent signal was not detected from any of the control mice that were orally administered with L. casei CJNU 0588 (pLuc3) (data not shown), whereas the signal was detected from all three mice that were administered with bioluminescent L. casei CJNU 0588 (pLuc2) at 1 h and one mouse at 3 h but was not detected thereafter (Fig. 3A). The experiment was repeated in the same conditions, and the signals were detected only at 1 h after oral administration (Fig. 3B). From the results, we have known that L. casei CJNU 0588 strain can pass through the murine stomach and reach the cecum at 1 h after oral administration. No detection of the signal 3 h after oral administration might be due to the weak signal rather than cell death because the strain could be detected in a murine small intestine 3 days after oral administration in a biopsy (unpublished data). The functional roles of L. casei CJNU 0588 in the GI tract have not been revealed even though it presents BGS activity in vitro tests (18). Before clarifying the functions, it is necessary to determine whether the strain can pass through stomach and colonize the intestine. In this study, we confirmed that the strain can survive in the stomach and successfully reach the murine intestines by both in vivo whole body and ex vivo imaging. In a previous study (17) the bioluminescent signals from luciferase-labeled Lactococcus
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Fig. 2. Linearity between cell density and bioluminescent signal. The samples (1-9) were serially diluted by 2-fold using original cell cultures where cell counts were 5.6×1010 CFU/0.2 mL for L. casei CJNU 0588 (pLuc3) and 4.5×1010 CFU/0.2 mL for L. casei CJNU 0588 (pLuc2).
lactis and Lactobacillus plantarum in the murine GI tracts were very strong 5 min after oral administration and maintained at a plateau but declined thereafter. Furthermore, those authors found that the strains reached the cecum and colon approximately 90 min after oral administration using ex vivo imaging, which is similar to our results. In conclusion, in vivo whole body and ex vivo bioluminescent imaging can be a powerful tool to monitor probiotic LAB in murine GI tracts and understand their intestinal behavior and final destination. The labeling system developed in this study was successfully used for L. casei CJNU 0588 and could be applied to other lactobacilli strains.
Fig. 3. In vivo whole body imaging for monitoring bioluminescent Lactobacillus casei CJNU 0588 (pLuc2) in murine intestines. Panel A, whole body imaging 1 and 3 h after oral administration; Panel B, a repeat experiment which whole body imaging 1 h after oral administration and ex-vivo imaging
Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (20110009597). The authors would like to thank Prof. Dae-Kyun Chung (Kyung Hee Univ.) and Prof. Jeong-Hwan Kim (Gyeongsang National Univ.) for kind gift of pCW4. Disclosure The authors declare no conflict of interest.
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