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Plasmid DNA Delivery by D-Alanine-Deficient Listeria monocytogenes Benjamin E. Simon,*,† Noel Ybarra,† Aime´ e O. Bonneval,† and Ronald A. Barry†,‡,§ Department of Veterans Affairs Medical Center, Portland, Oregan, and Department of Molecular Microbiology and Immunology and Department of Medicine, Oregon Health Sciences University, Portland, Oregon 97201
Optimal DNA vaccine efficacy requires circumventing several obstacles, including low immunogenicity, a need for adjuvant, and the costs of purifying injection grade plasmid DNA. Bacterial delivery of plasmid DNA may provide an efficient and low-cost alternative to plasmid purification and injection. Also, the bacterial vector may exhibit potential as an immune adjuvant in vivo. Thus, we elected to examine the use of cell-wall-deficient Listeria monocytogenes as a DNA delivery vehicle in vitro. First, the D-alanine-deficient (∆dal-dat) L. monocytogenes strain DP-L3506, which undergoes autolysis inside eukaryotic host cells in the absence of D-alanine, was transformed with a plasmid encoding green fluorescent protein (GFP) under control of the CMV promoter (pAM-EGFP). Then COS-7 and MC57G cell lines were infected with the transformed DP-L3506 at various multiplicities of infection (MOI) in the presence or absence of D-alanine. Subsequent GFP expression was observed in both cell lines by 24 h post-infection with DP-L3506(pAM-EGFP). Notably, no GFP positive cells were observed when D-alanine was omitted. Although transfection efficiency initially increased as a result of D-alanine supplementation, high concentration or long-term supplementation led to sustained bacterial growth that killed the infected host cells, resulting in fewer GFP-expressing cells. Thus, efficient DNA delivery by transformed bacteria must balance bacterial invasion and survival with target cell health and survival.
Introduction Despite nearly 2 decades of investigation and testing, only two DNA vaccines have been approved for veterinary use to date (1, 2), and none have yet been approved for use in humans. In practice, some DNA vaccines may have limited immunogenicity and require multiple doses or co-administration of an immuno-stimulatory adjuvant. Additionally, purification of high quality endotoxin-free plasmid DNA can be difficult for some low copy plasmid vectors. Bacterial delivery of plasmid DNA, a process called “bactofection”, has the potential to overcome some of these limitations, e.g., the bacterial vehicle may act as an adjuvant and may eliminate the need to purify plasmid DNA. Previous reports using attenuated bacterial delivery indicate that release of plasmid DNA into host cells is enhanced by bacterial death within the host cell (3). One strategy to achieve intracellular rupture of the bacteria is through the use of cell-walldeficient bacteria, an approach that has been examined in Gramnegative bacteria such as E. coli and Shigella (4-8). Due the intracellular life cycle of Listeria monocytogenes (9), this Grampositive bacterium also has been examined as a DNA delivery vector for immune modulation or gene therapy (10-15). These previous systems have used wild-type L. monocytogenes (10, 16), bacteria that express reduced hemolysin activity (12), or bacteria that express the lytic protein from bacteriophage A118 (11, 13-15). However, use of wild-type bacteria for DNA delivery in vivo could be impaired by any antibiotic treatment * To whom correspondence should be addressed. Mailing address: The Evergreen State College, Lab I 3003, Olympia, WA 98505. Ph: 360-8676912. Email:
[email protected]. † Department of Veterans Affairs Medical Center. ‡ Department of Molecular Microbiology and Immunology, Oregon Health Sciences University. § Department of Medicine, Oregon Health Sciences University.
Figure 1. A model of gene transfer to eukaryotic cells by attenuated L. monocytogenes. (1) The bacteria are engulfed by the eukaryotic cell. (2) Listeriolysin causes lysis of the phagolysosome and escape of the bacteria into the cytoplasm. (3) The bacteria undergo autolysis in the cytoplasm due to the attenuating mutation (D-alanine deficiency), releasing plasmid DNA. (4) Plasmid DNA is transported to the nucleus. (5) The eukaryotic cell expresses and processes the plasmid-encoded antigen.
used to limit the growth of the bacteria and to prevent morbidity from the clinical infection. This strategy also may carry considerable risk in immuno-compromised patients. Alternatively, bacterial vectors expressing the phage lysin protein driven by the actA promoter are severely attenuated in vivo, and the bacteria are designed to lyse as the bacteria are beginning to spread intercellularly (11, 13). Although this strategy restricts bacterial growth in vivo, the early death of the bacteria may limit efficiency of DNA transfer in vivo. In this study, the D-alanine-deficient strain of L. monocytogenes, DP-L3506 (17), was used to deliver plasmid DNA to cultured cells. When removed from a source of D-alanine (which is generally absent from animal cells), these bacteria cannot synthesize the peptide cross-bridges for peptidoglycan cell wall formation and thus undergo autolysis during subsequent growth. Infection with such an attenuated strain is likely to be safer in
10.1021/bp060177i This article not subject to U.S. Copyright. Published 2006 by the American Chemical Society and American Institute of Chemical Engineers Published on Web 09/15/2006
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Figure 2. Survival of bacteria and infected cells with or without D-alanine supplementation. COS-7 (a, c) and MC57G cells (b, d) were infected with DP-L3506(pAM-EGFP) at a MOI of 5, 25, or 125 bacteria per cell. D-Alanine was excluded from some wells or supplied at 200 µg/mL. After 1 h the wells were rinsed to remove extracellular bacteria, and the medium was replaced with RPMI-10 plus 5 µg/mL gentamicin, with or without D-alanine. Cell monolayers were lysed at the indicated timepoints and serial dilutions of the lysate were plated on BHI agar with supplemental D-alanine (a, b), or after 4 or 20 ((2) h incubation the wells were rinsed and the medium replaced with RPMI-10 plus gentamicin and at 48 h post-infection viable cells were counted for each well (c, d). Panels a and b are individual wells from one typical experiment. Similar results were observed in repeated experiments. The data in panels c and d represent duplicate wells from one typical experiment. Similar results were observed in repeated experiments.
vivo than infection with wild-type bacteria. Additionally, and in contrast to the phage lysin system, the survival of the bacteria in the host cell can be extended if necessary by addition of exogenous D-alanine. In this study we examined the effect of D-alanine supplementation on the ability of D-alanine-deficient L. monocytogenes to infect and deliver plasmid DNA to cultured cells in vitro. L. monocytogenes can infect and reside within non-phagocytic cells, such as hepatocytes, and these infected somatic cells have been shown to be targets of the adaptive cell-mediated immune response (18, 19). Hence, for these experiments we used two adherent non-phagocytic cell lines as targets for bactofection. The COS series of cell lines (COS-1, COS-3, and COS-7), derived from green monkey kidney (20), have been used extensively in transfection studies (10, 13, 21-23), and we used COS-7 cells in our studies due to the reported ease of transfection. MC57G, a fibrosarcoma cell line derived from C57BL/6 mice, expresses the H-2b haplotype of MHC-I (24) and will present H2-Kb restricted peptides such as the SIINFEKL peptide from ovalbumin. We chose this C57BL/6 derived cell line in anticipation of future in vivo studies using C57BL/6 mice and ovalbumin as a model antigen.
Materials and Methods
Figure 3. In vitro gene transfer to COS-7 and MC57G cultured cells. (a) Photomicrograph of COS-7 cells 48 h post-infection with DP-L3506(pAM-GFP). (b) Uninfected COS-7 cells. (c) MC57G cells 48 h postinfection with DP-L3506(pAM-GFP). (d) Uninfected MC57G cells.
Plasmid DNA Constructs and Bacteria. Plasmid pAMEGFP was constructed by inserting a fragment of pEGFPIRESpuro (Clontech) containing the CMV promoter and EGFP gene, into the broad host-range plasmid pAM401 (25) using standard methods (26). The negative control plasmid pAMUbOVA was constructed with an expression cassette encoding
the CMV promoter and a ubiquitin-ovalbumin fusion protein from pUb-OVA. The ubiquitin-ovalbumin fusion protein cassette of pUb-OVA was constructed similarly to our previously described ubiquitin vaccine plasmids (27). The plasmids were introduced into competent L. monocytogenes DP-L3506 by
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Figure 4. Short-term d-alanine supplementation increases the percentage of transfected cells in vitro. COS-7 and MC57G cells were infected with DP-L3506(pAM-EGFP) (as described in Figure 2). Four or 20 ((2) h post-infection the wells were rinsed to remove the D-alanine and the medium was replaced with RPMI-10 plus gentamicin. Cell transfection was assessed 48 h post-infection. These data represent the mean and standard error from 3-5 independent experiments. (a) Percentage of GFP positive COS-7 cells. (b) Relative transfection efficiency for COS-7 cells. (c) Percentage of GFP positive MC57G cells. (d) Relative transfection efficiency for MC57G cells
electroporation (28). For cell infection studies frozen stocks of bacteria were produced from overnight cultures of the L. monocytogenes strains, diluted 1:1 with 40% glycerol/BHI and stored in aliquots at -80 °C. Viable CFU/mL of the infection stocks was determined by serial dilution and plating on BHI agar plates plus D-alanine (200 µg/mL) and chloramphenicol (20 µg/mL). Cell Lines and Infection Studies. COS-7 or MC57G cell lines were maintained in RPMI-10 plus antibiotics (RPMI-1640 [Invitrogen, Carlsbad, CA] with 10% FBS, 100 µg/mL streptomycin, and 100 units/mL penicillin) and incubated at 37 °C in a 5% CO2 atmosphere. When the monolayers reached confluency (every 3-4 days), the monolayers were dissociated using trypsin-EDTA (Invitrogen) and 1/10 of the cells were transferred to new flasks to achieve a cell density of ∼105 per mL. For infection studies, cells were seeded into 24-well plates in antibiotic-free RPMI-10 1 day prior to infection at 5-10 × 104 cells per well. Bacterial suspensions were prepared in RPMI1640 from previously frozen and enumerated stocks of bacteria. Tissue culture cells were infected with varying multiplicities of infection (MOI) in RPMI-10 for 1 h, with or without supplemental D-alanine (200 µg/mL). After the 1-h infection period, the wells were rinsed three times and the medium was replaced with RPMI-10 plus 5 µg/mL gentamicin with or without D-alanine. In some experiments the cells were provided with supplemental D-alanine for an additional 4 h or overnight after addition of gentamicin, then the wells were washed once to remove the supplemental D-alanine, and the medium was replaced with RPMI-10 plus 5 µg/mL gentamicin. Positive control transfections of MC57G and COS-7 cells were performed in 24-well plates using ExGen500 (MBI Fermentas, Hanover, MD) according to manufacturers instructions using 1
µg of pAM-EGFP per well at DNA:ExGen500 ratios ranging from 1:1 to 1:9. Intracellular Survival of Bacteria. Replicate wells were infected at various MOI for 1 h with D-alanine, and then the monolayers were rinsed to remove extracellular bacteria and exogenous D-alanine. Infected monolayers were lysed at various time points after infection, and serial dilutions of the lysate were plated on BHI agar (supplemented with 200 µg/mL D-alanine) to determine colony forming units (CFU) per mL. Evaluation of GFP Expression. Two days post-infection, the cells were trypsinized to obtain single-cell suspensions. Samples from each well were counted on a hemacytometer to estimate total cell number for each well. The remainder of each sample was analyzed by flow cytometry to estimate the percentage of GFP positive cells. Relative transfection efficiency was calculated as (%GFP positive/maximum % GFP positive within the experiment) (viable cells in experimental/viable cells in uninfected control).
Results and Discussion Successful bactofection of eukaryotic cells requires several discrete events (Figure 1): First, the bacteria must enter the eukaryotic cell, next the bacteria must release the plasmid DNA, then the DNA must enter the nucleus to be transcribed, and finally the mRNA must be translated to produce the protein of interest. The D-alanine-deficient L. monocytogenes used in this study is capable of directing these events in vitro. Strain DP-L3506 was previously shown to infect and enter the cytoplasm of HeLa cells and J774 cells, as well as primary bone marrow derived macrophages, in the presence of D-alanine (17). As expected, we observed that the D-alanine-deficient
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Figure 5. Low concentration d-alanine supplementation increases the percentage of transfected cells in vitro. COS-7 and MC57G cells were infected with recombinant L. monocytogenes as described above. D-Alanine was supplied at 200 µg/mL during the 1 h infection period. After infection the wells were rinsed and the medium was replaced with RPMI-10 plus gentamicin (5 µg/mL) and D-alanine (0, 10, 25, or 50 µg/mL). Cell transfection was assessed 48 h post-infection. These data represent the mean and standard error from 2-5 independent experiments. (a) Percentage of GFP positive COS-7 cells. (b) Relative transfection efficiency for COS-7 cells. (c) Percentage of GFP positive MC57G cells. (d) Relative transfection efficiency for MC57G cells. “Add-back” refers to the D-alanine concentration in the culture medium after the 1 h infection period.
bacteria were also able to infect COS-7 and MC57G cell lines in the presence of D-alanine. Following such an infection, viable bacteria could be recovered from MC57G and COS-7 cells for at least 4 h after removal of D-alanine (Figure 2a,b). However, the number of recoverable bacteria was reduced at least 1000fold after 20 h without D-alanine. Bacterial survival was also enhanced by inclusion of intermediate concentrations of Dalanine. Inclusion of as little as 10 µg/mL D-alanine enhanced bacterial survival 10- to 1000-fold over a 24 h period (Figure 1, Supporting Information). Generally, a higher MOI or more D-alanine supplementation resulted in decreased survival of the MC57G or COS-7 host cells and coincided with enhanced intracellular growth of the infecting bacteria (Figure 2c,d). When MC57G or COS-7 cells were infected with DP-L3506 carrying the GFP expression plasmid pAM-EGFP in the presence of 200 µg/mL D-alanine, GFP expression was observed by 24 h post-infection (Figure 3). If D-alanine was omitted during the 1-h infection period, GFP expression was rarely observed from infected MC57G or COS-7 cells (less than 0.1%). GFP was never observed in COS-7 or MC57G cells infected with DP-L3506 carrying the control plasmid pAM-UbOVA. Transfection percentage and relative transfection efficiency varied with the duration of D-alanine supplementation and MOI (Figure 4). Presumably, short-term bacterial survival was required for DNA delivery to COS-7 cells, as exclusion of D-alanine during the 1 h infection phase did not lead to detectable numbers of GFP-positive cells (Figure 4a). D-Alanine supplementation during the infection phase resulted in a low but detectable number of GFP-positive COS-7 cells. Transfection frequency increased if the cells were incubated with D-alanine for 4 h; however, with longer D-alanine supplementation, heavy bacterial infection or intracellular growth of the bacteria eventually killed the infected cells (Figure 2c,d)
resulting in decreased numbers of GFP expressing cells (Figure 4a). Higher infectious doses (MOI > 25) also led to reduced numbers of viable COS-7 cells and lower overall transfection efficiency (Figure 4b). D-Alanine alone had no effect on COS-7 cell survival (data not shown). Similar results were observed with infected MC57G cells, although GFP transfection frequency was highest with 20 h of D-alanine supplementation (Figure 4c). Again, the highest MOI with long-term (>20 h) D-alanine supplementation resulted in reduced overall transfection efficiency (Figure 4d). The reduced survival of infected tissue culture cells at high D-alanine concentrations led us to ask whether continuous supplementation with lower concentrations of D-alanine might prolong intracellular bacterial survival and enhance transfection without substantial bacterial growth or damage to the infected tissue culture cells, thus resulting in higher overall transfection efficiency. We observed that transfection percentage and relative transfection efficiency did vary with the concentration of supplemental D-alanine (Figure 5). For COS-7 cells the optimal concentration of D-alanine for in vitro gene transfer was 10 µg/ mL (Figure 5a). Higher concentrations of D-alanine led to a lower frequency of transfected cells (Figure 5a) and a reduced overall transfection efficiency (Figure 5b). The highest MOI also resulted in fewer GFP-positive cells and reduced transfection efficiency (Figure 5a,b). For MC57G cells, supplementation with 25 µg/mL D-alanine was optimal for gene transfer (Figure 5c), possibly due to a lower infection level in these cells compared to COS-7 cells (see Figure 2b). At these concentrations of D-alanine, the highest MOI resulted in the highest percent of GFP-positive cells (Figure 5c), but at higher D-alanine concentrations the bacterial infection eventually killed the target cells, leading to decreased numbers of viable cells and reduced transfection efficiency (Figure 5d).
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In vitro gene transfer by strain DP-L3506 appears to be relatively efficient, but comparison of this system to the previously described phage-lysin system (13) would be of interest. In side-by-side comparisons of DP-L3506(pAM-EGFP), WL140(pSP118-EGFP) (encoding the phage-lysin under control of the actA promoter, kindly provided by Dr. Sabine Pilgrim, University of Wurzberg, Germany), and DP-L3506(pSP118EGFP), all three strains showed similar percentages of GFP positive cells when D-alanine was supplied at 200 µg/mL (Figure 2a, Supporting Information; note that strain WL140 is not D-alanine deficient). DP-L3506(pAM-EGFP) induced a higher percentage of GFP positive cells at an MOI of 25 with 10 µg/ mL D-alanine (Figure 2b, Supporting Information). In these experiments COS-7 cells were more susceptible to bactofection than were MC57G cells, likely due to several factors including basal transfection rate and structure of the monolayer. Specifically, the MC57G cells were sub-confluent at time of infection, whereas the COS-7 cells were confluent (data not shown). With adjacent cells in contact, L. monocytogenes can spread cell-to-cell without exposure to the gentamicin in the extracellular media (9). Similar cell to cell spread would be limited in sub-confluent monolayers. Additionally, we observed that MC57G cells were not as easily transfected with ExGen500 (MBI Fermentas, Hanover, MD) as were the COS-7 cells. We achieved peak transfection rates with plasmid pAMEGFP of 2-2.5% for MC57G compared to 15-18% for COS-7 cells. Gene transfer to tissue culture cells by these D-alaninedeficient bacteria appears to be relatively efficient in vitro but requires addition of D-alanine during the infection. Thus, D-alanine supplementation will likely be required for this delivery method to be functional in vivo. Despite limited toxicity information, D-alanine supplementation in vivo is unlikely to be harmful to the host animal due to rapid conversion of D-alanine to L-alanine by the activity of D-amino acid oxidase (29). Future studies will assess the optimal parameters for in vivo plasmid delivery by L. monocytogenes, including route of administration, infectious dose, and duration or concentration of D-alanine supplementation. Alternately, a system for regulated synthesis of D-alanine in the DP-L3506 strain of L. monocytogenes has recently been described by Li et al. (30). In the presence of micromolar quantities of the inducer IPTG, the bacteria express low levels of alanine racemase (dal) and are rescued from death. In the absence of IPTG, the bacteria are still dependent on exogenous D-alanine for cell wall synthesis. IPTG may have more favorable in vivo pharmacokinetics than D-alanine; however, little information is available regarding toxicity of this compound for animals.
Conclusions Cell-wall-deficient L. monocytogenes can deliver plasmid DNA to eukaryotic cells in vitro but this process is dependent on short-term bacterial survival after infecting the host cells. Transfection efficiency initially increased with increasing Dalanine supplementation or increasing MOI. Eventually, however, heavy bacterial infection or intracellular growth of the bacteria killed the infected cells, leading to decreased numbers of GFP expressing cells. This effect is likely to occur in vivo as well as in vitro, and therefore the ability to prolong or shorten bacterial survival may allow better control of DNA delivery in vivo. For bactofection to become a realistic gene therapy tool or immunization method, bacterial invasion and survival must be balanced with target cell health and survival. Future studies will focus on adapting this system to deliver genes in vivo.
Acknowledgment This work was funded by the Department of Veterans Affairs. L. monocytogenes strain DP-L3506 was generously provided by Fred Frankel, University of Pennsylvania. Supporting Information Available: Figures showing the survival of bacteria with varying concentrations of D-alanine supplementation and a comparison of DP-L3506(pAM-EGFP) to phage-lysin based bactofection strategies. This material is available free of charge via the Internet at http://pubs.acs.org.
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