- Email: [email protected]
Escherichia coli bactofection using Lipofectamine
Analytical Biochemistry 439 (2013) 142–144
Contents lists available at SciVerse ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Escherichia coli bactofection using Lipofectamine Kumaran Narayanan a,b,⇑, Choon Weng Lee c, Aurelian Radu d, Edmund Ui Hang Sim e a
School of Science, Monash University, 46150 Selangor Darul Ehsan, Malaysia Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA c Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia d Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA e Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan Sarawak, Malaysia b
a r t i c l e
i n f o
Article history: Received 24 March 2013 Received in revised form 6 April 2013 Accepted 8 April 2013 Available online 19 April 2013 Keywords: Bactofection E. coli Invasin DNA delivery Transfection reagent Lipofectamine
a b s t r a c t Successful gene delivery into mammalian cells using bactofection requires entry of the bacterial vector via cell surface integrin receptors followed by release of plasmid DNA into the cellular environment. We show, for the first time, that addition of the DNA transfection reagent Lipofectamine improves entry of invasive Escherichia coli into HeLa cells and enhances up to 2.8-fold green fluorescent protein (GFP) expression from a reporter plasmid. The addition of Lipofectamine may be applicable to other bacterial vectors to increase their DNA delivery efficiency into mammalian cells. Ó 2013 Elsevier Inc. All rights reserved.
Successful gene expression using bactofection requires entry of the bacterial vector via integrin receptors followed by release of plasmid DNA into the cellular environment. Integrins are transmembrane surface receptors found on the surface of most mammalian cells [1,2]. They are involved in cell–cell adhesion and enable interaction of mammalian cells and components of the extracellular matrix [1,2]. Integrin receptors have been exploited previously for targeting plasmid DNA into cells as part of nonviral gene transfer strategies. Remarkably, integrin-targeting efficiency of the DNA was improved by the simple addition of DNA transfection reagents, resulting in considerably higher gene expression. In one example, b-galactosidase reporter expression from integrin-targeted peptide/DNA complexes was enhanced from approximately 10% to more than 50% in melanoma cells (A375 M) and monkey kidney epithelial cells (COS-7) when Lipofectin was added [3]. In another work, the addition of the cationic liposome DOTAP or the polymer polyethylenimine to plasmid DNA attached to a histone H1derived DNA binding domain that targets the integrin receptor increased luciferase expression from the plasmid by 10- to 20-fold in HeLa cells compared with plasmid combined with the transfection reagents alone [4]. Although the actual mechanism is still
⇑ Corresponding author at: School of Science, Monash University, 46150 Selangor Darul Ehsan, Malaysia. Fax: +6 03 5514 6364. E-mail address: [email protected] (K. Narayanan). 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.04.010
unknown, these studies demonstrated convincingly that addition of DNA transfection reagents could enhance gene delivery via integrin receptors. Based on these observations, we hypothesized that DNA transfection reagents could increase the gene delivery efficiency of a bactofection vector that uses the integrin receptor. To test this hypothesis, we investigated the effect of the widely used, commercially available Lipofectamine reagent on the invasive asd– Escherichia coli DH10B vector that we developed previously [5]. This vector is a diaminopimelic acid-requiring auxotroph derived from the commercially available DH10B strain that is adapted to invade and deliver genes into mammalian cells when the Yersinia pseudotuberculosis invasin gene is coexpressed [5]. To investigate the effect of Lipofectamine on bactofection, we tested the ability of the vector to (i) internalize into cells and (ii) express the green fluorescent protein (GFP)1 reporter gene from a plasmid. In the internalization experiment, increasing concentrations of Lipofectamine were combined with a constant number of either invasive or noninvasive bacteria and presented to cells. The invasive E. coli vector contained the Y. pseudotuberculosis invasin gene, facilitating its entry using integrin receptors [5,6]. The noninvasive E. coli, isogenic to the invasive strain but without the invasin gene, was used to determine background internalization [5].
1
Abbreviations used: GFP, green fluorescent protein; MOI, multiplicity of infection.
Notes & Tips / Anal. Biochem. 439 (2013) 142–144
Fig.1. Bactofection of HeLa cells with E. coli using Lipofectamine transfection reagent. (A) Internalization of both invasive and noninvasive E. coli increased with the addition of Lipofectamine. (B) In the presence of Lipofectamine, the percentage of GFP expression from a plasmid was significantly higher for the invasive E. coli strain than for the noninvasive strain.
Several ratios of bacteria to lipid that could form the optimal complex for entry into cells were tested. Lipofectamine reagent was diluted and combined in increasing concentrations (0–2.0 lg) with a constant number of bacteria that represented a multiplicity of infection (MOI, ratio of bacteria to mammalian cells) of 1000 (Fig. 1A). The complex was overlaid onto 1 105 cells that had been seeded in a 6-well plate approximately 24 h earlier. The plate was centrifuged at 250g before coincubating the complex with the cells for 1 h at 37 °C in a 5% CO2 atmosphere. Following the incubation, cells were treated with gentamicin to kill extracellular bacteria that did not internalize and remained outside in the medium. Meanwhile, as the internalized bacteria remained protected from killing, they were rescued and quantified to determine the frequency of internalization as follows [5,6]. The cells were washed three times with 1 phosphate-buffered saline and incubated with 0.1% Triton X-100 for 5 min at 37 °C. Following this, the cells were scraped and plated on selective plates. The results showed that Lipofectamine strongly increased internalization of both the invasive and noninvasive E. coli vector. For the invasive E. coli, as the concentration of Lipofectamine increased from 0.1 to 2.0 lg, the number of bacteria internalized per HeLa cell grew as high as 9.18 (Fig. 1A, black bar, 0.2 lg), a rise of approximately 2.8-fold compared with when no transfection reagent was used (Fig. 1A, black bar, 0.0 lg). Within the range of the Lipofectamine concentrations used (0.1–2.0 lg), the increase in the count of internalized bacteria per HeLa cell with the Lipofectamine concentration was statistically insignificant, suggesting that a very low amount of the transfection reagent (0.1 lg in this
143
case) was sufficient to boost entry of the invasive E. coli into HeLa cells (Fig. 1). The noninvasive E. coli showed the highest entry of 10.28 bacteria/HeLa cell at the maximum Lipofectamine concentration tested (Fig. 1A, white bar, 2.0 lg), an astonishing rise of 1028-fold compared with noninvasive E. coli alone (Fig. 1A, white bar, 0.0 lg). The entry of the noninvasive E. coli into HeLa cells (Fig. 1A, white bar, 2.0 lg) ultimately reached a level as high as the invasive E. coli (Fig. 1A, black bar, 2.0 lg). In other words, Lipofectamine was able to significantly boost the invasion of the noninvasive E. coli strain to match that of the invasive E. coli strain (Fig. 1A). However, as will be seen in the next experiment, the gene delivery efficiency of the noninvasive strain never reached anywhere near the level achieved by the invasive strain (Fig. 1B). Next, we investigated whether combining Lipofectamine with the invasive E. coli vector was able to improve gene expression, the ultimate test of a vector’s efficacy. For this experiment, the invasive E. coli vector was transformed with the pEGFP-N2 (Invitrogen) plasmid that carried the reporter GFP [5]. Lipofectamine was added to the bacterial vector using the same protocol used to determine entry, but this time the cells were incubated for 48 h postinvasion before GFP expression was measured [5]. Briefly, Lipofectamine at increasing concentrations was combined with bacteria at an MOI of 1000. The complex was then centrifuged onto cells and coincubated for 1 h at 37 °C in a 5% CO2 incubator followed by gentamicin treatment. Finally, the cells were incubated for 2 days at 37 °C in a 5% CO2 incubator before GFP was quantified using a fluorescence-activated cell sorter. For the invasive E. coli, the background GFP expression was 2.86% without the addition of Lipofectamine (Fig. 1B, black bar, 0.0 lg) but increased to 8.14% at 2.0 lg Lipofectamine (Fig. 1B, black bar, 2.0 lg). This represented a rise of 2.8-fold in gene expression for this strain, matching its increase in entry in the presence of Lipofectamine (Fig. 1A). For the noninvasive E. coli, the background GFP expression was as low as 0.01% without Lipofectamine (Fig. 1B, white bar, 0.0 lg) but moved up to 0.24% at the highest Lipofectamine concentration used (Fig. 1B, white bar, 2.0 lg). Although this was a 24-fold increase in GFP expression, this value was 12-fold below the basal level expression obtained with the invasive E. coli alone minus Lipofectamine (Fig. 1B, black bar, 0.0 lg). The diminished GFP expression seen with the noninvasive strain suggests that the expression of invasin is a prerequisite for the amplification effect of Lipofectamine to take place, perhaps through a synergistic or additive effect of invasin and the transfection reagent. Our laboratory is currently investigating the mechanism of this interaction in more detail. The Lipofectamine-assisted method described in this work should be valuable to improve E. coli bactofection into cells, especially for the transfer of large intact DNA for gene expression and for assembly of de novo chromosomes [5,7]. Furthermore, this protocol could be easily adapted to examine whether Lipofectamine is similarly effective on other widely used bacterial vectors for gene delivery, including Listeria monocytogenes [8] and Salmonella typhimurium [9].
References [1] R.R. Isberg, J.M. Leong, Multiple b1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells, Cell 60 (1990) 861–871. [2] J. Pizarro-Cerdá, P. Cossart, Bacterial adhesion and entry into host cells, Cell 124 (2006) 715–727. [3] S.L. Hart, C.V. Arancibia-Cárcamo, M.A. Wolfert, C. Mailhos, N.J. O’Reilly, R.R. Ali, C. Coutelle, A.J. George, R.P. Harbottle, A.M. Knight, D.F. Larkin, R.J. Levinsky, L.W. Seymour, A.J. Thrasher, C. Kinnon, Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector, Hum. Gene Ther. 9 (1998) 575–585.
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Notes & Tips / Anal. Biochem. 439 (2013) 142–144
[4] E. Fortunati, E. Ehlert, N.D. van Loo, C. Wyman, J.A. Eble, F. Grosveld, B.J. Scholte, A multi-domain protein for b1 integrin-targeted DNA delivery, Gene Ther. 7 (2000) 1505–1515. [5] K. Narayanan, P.E. Warburton, DNA modification and functional delivery into human cells using Escherichia coli DH10B, Nucleic Acids Res. 31 (2003) e51. [6] C. Grillot-Courvalin, S. Goussard, F. Huetz, D.M. Ojcius, P. Courvalin, Functional gene transfer from intracellular bacteria to mammalian cells, Nat. Biotechnol. 16 (1998) 862–866. [7] A. Laner, S. Goussard, A.S. Ramalho, T. Schwarz, M.D. Amaral, P. Courvalin, D. Schindelhauer, C. Grillot-Courvalin, Bacterial transfer of large functional genomic DNA into human cells, Gene Ther. 12 (2005) 1559–1572.
[8] J.P. van Pijkeren, D. Morrissey, I.R. Monk, M. Croni, S. Rajendran, G.C. O’Sullivan, C.G. Gahan, M. Tangney, A novel Listeria monocytogenes-based DNA delivery system for cancer gene therapy, Hum. Gene Ther. 21 (2010) 405–416. [9] Y. Tian, B. Guo, H. Jia, K. Ji, Y. Sun, Y. Li, T. Zhao, L. Gao, Y. Meng, D.V. Kalvakolanu, D.J. Kopecko, X. Zhao, L. Zhang, D. Xu, Targeted therapy via oral administration of attenuated Salmonella expression plasmid-vectored Stat3– shRNA cures orthotopically transplanted mouse HCC, Cancer Gene Ther. 19 (2012) 393–401.
Contents lists available at SciVerse ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Escherichia coli bactofection using Lipofectamine Kumaran Narayanan a,b,⇑, Choon Weng Lee c, Aurelian Radu d, Edmund Ui Hang Sim e a
School of Science, Monash University, 46150 Selangor Darul Ehsan, Malaysia Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA c Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia d Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA e Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan Sarawak, Malaysia b
a r t i c l e
i n f o
Article history: Received 24 March 2013 Received in revised form 6 April 2013 Accepted 8 April 2013 Available online 19 April 2013 Keywords: Bactofection E. coli Invasin DNA delivery Transfection reagent Lipofectamine
a b s t r a c t Successful gene delivery into mammalian cells using bactofection requires entry of the bacterial vector via cell surface integrin receptors followed by release of plasmid DNA into the cellular environment. We show, for the first time, that addition of the DNA transfection reagent Lipofectamine improves entry of invasive Escherichia coli into HeLa cells and enhances up to 2.8-fold green fluorescent protein (GFP) expression from a reporter plasmid. The addition of Lipofectamine may be applicable to other bacterial vectors to increase their DNA delivery efficiency into mammalian cells. Ó 2013 Elsevier Inc. All rights reserved.
Successful gene expression using bactofection requires entry of the bacterial vector via integrin receptors followed by release of plasmid DNA into the cellular environment. Integrins are transmembrane surface receptors found on the surface of most mammalian cells [1,2]. They are involved in cell–cell adhesion and enable interaction of mammalian cells and components of the extracellular matrix [1,2]. Integrin receptors have been exploited previously for targeting plasmid DNA into cells as part of nonviral gene transfer strategies. Remarkably, integrin-targeting efficiency of the DNA was improved by the simple addition of DNA transfection reagents, resulting in considerably higher gene expression. In one example, b-galactosidase reporter expression from integrin-targeted peptide/DNA complexes was enhanced from approximately 10% to more than 50% in melanoma cells (A375 M) and monkey kidney epithelial cells (COS-7) when Lipofectin was added [3]. In another work, the addition of the cationic liposome DOTAP or the polymer polyethylenimine to plasmid DNA attached to a histone H1derived DNA binding domain that targets the integrin receptor increased luciferase expression from the plasmid by 10- to 20-fold in HeLa cells compared with plasmid combined with the transfection reagents alone [4]. Although the actual mechanism is still
⇑ Corresponding author at: School of Science, Monash University, 46150 Selangor Darul Ehsan, Malaysia. Fax: +6 03 5514 6364. E-mail address: [email protected] (K. Narayanan). 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.04.010
unknown, these studies demonstrated convincingly that addition of DNA transfection reagents could enhance gene delivery via integrin receptors. Based on these observations, we hypothesized that DNA transfection reagents could increase the gene delivery efficiency of a bactofection vector that uses the integrin receptor. To test this hypothesis, we investigated the effect of the widely used, commercially available Lipofectamine reagent on the invasive asd– Escherichia coli DH10B vector that we developed previously [5]. This vector is a diaminopimelic acid-requiring auxotroph derived from the commercially available DH10B strain that is adapted to invade and deliver genes into mammalian cells when the Yersinia pseudotuberculosis invasin gene is coexpressed [5]. To investigate the effect of Lipofectamine on bactofection, we tested the ability of the vector to (i) internalize into cells and (ii) express the green fluorescent protein (GFP)1 reporter gene from a plasmid. In the internalization experiment, increasing concentrations of Lipofectamine were combined with a constant number of either invasive or noninvasive bacteria and presented to cells. The invasive E. coli vector contained the Y. pseudotuberculosis invasin gene, facilitating its entry using integrin receptors [5,6]. The noninvasive E. coli, isogenic to the invasive strain but without the invasin gene, was used to determine background internalization [5].
1
Abbreviations used: GFP, green fluorescent protein; MOI, multiplicity of infection.
Notes & Tips / Anal. Biochem. 439 (2013) 142–144
Fig.1. Bactofection of HeLa cells with E. coli using Lipofectamine transfection reagent. (A) Internalization of both invasive and noninvasive E. coli increased with the addition of Lipofectamine. (B) In the presence of Lipofectamine, the percentage of GFP expression from a plasmid was significantly higher for the invasive E. coli strain than for the noninvasive strain.
Several ratios of bacteria to lipid that could form the optimal complex for entry into cells were tested. Lipofectamine reagent was diluted and combined in increasing concentrations (0–2.0 lg) with a constant number of bacteria that represented a multiplicity of infection (MOI, ratio of bacteria to mammalian cells) of 1000 (Fig. 1A). The complex was overlaid onto 1 105 cells that had been seeded in a 6-well plate approximately 24 h earlier. The plate was centrifuged at 250g before coincubating the complex with the cells for 1 h at 37 °C in a 5% CO2 atmosphere. Following the incubation, cells were treated with gentamicin to kill extracellular bacteria that did not internalize and remained outside in the medium. Meanwhile, as the internalized bacteria remained protected from killing, they were rescued and quantified to determine the frequency of internalization as follows [5,6]. The cells were washed three times with 1 phosphate-buffered saline and incubated with 0.1% Triton X-100 for 5 min at 37 °C. Following this, the cells were scraped and plated on selective plates. The results showed that Lipofectamine strongly increased internalization of both the invasive and noninvasive E. coli vector. For the invasive E. coli, as the concentration of Lipofectamine increased from 0.1 to 2.0 lg, the number of bacteria internalized per HeLa cell grew as high as 9.18 (Fig. 1A, black bar, 0.2 lg), a rise of approximately 2.8-fold compared with when no transfection reagent was used (Fig. 1A, black bar, 0.0 lg). Within the range of the Lipofectamine concentrations used (0.1–2.0 lg), the increase in the count of internalized bacteria per HeLa cell with the Lipofectamine concentration was statistically insignificant, suggesting that a very low amount of the transfection reagent (0.1 lg in this
143
case) was sufficient to boost entry of the invasive E. coli into HeLa cells (Fig. 1). The noninvasive E. coli showed the highest entry of 10.28 bacteria/HeLa cell at the maximum Lipofectamine concentration tested (Fig. 1A, white bar, 2.0 lg), an astonishing rise of 1028-fold compared with noninvasive E. coli alone (Fig. 1A, white bar, 0.0 lg). The entry of the noninvasive E. coli into HeLa cells (Fig. 1A, white bar, 2.0 lg) ultimately reached a level as high as the invasive E. coli (Fig. 1A, black bar, 2.0 lg). In other words, Lipofectamine was able to significantly boost the invasion of the noninvasive E. coli strain to match that of the invasive E. coli strain (Fig. 1A). However, as will be seen in the next experiment, the gene delivery efficiency of the noninvasive strain never reached anywhere near the level achieved by the invasive strain (Fig. 1B). Next, we investigated whether combining Lipofectamine with the invasive E. coli vector was able to improve gene expression, the ultimate test of a vector’s efficacy. For this experiment, the invasive E. coli vector was transformed with the pEGFP-N2 (Invitrogen) plasmid that carried the reporter GFP [5]. Lipofectamine was added to the bacterial vector using the same protocol used to determine entry, but this time the cells were incubated for 48 h postinvasion before GFP expression was measured [5]. Briefly, Lipofectamine at increasing concentrations was combined with bacteria at an MOI of 1000. The complex was then centrifuged onto cells and coincubated for 1 h at 37 °C in a 5% CO2 incubator followed by gentamicin treatment. Finally, the cells were incubated for 2 days at 37 °C in a 5% CO2 incubator before GFP was quantified using a fluorescence-activated cell sorter. For the invasive E. coli, the background GFP expression was 2.86% without the addition of Lipofectamine (Fig. 1B, black bar, 0.0 lg) but increased to 8.14% at 2.0 lg Lipofectamine (Fig. 1B, black bar, 2.0 lg). This represented a rise of 2.8-fold in gene expression for this strain, matching its increase in entry in the presence of Lipofectamine (Fig. 1A). For the noninvasive E. coli, the background GFP expression was as low as 0.01% without Lipofectamine (Fig. 1B, white bar, 0.0 lg) but moved up to 0.24% at the highest Lipofectamine concentration used (Fig. 1B, white bar, 2.0 lg). Although this was a 24-fold increase in GFP expression, this value was 12-fold below the basal level expression obtained with the invasive E. coli alone minus Lipofectamine (Fig. 1B, black bar, 0.0 lg). The diminished GFP expression seen with the noninvasive strain suggests that the expression of invasin is a prerequisite for the amplification effect of Lipofectamine to take place, perhaps through a synergistic or additive effect of invasin and the transfection reagent. Our laboratory is currently investigating the mechanism of this interaction in more detail. The Lipofectamine-assisted method described in this work should be valuable to improve E. coli bactofection into cells, especially for the transfer of large intact DNA for gene expression and for assembly of de novo chromosomes [5,7]. Furthermore, this protocol could be easily adapted to examine whether Lipofectamine is similarly effective on other widely used bacterial vectors for gene delivery, including Listeria monocytogenes [8] and Salmonella typhimurium [9].
References [1] R.R. Isberg, J.M. Leong, Multiple b1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells, Cell 60 (1990) 861–871. [2] J. Pizarro-Cerdá, P. Cossart, Bacterial adhesion and entry into host cells, Cell 124 (2006) 715–727. [3] S.L. Hart, C.V. Arancibia-Cárcamo, M.A. Wolfert, C. Mailhos, N.J. O’Reilly, R.R. Ali, C. Coutelle, A.J. George, R.P. Harbottle, A.M. Knight, D.F. Larkin, R.J. Levinsky, L.W. Seymour, A.J. Thrasher, C. Kinnon, Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector, Hum. Gene Ther. 9 (1998) 575–585.
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Notes & Tips / Anal. Biochem. 439 (2013) 142–144
[4] E. Fortunati, E. Ehlert, N.D. van Loo, C. Wyman, J.A. Eble, F. Grosveld, B.J. Scholte, A multi-domain protein for b1 integrin-targeted DNA delivery, Gene Ther. 7 (2000) 1505–1515. [5] K. Narayanan, P.E. Warburton, DNA modification and functional delivery into human cells using Escherichia coli DH10B, Nucleic Acids Res. 31 (2003) e51. [6] C. Grillot-Courvalin, S. Goussard, F. Huetz, D.M. Ojcius, P. Courvalin, Functional gene transfer from intracellular bacteria to mammalian cells, Nat. Biotechnol. 16 (1998) 862–866. [7] A. Laner, S. Goussard, A.S. Ramalho, T. Schwarz, M.D. Amaral, P. Courvalin, D. Schindelhauer, C. Grillot-Courvalin, Bacterial transfer of large functional genomic DNA into human cells, Gene Ther. 12 (2005) 1559–1572.
[8] J.P. van Pijkeren, D. Morrissey, I.R. Monk, M. Croni, S. Rajendran, G.C. O’Sullivan, C.G. Gahan, M. Tangney, A novel Listeria monocytogenes-based DNA delivery system for cancer gene therapy, Hum. Gene Ther. 21 (2010) 405–416. [9] Y. Tian, B. Guo, H. Jia, K. Ji, Y. Sun, Y. Li, T. Zhao, L. Gao, Y. Meng, D.V. Kalvakolanu, D.J. Kopecko, X. Zhao, L. Zhang, D. Xu, Targeted therapy via oral administration of attenuated Salmonella expression plasmid-vectored Stat3– shRNA cures orthotopically transplanted mouse HCC, Cancer Gene Ther. 19 (2012) 393–401.