- Email: [email protected]
Bacteria as Gene Delivery Vectors for Mammalian Cells
C
H
A
P
T
E
R
23
Bacteria as Gene Delivery Vectors for Mammalian Cells Catherine Grillot-Courvalin, Sylvie Goussard and Patrice Courvalin
IN VITRO GENE TRANSFER
Gene transfer can occur from bacteria to a very broad host range of recipient cells. It has been reported between distantly related bacterial genera, as demonstrated by Trieu-Cuot et al. (1987), between bacteria and yeast as demonstrated by Heinemann and Sprague (1989) and from bacteria to plants, as demonstrated by Buchanan-Wollaston et al. (1987). More recently, several laboratories have reported that bacteria can also transfer functional genetic information into mammalian cells. Transfer of replicons has been described after either in vitro co-incubation of Shigella or Listeria with phagocytic or nonphagocytic cells or in vivo administration of attenuated Shigella or Salmonella. We have developed a non-pathogenic bacterium, an invasive Escherichia coli deficient in cell wall synthesis, to study this type of transfer in vitro. This process is known as abortive or suicidal invasion since the bacteria have to lyse for DNA delivery to occur. The bacterial species which can transfer genes to professional and non-professional phagocytes have in common the ability to invade these cells. The plasmid DNA released by intracellular bacteria is transferred from the cytoplasm to the nucleus resulting in cellular expression of the transfected gene(s). Bacterial transfer of DNA can result in stimulation of humoral and cellular responses after in vivo administration. Gene delivery by abortive invasion of eukaryotic cells by bacteria may be valuable for in vivo and ex vivo gene therapy and for stimulation of mucosal immunity. Horizontal Gene Transfer ISBN: 0-12-680126-6
Mammalian cells can transiently express genes delivered intracellularly by strains of Shigella flexneri impaired in peptidoglycan synthesis, as demonstrated by Sizemore et al. (1995) and by Courvalin et al. (1995). A diaminopimelate auxotroph (dap–) mutant of Shigella, which undergoes lysis upon entry into mammalian cells because of impaired cell wall synthesis, was transformed with plasmid pCMVβ which directs the synthesis of E. coli β-galactosidase under the control of a eukaryotic promoter. Forty-eight hours after a 90 minute co-incubation of this suicidal bacterial vector with cultured BHK cells, 1% to 2% of cells expressed βgalactosidase. Similar results were obtained with the murine cell line P815. The genes responsible for entry, intra- and intercellular mobility of S. flexneri are borne by the ca. 200 kb virulence plasmid pWR100. Transfer of the plasmid to E. coli confers to this otherwise extracellular bacterial species the ability to invade epithelial cells. Using this invasive strain of E. coli, designated BM 2710, we have shown that bacteria that undergo lysis upon entry into mammalian cells (because of impaired cell wall synthesis due to diaminopimelate (dap) auxotrophy) can deliver plasmid DNA to their hosts. L. monocytogenes is able to invade a wide range of mammalian cell types. After internalization, these bacteria rapidly escape from the primary
261
279
Copyright © 2002 by Academic Press. All rights of reproduction in any form reserved.
262
C. GRILLOT-COURVALIN, S. GOUSSARD AND P. COURVALIN
vacuole into the cytosol where they replicate. An attenuated strain, impaired in intra- and intercellular movements has been engineered to undergo self-destruction upon entry into mammalian cells by production of a phage lysin under the control of the promoter of the actA gene which is preferentially activated in the cytosol. This bacterial vector was able, as demonstrated by Dietrich et al. (1998), to deliver a plasmid carrying gfp (green fluorescent protein), cat (chloramphenicol acetyltransferase) or a portion of the ova (ovalbumine) gene under the control of the CMV promoter in the P388D macrophage cell line. Functional transfer was dramatically increased (from 0.001% to 0.2%) if there was lysis of the internalized Listeria in the cytoplasm, either by selfkilling by lysin production or after antibiotic treatment. Efficient expression of chloramphenicol acetyltransferase or antigen presentation was observed in P388D macrophages. Plasmid DNA, from macrophage clones cultured in selective medium for more than 12 weeks, was found to be integrated into the genome of the new host at a frequency of approximately 10–7. In order to design a genetically more defined bacterial vector, we have cloned the 3.2 kb inv locus encoding the invasin of Yersinia pseudotuberculosis alone or combined with the 1.5 kb hly gene coding for the listeriolysin O from L. monocytogenes in the stable dap– auxotroph E. coli BM2710 (Grillot-Courvalin et al., 1998). We also introduced by transformation in that strain plasmid pEGFP-C1 which directs synthesis of the green fluorescent protein (GFP) in mammalian cells but not in bacteria. Between 5 and 20% of HeLa, CHO, and COS-1 cells producing GFP were observed 2 days after incubation with invasive bacteria. Co-expression in E. coli of the gene for listeriolysin enhanced transfer efficiency. Expression of the acquired genes occurred both in dividing and in quiescent cells, albeit at a lower efficiency in the latter. Transfer of functional plasmid DNA could be observed in J774 macrophages but not in a mouse dendritic cell line. Expression of acquired DNA was very stable, as tested after 2 months of culture. The number of viable intracellular bacteria decreased rapidly with no survivors after 24 to 72 h, and chromosomal DNA of the donor could not be detected after 23
to 48 days. Plasmid transfer was obtained, although at a lower frequency, from E. coli BM2711, the dap prototroph parental strain counterpart of BM2710, indicating that bacteria may transfer functional genetic information to non-professional phagocytes provided they can induce their own internalization. Agrobacterium tumefaciens is a soil phytopathogen that elicits neoplastic growths on the host plant species. It has very recently been shown that Agrobacterium can also transfer DNA to mammalian cells, as demonstrated by Kunick et al. (2001). Agrobacterium infection requires two genetic components that are carried by the tumor-inducing plasmid: the transferred DNA (T-DNA) which is introduced into the plant genome and the virulence region which specifies the protein apparatus for T-DNA transfer. Experiments in vitro indicated that A. tumefaciens attaches to and transfers DNA to several types of human cells, by conjugation, the mechanism it uses to transform plant cells. Study of stable transfected HeLa cells indicate integration of T-DNA into their genome.
IN VIVO GENE TRANSFER IN ANIMAL MODELS The ability of Shigella to enter intestinal epithelial cells and to evade from the endocytic vesicle was exploited to develop an in vivo gene delivery system as demonstrated by Sizemore et al. (1995). Mice were inoculated twice intranasally with 106 to 107 bacteria transformed with plasmid pCMVβ, 4 weeks apart; splenocytes from these mice proliferated after a 3 day culture in vitro in the presence of βgalactosidase and anti-β-galactosidase antibodies were detected in their sera as demonstrated by Sizemore et al. (1997). These results provided the first evidence that delivery of plasmid DNA by Shigella in vivo could elicit an immune response to the plasmid-encoded antigen. According to recent findings (Fennelly et al., 1999), after intranasal inoculation of mice, a highly attenuated strain of ∆asd Shigella harboring a DNA measles vaccine plasmid induced a vigorous measles-specific immune-response of both Th1 and Th2 types.
280
BACTERIA AS GENE DELIVERY VECTORS FOR MAMMALIAN CELLS
Attenuated Salmonella expressing heterologous antigens have been used for oral immunization in mice, farm animals, and in humans resulting in efficient stimulation of mucosal and systemic immune responses. An attenuated strain of S. typhimurium was used successfully by Darji et al. (1997) as a vector for oral genetic immunization. In this work, oral administration of an aroA– auxotrophic mutant harboring plasmids encoding E. coli β-galactosidase or truncated ActA and listeriolysin (two virulence factors of L. monocytogenes), each under the control of an eukaryotic promoter, led to excellent humoral and cellular immune responses in mice. The immunized mice were protected from a lethal challenge with virulent L. monocytogenes, even after a single administration. β-galactosidase activity was detected in splenic macrophages 5 weeks after oral administration of S. typhimurium carrying plasmid pCMVβ. Plasmid DNA transfer was studied in vitro after 1 h infection of mouse primary peritoneal macrophages with attenuated S. typhimurium carrying pCMVβ. Evidence of β-galactosidase synthesis by the infected macrophages was provided but tetracycline had to be added during the entire culture period to inhibit endogenous residual synthesis of β-galactosidase by bacteria. In a similar study by Paglia et al. (1998), oral administration to mice of an attenuated Salmonella harboring pCMVβ could generate humoral and cellular immune responses to β-galactosidase and a protective response against an aggressive murine fibrosarcoma transduced with the βgalactosidase gene. Expression of the transgene by antigen presenting cells after per os administration was documented with an eukaryotic vector expressing GFP. Among the 19% of splenocytes expressing GFP 28 days after oral administration, 50% were dendritic cells and 30% expressed macrophage markers. This provides evidence of direct in vivo gene transfer by orally administered bacteria to dendritic cells. According to more recent findings by Paglia et al. (2000), oral administration of an attenuated Salmonella, carrier for an eukaryotic expression vector encoding the murine INFγ gene, resulted in the production of this cytokine in INFγ-deficient mice. This provides evidences that attenuated Salmonella can be used
263
in vivo as a DNA delivery system for the correction of a genetic defect.
DISCUSSION In very few instances, direct introduction of DNA from bacteria to mammalian cells has been reported, as demonstrated by Schaffner (1980), that plasmids carrying tandem copies of the SV40 virus genome could be transferred from E. coli to mammalian cells by exposing the cell culture to a bacterial suspension. However, transfer was found to occur at a very low frequency. Similar observations were made with E. coli harboring plasmids carrying poliovirus 1 cDNA and transfer took place in the presence of high concentrations of DNAse in the culture medium, as demonstrated by Heitmann and Lópes-Pila (1993). In these experiments, the vectors were human pathogens that could not be employed outside an experimental setting. Attenuated intracellular bacteria, such as Shigella, Salmonella, Listeria, and invasive E. coli have been found to act as efficient gene delivery vectors in both phagocytic and non-phagocytic mammalian cells (Table 23.1). Observations from in vitro and in vivo experiments are consistent with the hypothesis that, after internalization into a primary vacuole, bacteria or their plasmid content have to escape into the cytosol to gain access to the nucleus. Studies on microbial pathogenesis have expanded our understanding of the mechanisms designed by bacteria to achieve entry into host cells and to gain access to intracellular compartments. There are two major strategies for bacteria to gain access to host cells, as reviewed by Marra and Isberg (1996). For certain genera like Salmonella or Shigella, contact between the bacteria and the host cells results in the secretion, by the bacteria, of a set of invasion proteins that triggers signalling events into the cells, leading to cytoskeletal rearrangement, membrane ruffling and bacterial uptake by micropinocytose. For other genera like Yersinia or Listeria, binding of a single bacterial protein to a particular ligand on the host cell surface is necessary and sufficient to trigger entry into phagocytic and nonphagocytic cells by a zipper-like mechanism.
281
264
C. GRILLOT-COURVALIN, S. GOUSSARD AND P. COURVALIN
TABLE 23.1 Main studies on bacteria to eukaryote gene transfer Recipient Donor bacterial species
Transferred plasmid(s)
In vitro: cell lines transfected
In vivo: route of administration
Shigella flexneri dapA–
pCMVβ
BHK P815
Intranasal Intracorneal Oral
HeLa COS-1 CHO A549 J774 Primary mouse macrophage
Not determined
Invasive E. coli dapA–
Salmonella typhimurium aroA– Listeria monocytogenes ∆ mpl actA plcB
Measles-vaccine plasmid β-gal replicative and integrative eukaryotic vectors pEGFP-C1 pCMVβ pCMV actA-hly INFγ gene pCMVgfp pCMVcat pCMVova
J774 P388D
Binding of Yersinia invasin to β1 integrins results in entry into cells expressing this integrin at the surface; binding of internalin with E-cadherin mediates entry of Listeria into certain cell types. Similarly, introduction in E. coli of the virulence plasmid (pWR100) of S. flexneri or of the inv gene of Y. pseudotuberculosis confers to this otherwise extracellular bacteria the ability to invade nonphagocytic cells. Access to the cell cytosol depends on the mechanisms by which bacteria survive inside the cells. Listeria and Shigella escape rapidly from the vacuole of entry after lysis of its membrane, by production of listeriolysin in the case of Listeria, and replicate in the cytoplasm of the cell. Other bacteria, like Salmonella, remain in the phagosomal vacuole and replicate within this compartment. The successful delivery of genes by Salmonella is therefore surprising since Salmonella-containing vacuoles have a unique trafficking pathway, uncoupled from the normal endocytic degradation pathway. Interestingly, infection by Salmonella resulted in functional DNA delivery into primary macrophages but not in macrophage cell lines, as demonstrated by Sizemore et al. (1995) and Darji et al. (1997). DNA entering host cell cytoplasm by phagocytosis is less efficiently routed to the nucleus
Oral Oral Intraperitoneally
Reference Sizemore et al. (1995, 1997) Fennelly et al. (1999) Courvalin et al. (1995), GrillotCourvalin et al. (1998) Darji et al. (1997), Paglia et al. (1998) Paglia et al. (2000) Dietrich et al. (1998), Spreng et al. (2000)
than when introduced directly into the cytoplasm (e.g. by gene gun). Consistent with this finding are the observations that more efficient gene transfer was observed when Listeria was destroyed in the cytoplasm of the cell (Dietrich et al., 1987) or if the invasive E. coli produced listeriolysin that triggers pore formation in the vacuolar membrane (Grillot-Courvalin et al., 1998). However, it cannot be excluded that DNA may gain access to the mammalian cell cytoplasm via leakage from host cell phagosomes, as has been proposed for transfer of certain antigens by Kovacsovics-Bankowski and Rock (1995). The property of bacteria to act as a gene delivery system has been mainly exploited for DNA vaccination. Attenuated auxotroph mutants of Salmonella are already in use as live vaccines in man and in animals, and effective gene transfer to dendritic cells after oral administration of mice with these bacteria has been documented by Paglia et al. (1998). In these in vivo studies, the number of transfected cells varies greatly depending on the type of bacterial vector and on the cell type. However, dendritic cells are highly efficient antigen presenting cells and a small number of transfected dendritic cells has been shown to be sufficient to stimulate both primary and secondary T and B
282
BACTERIA AS GENE DELIVERY VECTORS FOR MAMMALIAN CELLS
cell responses and to process and present antigens efficiently, as demonstrated by Banchereau and Steinman (1998). Moreover, per os administration of bacterial vectors could lead to transfection of gut-associated lymphoid cells and stimulation of mucosal immunity, as demonstrated by Darji et al. (1997), Paglia et al. (1998), and Fennelly et al. (1999). The potential use of bacterial vectors for in vivo or ex vivo gene therapy has only been tested once (Paglia et al., 2000) for a genetic defect associated with monocyte/macrophage cell type. Despite the high numbers of bacteria and plasmids internalized by the host cells, transgene expression remains low and comparable to the levels obtained with other non-viral delivery systems such as polycation-DNA complexes. Following bacterial internalization, plasmid DNA has to be released into the cytosol before its nuclear entry can occur. The turnover of plasmid DNA delivered by microinjection in the cytosol has been shown by Lechardeur et al. (1999) to be rapid, with an apparent half-life of 50 to 90 min in HeLa and COS cells. Direct delivery into the cytosol of native plasmids by intracellular bacteria may constitute a means of protecting this DNA from degradation by cytosolic nucleases.
REFERENCES Banchereau, J. and Steinman, R.M. (1998) Dendritic cells and the control of immunity. Nature 392: 245–252. Buchanan-Wollaston, V., Passiatore, J.E. and Cannon, F. (1987) The mob and oriT mobilization functions of a bacterial plasmid promote its transfer to plants. Nature 328: 172–175. Courvalin, P., Goussard, S. and Grillot-Courvalin, C. (1995) Gene transfer from bacteria to mammalian cells. CR Acad. Sci. 318: 1207–1212. Darji, A., Guzman, C.A., Gerstel, B. et al. (1997) Oral somatic transgene vaccination using attenuated S. typhimurium. Cell 91: 765–775. Dietrich, G., Bubert, A., Gentschev, I. et al. (1998) Delivery of antigen-encoding plasmid DNA into the cytosol of
265
macrophages by attenuated suicide Listeria monocytogenes. Nat. Biotechnol. 16: 181–185. Fennelly, G.J., Khan, S.A., Abadi, M.A. et al. (1999) Mucosal DNA vaccine immunization against measles with a highly attenuated Shigella flexneri vector. J. Immuno. 162: 1603–1610. Grillot-Courvalin, C., Goussard, S., Huetz, F. et al. (1998) Functional gene transfer from intracellular bacteria to mammalian cells. Nat. Biotechnol. 16: 862–866. Heinemann, J.A. and Sprague, G.F. Jr. (1989) Bacterial conjugative plasmids mobilize DNA transfer between bacteria and yeast. Nature 340: 205–209. Heitmann, D. and Lópes-Pila, J.M. (1993) Frequency and conditions of spontaneous plasmid transfer from E. coli to cultured mammalian cells. Biosystems 29: 37–48. Kovacsovics-Bankowski, M. and Rock, K.L. (1995) A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules. Science 267: 243–246. Kunik, T., Tzfira, T., Kapulnik, Y. et al. (2001) Genetic transformation of HeLa cells by Agrobacterium. Proc. Natl Acad. Sci. USA 98: 1871–1876. Lechardeur, D., Sohn, K.J., Haardt, M. et al. (1999) Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Ther. 6: 482–497. Marra, A. and Isberg, R.R. (1996) Common entry mechanisms. Bacterial pathogenesis. Curr. Biol. 6: 1084–1086. Paglia, P., Medina, E., Arioli, I. et al. (1998) Gene transfer in dendritic cells, induced by oral DNA vaccination with Salmonella typhimurium, results in protective immunity against a murine fibrosarcoma. Blood 92: 3172–3176. Paglia, P., Terrazzini, N., Schulze, K. et al. (2000) In vivo correction of genetic defects of monocyte/macrophages using attenuated Salmonella as oral vectors for targeted gene delivery. Gene. Ther. 7: 1725–1730. Schaffner, W. (1980) Direct transfer of cloned genes from bacteria to mammalian cells. Proc. Natl Acad. Sci. USA 77: 2163–2167. Sizemore, D.R., Branstrom, A.A, Sadoff and J.C. (1995) Attenuated Shigella as a DNA delivery vehicle for DNAmediated immunization. Science 270: 299–302. Sizemore, D.R., Branstrom, A.A and Sadoff, J.C. (1997) Attenuated bacteria as a DNA delivery vehicle for DNAmediated immunization. Vaccine 15: 804–807. Spreng, S., Dietrich, G., Niewiesk, S. et al. (2000) Novel bacterial systems for the delivery of recombinant protein or DNA. FEMS Immuno. Med. Microbiol. 27: 299–304. Trieu-Cuot, P., Carlier, C., Martin, P. and Courvalin, P. (1987) Plasmid transfer by conjugation from Escherichia coli to Gram-positive bacteria. FEMS Microbiol. Lett. 48: 289–294.
283
H
A
P
T
E
R
23
Bacteria as Gene Delivery Vectors for Mammalian Cells Catherine Grillot-Courvalin, Sylvie Goussard and Patrice Courvalin
IN VITRO GENE TRANSFER
Gene transfer can occur from bacteria to a very broad host range of recipient cells. It has been reported between distantly related bacterial genera, as demonstrated by Trieu-Cuot et al. (1987), between bacteria and yeast as demonstrated by Heinemann and Sprague (1989) and from bacteria to plants, as demonstrated by Buchanan-Wollaston et al. (1987). More recently, several laboratories have reported that bacteria can also transfer functional genetic information into mammalian cells. Transfer of replicons has been described after either in vitro co-incubation of Shigella or Listeria with phagocytic or nonphagocytic cells or in vivo administration of attenuated Shigella or Salmonella. We have developed a non-pathogenic bacterium, an invasive Escherichia coli deficient in cell wall synthesis, to study this type of transfer in vitro. This process is known as abortive or suicidal invasion since the bacteria have to lyse for DNA delivery to occur. The bacterial species which can transfer genes to professional and non-professional phagocytes have in common the ability to invade these cells. The plasmid DNA released by intracellular bacteria is transferred from the cytoplasm to the nucleus resulting in cellular expression of the transfected gene(s). Bacterial transfer of DNA can result in stimulation of humoral and cellular responses after in vivo administration. Gene delivery by abortive invasion of eukaryotic cells by bacteria may be valuable for in vivo and ex vivo gene therapy and for stimulation of mucosal immunity. Horizontal Gene Transfer ISBN: 0-12-680126-6
Mammalian cells can transiently express genes delivered intracellularly by strains of Shigella flexneri impaired in peptidoglycan synthesis, as demonstrated by Sizemore et al. (1995) and by Courvalin et al. (1995). A diaminopimelate auxotroph (dap–) mutant of Shigella, which undergoes lysis upon entry into mammalian cells because of impaired cell wall synthesis, was transformed with plasmid pCMVβ which directs the synthesis of E. coli β-galactosidase under the control of a eukaryotic promoter. Forty-eight hours after a 90 minute co-incubation of this suicidal bacterial vector with cultured BHK cells, 1% to 2% of cells expressed βgalactosidase. Similar results were obtained with the murine cell line P815. The genes responsible for entry, intra- and intercellular mobility of S. flexneri are borne by the ca. 200 kb virulence plasmid pWR100. Transfer of the plasmid to E. coli confers to this otherwise extracellular bacterial species the ability to invade epithelial cells. Using this invasive strain of E. coli, designated BM 2710, we have shown that bacteria that undergo lysis upon entry into mammalian cells (because of impaired cell wall synthesis due to diaminopimelate (dap) auxotrophy) can deliver plasmid DNA to their hosts. L. monocytogenes is able to invade a wide range of mammalian cell types. After internalization, these bacteria rapidly escape from the primary
261
279
Copyright © 2002 by Academic Press. All rights of reproduction in any form reserved.
262
C. GRILLOT-COURVALIN, S. GOUSSARD AND P. COURVALIN
vacuole into the cytosol where they replicate. An attenuated strain, impaired in intra- and intercellular movements has been engineered to undergo self-destruction upon entry into mammalian cells by production of a phage lysin under the control of the promoter of the actA gene which is preferentially activated in the cytosol. This bacterial vector was able, as demonstrated by Dietrich et al. (1998), to deliver a plasmid carrying gfp (green fluorescent protein), cat (chloramphenicol acetyltransferase) or a portion of the ova (ovalbumine) gene under the control of the CMV promoter in the P388D macrophage cell line. Functional transfer was dramatically increased (from 0.001% to 0.2%) if there was lysis of the internalized Listeria in the cytoplasm, either by selfkilling by lysin production or after antibiotic treatment. Efficient expression of chloramphenicol acetyltransferase or antigen presentation was observed in P388D macrophages. Plasmid DNA, from macrophage clones cultured in selective medium for more than 12 weeks, was found to be integrated into the genome of the new host at a frequency of approximately 10–7. In order to design a genetically more defined bacterial vector, we have cloned the 3.2 kb inv locus encoding the invasin of Yersinia pseudotuberculosis alone or combined with the 1.5 kb hly gene coding for the listeriolysin O from L. monocytogenes in the stable dap– auxotroph E. coli BM2710 (Grillot-Courvalin et al., 1998). We also introduced by transformation in that strain plasmid pEGFP-C1 which directs synthesis of the green fluorescent protein (GFP) in mammalian cells but not in bacteria. Between 5 and 20% of HeLa, CHO, and COS-1 cells producing GFP were observed 2 days after incubation with invasive bacteria. Co-expression in E. coli of the gene for listeriolysin enhanced transfer efficiency. Expression of the acquired genes occurred both in dividing and in quiescent cells, albeit at a lower efficiency in the latter. Transfer of functional plasmid DNA could be observed in J774 macrophages but not in a mouse dendritic cell line. Expression of acquired DNA was very stable, as tested after 2 months of culture. The number of viable intracellular bacteria decreased rapidly with no survivors after 24 to 72 h, and chromosomal DNA of the donor could not be detected after 23
to 48 days. Plasmid transfer was obtained, although at a lower frequency, from E. coli BM2711, the dap prototroph parental strain counterpart of BM2710, indicating that bacteria may transfer functional genetic information to non-professional phagocytes provided they can induce their own internalization. Agrobacterium tumefaciens is a soil phytopathogen that elicits neoplastic growths on the host plant species. It has very recently been shown that Agrobacterium can also transfer DNA to mammalian cells, as demonstrated by Kunick et al. (2001). Agrobacterium infection requires two genetic components that are carried by the tumor-inducing plasmid: the transferred DNA (T-DNA) which is introduced into the plant genome and the virulence region which specifies the protein apparatus for T-DNA transfer. Experiments in vitro indicated that A. tumefaciens attaches to and transfers DNA to several types of human cells, by conjugation, the mechanism it uses to transform plant cells. Study of stable transfected HeLa cells indicate integration of T-DNA into their genome.
IN VIVO GENE TRANSFER IN ANIMAL MODELS The ability of Shigella to enter intestinal epithelial cells and to evade from the endocytic vesicle was exploited to develop an in vivo gene delivery system as demonstrated by Sizemore et al. (1995). Mice were inoculated twice intranasally with 106 to 107 bacteria transformed with plasmid pCMVβ, 4 weeks apart; splenocytes from these mice proliferated after a 3 day culture in vitro in the presence of βgalactosidase and anti-β-galactosidase antibodies were detected in their sera as demonstrated by Sizemore et al. (1997). These results provided the first evidence that delivery of plasmid DNA by Shigella in vivo could elicit an immune response to the plasmid-encoded antigen. According to recent findings (Fennelly et al., 1999), after intranasal inoculation of mice, a highly attenuated strain of ∆asd Shigella harboring a DNA measles vaccine plasmid induced a vigorous measles-specific immune-response of both Th1 and Th2 types.
280
BACTERIA AS GENE DELIVERY VECTORS FOR MAMMALIAN CELLS
Attenuated Salmonella expressing heterologous antigens have been used for oral immunization in mice, farm animals, and in humans resulting in efficient stimulation of mucosal and systemic immune responses. An attenuated strain of S. typhimurium was used successfully by Darji et al. (1997) as a vector for oral genetic immunization. In this work, oral administration of an aroA– auxotrophic mutant harboring plasmids encoding E. coli β-galactosidase or truncated ActA and listeriolysin (two virulence factors of L. monocytogenes), each under the control of an eukaryotic promoter, led to excellent humoral and cellular immune responses in mice. The immunized mice were protected from a lethal challenge with virulent L. monocytogenes, even after a single administration. β-galactosidase activity was detected in splenic macrophages 5 weeks after oral administration of S. typhimurium carrying plasmid pCMVβ. Plasmid DNA transfer was studied in vitro after 1 h infection of mouse primary peritoneal macrophages with attenuated S. typhimurium carrying pCMVβ. Evidence of β-galactosidase synthesis by the infected macrophages was provided but tetracycline had to be added during the entire culture period to inhibit endogenous residual synthesis of β-galactosidase by bacteria. In a similar study by Paglia et al. (1998), oral administration to mice of an attenuated Salmonella harboring pCMVβ could generate humoral and cellular immune responses to β-galactosidase and a protective response against an aggressive murine fibrosarcoma transduced with the βgalactosidase gene. Expression of the transgene by antigen presenting cells after per os administration was documented with an eukaryotic vector expressing GFP. Among the 19% of splenocytes expressing GFP 28 days after oral administration, 50% were dendritic cells and 30% expressed macrophage markers. This provides evidence of direct in vivo gene transfer by orally administered bacteria to dendritic cells. According to more recent findings by Paglia et al. (2000), oral administration of an attenuated Salmonella, carrier for an eukaryotic expression vector encoding the murine INFγ gene, resulted in the production of this cytokine in INFγ-deficient mice. This provides evidences that attenuated Salmonella can be used
263
in vivo as a DNA delivery system for the correction of a genetic defect.
DISCUSSION In very few instances, direct introduction of DNA from bacteria to mammalian cells has been reported, as demonstrated by Schaffner (1980), that plasmids carrying tandem copies of the SV40 virus genome could be transferred from E. coli to mammalian cells by exposing the cell culture to a bacterial suspension. However, transfer was found to occur at a very low frequency. Similar observations were made with E. coli harboring plasmids carrying poliovirus 1 cDNA and transfer took place in the presence of high concentrations of DNAse in the culture medium, as demonstrated by Heitmann and Lópes-Pila (1993). In these experiments, the vectors were human pathogens that could not be employed outside an experimental setting. Attenuated intracellular bacteria, such as Shigella, Salmonella, Listeria, and invasive E. coli have been found to act as efficient gene delivery vectors in both phagocytic and non-phagocytic mammalian cells (Table 23.1). Observations from in vitro and in vivo experiments are consistent with the hypothesis that, after internalization into a primary vacuole, bacteria or their plasmid content have to escape into the cytosol to gain access to the nucleus. Studies on microbial pathogenesis have expanded our understanding of the mechanisms designed by bacteria to achieve entry into host cells and to gain access to intracellular compartments. There are two major strategies for bacteria to gain access to host cells, as reviewed by Marra and Isberg (1996). For certain genera like Salmonella or Shigella, contact between the bacteria and the host cells results in the secretion, by the bacteria, of a set of invasion proteins that triggers signalling events into the cells, leading to cytoskeletal rearrangement, membrane ruffling and bacterial uptake by micropinocytose. For other genera like Yersinia or Listeria, binding of a single bacterial protein to a particular ligand on the host cell surface is necessary and sufficient to trigger entry into phagocytic and nonphagocytic cells by a zipper-like mechanism.
281
264
C. GRILLOT-COURVALIN, S. GOUSSARD AND P. COURVALIN
TABLE 23.1 Main studies on bacteria to eukaryote gene transfer Recipient Donor bacterial species
Transferred plasmid(s)
In vitro: cell lines transfected
In vivo: route of administration
Shigella flexneri dapA–
pCMVβ
BHK P815
Intranasal Intracorneal Oral
HeLa COS-1 CHO A549 J774 Primary mouse macrophage
Not determined
Invasive E. coli dapA–
Salmonella typhimurium aroA– Listeria monocytogenes ∆ mpl actA plcB
Measles-vaccine plasmid β-gal replicative and integrative eukaryotic vectors pEGFP-C1 pCMVβ pCMV actA-hly INFγ gene pCMVgfp pCMVcat pCMVova
J774 P388D
Binding of Yersinia invasin to β1 integrins results in entry into cells expressing this integrin at the surface; binding of internalin with E-cadherin mediates entry of Listeria into certain cell types. Similarly, introduction in E. coli of the virulence plasmid (pWR100) of S. flexneri or of the inv gene of Y. pseudotuberculosis confers to this otherwise extracellular bacteria the ability to invade nonphagocytic cells. Access to the cell cytosol depends on the mechanisms by which bacteria survive inside the cells. Listeria and Shigella escape rapidly from the vacuole of entry after lysis of its membrane, by production of listeriolysin in the case of Listeria, and replicate in the cytoplasm of the cell. Other bacteria, like Salmonella, remain in the phagosomal vacuole and replicate within this compartment. The successful delivery of genes by Salmonella is therefore surprising since Salmonella-containing vacuoles have a unique trafficking pathway, uncoupled from the normal endocytic degradation pathway. Interestingly, infection by Salmonella resulted in functional DNA delivery into primary macrophages but not in macrophage cell lines, as demonstrated by Sizemore et al. (1995) and Darji et al. (1997). DNA entering host cell cytoplasm by phagocytosis is less efficiently routed to the nucleus
Oral Oral Intraperitoneally
Reference Sizemore et al. (1995, 1997) Fennelly et al. (1999) Courvalin et al. (1995), GrillotCourvalin et al. (1998) Darji et al. (1997), Paglia et al. (1998) Paglia et al. (2000) Dietrich et al. (1998), Spreng et al. (2000)
than when introduced directly into the cytoplasm (e.g. by gene gun). Consistent with this finding are the observations that more efficient gene transfer was observed when Listeria was destroyed in the cytoplasm of the cell (Dietrich et al., 1987) or if the invasive E. coli produced listeriolysin that triggers pore formation in the vacuolar membrane (Grillot-Courvalin et al., 1998). However, it cannot be excluded that DNA may gain access to the mammalian cell cytoplasm via leakage from host cell phagosomes, as has been proposed for transfer of certain antigens by Kovacsovics-Bankowski and Rock (1995). The property of bacteria to act as a gene delivery system has been mainly exploited for DNA vaccination. Attenuated auxotroph mutants of Salmonella are already in use as live vaccines in man and in animals, and effective gene transfer to dendritic cells after oral administration of mice with these bacteria has been documented by Paglia et al. (1998). In these in vivo studies, the number of transfected cells varies greatly depending on the type of bacterial vector and on the cell type. However, dendritic cells are highly efficient antigen presenting cells and a small number of transfected dendritic cells has been shown to be sufficient to stimulate both primary and secondary T and B
282
BACTERIA AS GENE DELIVERY VECTORS FOR MAMMALIAN CELLS
cell responses and to process and present antigens efficiently, as demonstrated by Banchereau and Steinman (1998). Moreover, per os administration of bacterial vectors could lead to transfection of gut-associated lymphoid cells and stimulation of mucosal immunity, as demonstrated by Darji et al. (1997), Paglia et al. (1998), and Fennelly et al. (1999). The potential use of bacterial vectors for in vivo or ex vivo gene therapy has only been tested once (Paglia et al., 2000) for a genetic defect associated with monocyte/macrophage cell type. Despite the high numbers of bacteria and plasmids internalized by the host cells, transgene expression remains low and comparable to the levels obtained with other non-viral delivery systems such as polycation-DNA complexes. Following bacterial internalization, plasmid DNA has to be released into the cytosol before its nuclear entry can occur. The turnover of plasmid DNA delivered by microinjection in the cytosol has been shown by Lechardeur et al. (1999) to be rapid, with an apparent half-life of 50 to 90 min in HeLa and COS cells. Direct delivery into the cytosol of native plasmids by intracellular bacteria may constitute a means of protecting this DNA from degradation by cytosolic nucleases.
REFERENCES Banchereau, J. and Steinman, R.M. (1998) Dendritic cells and the control of immunity. Nature 392: 245–252. Buchanan-Wollaston, V., Passiatore, J.E. and Cannon, F. (1987) The mob and oriT mobilization functions of a bacterial plasmid promote its transfer to plants. Nature 328: 172–175. Courvalin, P., Goussard, S. and Grillot-Courvalin, C. (1995) Gene transfer from bacteria to mammalian cells. CR Acad. Sci. 318: 1207–1212. Darji, A., Guzman, C.A., Gerstel, B. et al. (1997) Oral somatic transgene vaccination using attenuated S. typhimurium. Cell 91: 765–775. Dietrich, G., Bubert, A., Gentschev, I. et al. (1998) Delivery of antigen-encoding plasmid DNA into the cytosol of
265
macrophages by attenuated suicide Listeria monocytogenes. Nat. Biotechnol. 16: 181–185. Fennelly, G.J., Khan, S.A., Abadi, M.A. et al. (1999) Mucosal DNA vaccine immunization against measles with a highly attenuated Shigella flexneri vector. J. Immuno. 162: 1603–1610. Grillot-Courvalin, C., Goussard, S., Huetz, F. et al. (1998) Functional gene transfer from intracellular bacteria to mammalian cells. Nat. Biotechnol. 16: 862–866. Heinemann, J.A. and Sprague, G.F. Jr. (1989) Bacterial conjugative plasmids mobilize DNA transfer between bacteria and yeast. Nature 340: 205–209. Heitmann, D. and Lópes-Pila, J.M. (1993) Frequency and conditions of spontaneous plasmid transfer from E. coli to cultured mammalian cells. Biosystems 29: 37–48. Kovacsovics-Bankowski, M. and Rock, K.L. (1995) A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules. Science 267: 243–246. Kunik, T., Tzfira, T., Kapulnik, Y. et al. (2001) Genetic transformation of HeLa cells by Agrobacterium. Proc. Natl Acad. Sci. USA 98: 1871–1876. Lechardeur, D., Sohn, K.J., Haardt, M. et al. (1999) Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Ther. 6: 482–497. Marra, A. and Isberg, R.R. (1996) Common entry mechanisms. Bacterial pathogenesis. Curr. Biol. 6: 1084–1086. Paglia, P., Medina, E., Arioli, I. et al. (1998) Gene transfer in dendritic cells, induced by oral DNA vaccination with Salmonella typhimurium, results in protective immunity against a murine fibrosarcoma. Blood 92: 3172–3176. Paglia, P., Terrazzini, N., Schulze, K. et al. (2000) In vivo correction of genetic defects of monocyte/macrophages using attenuated Salmonella as oral vectors for targeted gene delivery. Gene. Ther. 7: 1725–1730. Schaffner, W. (1980) Direct transfer of cloned genes from bacteria to mammalian cells. Proc. Natl Acad. Sci. USA 77: 2163–2167. Sizemore, D.R., Branstrom, A.A, Sadoff and J.C. (1995) Attenuated Shigella as a DNA delivery vehicle for DNAmediated immunization. Science 270: 299–302. Sizemore, D.R., Branstrom, A.A and Sadoff, J.C. (1997) Attenuated bacteria as a DNA delivery vehicle for DNAmediated immunization. Vaccine 15: 804–807. Spreng, S., Dietrich, G., Niewiesk, S. et al. (2000) Novel bacterial systems for the delivery of recombinant protein or DNA. FEMS Immuno. Med. Microbiol. 27: 299–304. Trieu-Cuot, P., Carlier, C., Martin, P. and Courvalin, P. (1987) Plasmid transfer by conjugation from Escherichia coli to Gram-positive bacteria. FEMS Microbiol. Lett. 48: 289–294.
283