RESEARCH ARTICLE

Bactofection of mammalian cells by Listeriamonocytogenes: improvement and mechanismof DNA delivery

S Pilgrim1,4, J Stritzker1,4, C Schoen1, A Kolb-Maurer1, G Geginat2, MJ Loessner3, I Gentschev1

and W Goebel1

1Theodor-Boveri-Institut (Biozentrum) der Universitat Wurzburg, Lehrstuhl fur Mikrobiologie, Am Hubland, Wurzburg, Germany;2Institut fur Medizinische Mikrobiologie und Hygiene, Fakultat fur Klinische Medizin Mannheim der Universitat Heidelberg,Mannheim, Germany; and 3Institut fur Lebensmittel- und Ernahrungswissenschaften, ETH Zuerich, Zuerich, Switzerland

Bacteria-mediated transfer of plasmid DNA into mammaliancells (bactofection) is a potent approach to express plasmid-encoded heterologous proteins (protein antigens, toxins orenzymes) in a large set of different cell types includingphagocytic and nonphagocytic mammalian cells. Previously,we have described a Listeria monocytogenes-mediated DNAdelivery system, which releases plasmid DNA directly intothe cytosol of mammalian cells by partial self-destruction ofthe carrier bacteria. Here we report on a second generationof this phage lysin supported bactofection system, which is

greatly improved with respect to plasmid stability, transferefficacy and biosafety. In this case, DNA release is initiatedby spontaneous bacterial lysis in the infected cells cytosolwhich is subsequently enhanced by the simultaneouslyreleased phage lysin produced by the intracellular carrierbacteria. Bacteria that are capable of cell-to-cell spread arefound to be much more efficient in bactofection than theirnonspreading counterparts.Gene Therapy (2003) 10, 2036–2045. doi:10.1038/sj.gt.3302105

Keywords: DNA delivery; bactofection; Listeria monocytogenes

Introduction

Naked plasmid DNA encoding antigens under thecontrol of strong eucaryotic promoters can induceantigen-specific humoral and cellular immune responseswhen injected intramuscularly1 or intradermally. 2 Usingthis vaccination approach, protection of mammalianhosts against a variety of viral, bacterial and parasiticagents and even tumors has been demonstrated (forrecent reviews see Donnelly et al3 and G�uur�uunathan et al 4).However, this method requires highly purified DNA,and when applied without adjuvants leads to ratherweak or even adverse immune responses probably dueto limited costimulatory activity.5

Recent studies have indicated that bacteria, likeShigella flexneri,6–9 Salmonella spp,9,10 E. coli,7,11 Yersiniaenterocolitica12 and Listeria monocytogenes,9,13,14 can beused as carriers for transporting plasmids similar tothose used as DNA vaccines into a variety of mammaliancells including antigen-presenting cells (APC). The term‘bactofection’ has been recently coined for this bacteria-mediated DNA delivery into mammalian cells.15 Inaddition to the easy manufacture and application, the

carrier bacteria can also provide the necessary costimu-latory effects when used as live vaccines.

Furthermore, intracellular bacteria can be applied todeliver eucaryotic expression plasmids to introducerelevant transgenes into somatic tissues to treat, cure orprevent diseases that result from genetic disorders.16 Onealready reported example is the transfer of the humancystic fibrosis transmembrane conductance regulator(cftr) into epithelial cells, which recently was shown byusing L. monocytogenes as carrier.17 Likewise, Salmonellatyphimurium were used to deliver genes encodingtherapeutic proteins like IL-12 or GM-CSF to achieveantitumor effects in mice.18

The use of bacterial systems for gene therapy has theadded advantage that bacteria often have an innatetropism for specific target tissues.

Shigella spp and L. monocytogenes reach the cytosol ofinfected host cells and deliver the DNA directly into thiscompartment upon lysis. Salmonella, Yersinia and E. coliremain in phagosomal compartments of infected hostcells and it is basically unknown how the released DNAreaches the host cells nuclei where transcription of theantigen-encoding DNA occurs. In most reported cases,disruption of the bacterial carriers with subsequentrelease of the plasmid DNA occurs either spontaneously9

within the infected cell or is facilitated by treatment withantibiotics14 or use of specific auxotrophic mutants6–8,10,11

that have a higher tendency of disintegration in the hostcell intracellular milieu. The suicide L. monocytogenescarrier system that we have recently developed for DNAReceived 16 December 2002; accepted 04 June 2003

Correspondence: Dr W Goebel, Theodor-Boveri-Institut (Biozentrum) derUniversitat Wurzburg, Lehrstuhl fur Mikrobiologie, Am Hubland, 97074Wurzburg, Germany4Both these authors contributed equally to this work

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delivery into mammalian cells takes advantage of aphage lysin19 that is specifically expressed once thebacteria enter the host cell cytosol.13

The highly improved system reported here is at leastas efficient as all other previously described bacteria-mediated DNA delivery systems6–14 with respect to theefficacy of bactofection and by far superior with respectto stability and biosafety.

Results

Construction of a novel balanced-lethal systemfor bactofection of mammalian cellsWhen applying the previously constructed suicide L.monocytogenes DNA delivery system13 to BALB/c mice,we noticed high loss of the expression plasmid leading toa large population of ‘empty’ carrier bacteria. To stabilizethe plasmid in the L. monocytogenes carrier strain, we firsttested a number of different replication origins for stablereplication and found that the one from pAMb120 wasmost stable. This origin was taken for the construction ofpUNK1 – the new parental plasmid for all furtherderivatives. Next, the trpS gene encoding tryptophanyl-tRNA synthetase was introduced into pUNK1, resultingin plasmid pSP0. We then inserted into pSP0 thepreviously described listerial autolysis cassette consist-ing of the lysis gene of phage A118 (ply118)19 under thecontrol of the actA promoter (PactA) which is activated inthe cytosol of infected mammalian host cells;13 thisplasmid is called pSP118. The two plasmids (pSP0,pSP118) are present in 95–100 copies per listerial cell asdetermined by real-time PCR (data not shown).

Additionally, we deleted the chromosomal trpS genecopy of the carrier strain L. monocytogenes EGD-e21 whichwas only possible in the presence of the trpS-carryingplasmid pTRPS (for details, see Experimental protocol)indicating the expected essential function of this gene(Figure 1a and b). After growth of this strain (WL-140)

for 50 generations in different culture media, weobserved no plasmid loss as shown by replica-platingof colonies obtained from a brain–heart-infusion (BHI)-grown culture on tetracycline-containing agar plates (tetgene is carried by the plasmid). After insertion of variouseucaryotic expression cassettes, all under the control ofthe immediate-early promoter of hCMV (PCMV) into pSP0and pSP118, the generated expression plasmids weretransformed into WL-140 with subsequent removal ofpTRPS (Figure 1c) by cultivation of WL-140 in mediumwithout tetracycline.

This trpS-based balanced-lethal plasmid carrier systemwas also found to be stable after a 4-day passage throughBALB/c mice in contrast to the isogenic wild-type strain,both carrying the plasmid pSP0. The DtrpS bacteria werefound in equal amounts in liver and spleen as the wild-type bacteria indicating that the displacement of the trpSgene from the chromosome to the plasmid does notconsiderably affect the intracellular growth rate of thisstrain (see also Figure 6). While 37% of wild-type carrierbacteria lost this plasmid in vivo, all isolated DtrpSbacteria still harbored plasmid pSP0.

For additional attenuation of the L. monocytogenesDtrpS carrier strain, we deleted the aroA gene, whichencodes the first enzyme in the biosynthesis of aromaticamino acids, rendering this strain dependent on allaromatic amino acids, resulting in strain WL-141. Allstrains and plasmids used in this study are summarizedin Table 1.

Efficacy of bactofection of nonphagocytic cells byphage lysin-positive and -negative L. monocytogenescarrier strainsL. monocytogenes DtrpS or D(trpS aroA) strains harboringeither pSP0-EGFP or pSP118-EGFP (each carrying thecDNA for enhanced green fluorescent protein (EGFP)adapted to eucaryotic cells as a model antigen under thecontrol of PCMV) were used for bactofection of severalnonphagocytic cell lines including Caco-2, HeLa, HepG2(all epithelial cells) and COS-1 (fibroblasts). Efficacy ofbactofection was determined by the number of EGFP-expressing cells, and the fate of the carrier bacteria inside

Figure 1 Deletion of the trpS-containing region on the chromosome of the(a) L. monocytogenes strain EGD-e, (b) complementation of this region inthe balanced-lethal DtrpS mutant strain WL-140 with plasmid pTRPS.The plasmid pTRPS contains a chromosomal fragment that comprises thepromotor region of trpS, the trpS gene and its transcription terminator. (c)This plasmid can be replaced by the eucaryotic expression plasmids pSP0and pSP118 due to different resistance markers: pTRPS – RTet, pSP0 andpSP118 – REm.

Table 1 Bacterial strains and plasmids

Strains andplasmids

Relevant genotype Reference orsource

Listeria monocytogenesSv1/2a EGD-e Glaser et al21

WL-140 DtrpS/pTRPS This workWL-141 D(trpS aroA)/pTRPS J StritzkerWL-142 D(trpS actA)/pTRPS This work

PlasmidspTRPS TetR, trpS This workpUNK1 EmR, oriE1, ori pAMb1 This workpSP0 EmR, trpS This workpSP118 EmR, trpS, PactA-ply118 This workpSP0-EGFP EmR, trpS, PCMV-egfp This workpSP118-EGFP EmR, trpS, PactA-ply118, PCMV-egfp This workpSP0-KMP EmR, trpS, PCMV-KMP-11 This workpSP118-DsRed EmR, trpS, PactA-ply118, PCMV-rfp This workPactA-gfp TetR, PactA-gfp Dietrich et al13

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infected HeLa cells was followed by staining the bacteriawith FITC-labelled anti-ActA antibodies. The highestnumber of EGFP-expressing cells (defined as bactofec-tion rate) was observed with all cell lines 3 days afterinfection (Figure 2). All bactofections were carried outwith a multiplicity of infection (MOI) of 5 since optimalbactofection rates were achieved using this ratio.

The D(trpS aroA) carrier strain harboring pSP118-EGFPyielded the highest bactofection rate in Caco-2 and COS-1 cells, while the aroAþ carrier strain was more efficientin HeLa and HepG2 cells possibly due to the ratherlow growth rate of the D(trpS aroA) strain in these hostcells (Figure 2a). Figure 2b shows also the rate of viabletarget cells (measured by the colorimetric reactionwith MTT) after infection compared to uninfected cellswhich was highest for all four cell lines with the D(trpSaroA) carrier strain harboring pSP118-EGFP. Thus thiscarrier strain not only yields high bactofection ratesbut is also least harmful to the different mammalian celllines tested.

EGFP expression and the fate of the intracellularcarrier bacteria harboring either pSP0-EGFP or pSP118-EGFP were followed in more detail in HeLa cells(Figure 3). Uptake of both bacterial strains was similar(about 20% of the cell population were infected) wheninfection was performed with 5 bacteria/HeLa cell; theinternalized L. monocytogenes pSP0-EGFP bacteria multi-plied extensively within the next 24 h as visualized bythe FITC-mediated fluorescence. Interestingly, spreadingof these carrier bacteria was rather restricted in the HeLacells (indicated by the rather small spreading halosshown in Figure 3c) compared to the other threenonphagocytic mammalian cell lines used (not shown).After 24 h, EGFP expression was observed in about 1% ofthe infected cells (Figure 3a) apparently by spontaneouslysis of the carrier bacteria in the infected HeLa cell.An approximately three-fold higher number of EGFP-expressing cells was obtained with L. monocytogenescarrying pSP118-EGFP (Figure 3b); in other experiments,we could also observe higher numbers of EGFP-expres-sing cells (Figure 2). Only weakly fluorescent bacteriawere observed in the infected cells (Figure 3d), but thenumber of colony-forming bacteria was comparable tothat of L. monocytogenes carrying pSP0-EGFP (data notshown) indicating surprisingly efficient replication ofthese phage lysin-expressing bacteria inside HeLa cells.We assume that the weak staining of the pSP118-EGFP-carrying bacteria is caused by reduced production ofActA due to titration of PrfA by the high copies of theactA promoter in front of ply118 carried on pSP118-EGFPrather than being a sign of bacterial lysis.

Bactofection of phagocytic cells and presentationof EGFP epitopes by P338.D1 macrophagesIn vitro bactofection of macrophages was unsuccessful orwas obtained with very low efficacy using the previouslydescribed listerial carrier systems9,14 including our firstL. monocytogenes suicide system.13

Figure 3 Bactofection of HeLa cells with the L. monocytogenes DtrpSstrain carrying the EGFP expression plasmids pSP0-EGFP (aþ c) andpSP118-EGFP (bþ d); plasmid pSP0-EGFP is without the phage lysingene (ply118) and pSP118-EGFP harbors the lysin gene cassette PactA-ply118. (aþ b) EGFP expression in HeLa cells 24 h after bactofection; cellswere infected with 5 bacteria per cell. (cþ d) Staining of the intracellularcarrier bacteria with FITC-labelled anti-ActA antibodies 24 h afterbactofection; infection was performed with 0.1 bacteria per cell.

Figure 2 Bactofection efficiency and cytotoxic effect of the applied L.monocytogenes strains carrying plasmids with or without phage lysin(pSP0-EGFP and pSP118-EGFP, respectively) in various cell lines (bothparameters were determined 3 days after infection). (a) Propidium iodide(PI)-negative, EGFP-expressing cells (calculated as percentage of PI-negative cells), measured by flow cytometry. (b) Rate of viable cells afterinfection in comparison with uninfected cells (set to 1), measured withMTT. (c) Owing to the unequal cell toxicity of the different bacterialstrains, the bactofection efficiencies were compared accurately by multi-plying the percentage of PI-negative (viable), EGFP-expressing cells withthe ratio of total viable cells.

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In contrast, bactofection of different phagocytic cellscould be successfully achieved with the new L. mono-cytogenes balanced-lethal system. The efficiencies ofEGFP expression were always considerably higher (10-fold and more) with the L. monocytogenes strains carryingpSP118-EGFP than with the same carrier strain carryingpSP0-EGFP (Figure 4a and b), while the numbers ofintracellular bacteria producing phage lysin werestrongly reduced (Figure 4c). With this improved system,EGFP expression could be obtained in up to 2.5% ofinfected P388.D1 macrophages which is more than 10-fold higher than with the previously described system.13

As shown in Figure 4b, the number of EGFP-expressingmacrophages, infected with the carrier strain harboringpSP118-EGFP, increases within 3 days after infection. Thehighest bactofection rate (about 1–2%) was obtained withthe macrophage line P388.D1, while isolated humanblood-derived dendritic cells (DC) yielded bactofectionrates of about 0.3–1% (Figure 5a and c). In the latter case,the obtained bactofection rates varied considerably anddepended strongly on the individual donator of the DCs.The bactofection rate always seems to coincide with theefficiency of infection of the DC by the carrier bacteria

and their rate of survival within the DC (data notshown).

Remarkably, DC infected with bacteria carryingpSP0-EGFP showed virtually no EGFP expression. EGFPexpression in DC was notably stronger compared toother cell types (Figure 5b) and was already observed20 h after infection with WL-140 carrying pSP118-EGFP.The viability of DC after infection was comparable to thatof uninfected DC and was independent of the listerialstrain used (Figure 5d). Primary murine bone marrowmacrophages (BMM) also yielded bactofection rates ofabout 1% with this carrier strain (data not shown).

Using an EGFP-specific CD8 T cell line recognizing theepitope HYLSTQSAL,22 we could show that P388.D1macrophages bactofected with L. monocytogenes contain-ing pSP118-EGFP are recognized by T cells (measured bythe production of gamma interferon (IFN-g); Figure 4d)indicating that EGFP-expressing macrophages can pre-sent EGFP epitopes after bactofection as expected in thecontext of MHC class I molecules. Optimal activation ofthe EGFP-specific CD8 T cell line was obtained when themacrophages were infected at a ratio of 10 carrierbacteria per macrophage (Figure 4d). Lower IFN-g valueswere obtained when higher (MOI of 100) or lowerinfection ratios (MOI of 100) were used which isprobably due to increased cell toxicity in the former case

Figure 4 Bactofection of phagocytic cells. (a) EGFP expression afterbactofection of P388.D1 macrophages with L. monocytogenes DtrpScarrying plasmids pSP0-EGFP or pSP118-EGFP, 72 h postinfection (p.i.).(b) PI-negative, EGFP-expressing P388.D1, 24–72 h p.i. (calculated aspercentage of PI-negative cells measured by flow cytometry). (c) Number ofintracellular bacteria per well, 2–72 h p.i. (d) MHC class I restrictedpresentation of the EGFP-derived CD8 T cell epitope HYLSTQSAL byP388.D1 macrophages after bactofection with L. monocytogenes DtrpScarrying plasmids pSP0-EGFP or pSP118-EGFP. Activation of the T cellsby the bactofected macrophages was determined by the amount of releasedIFN-g.

Figure 5 Bactofection of human dendritic cells (DC). (a,b) EGFPexpression after bactofection of human DC with L. monocytogenes DtrpScarrying plasmids pSP0-EGFP or pSP118-EGFP, 24 h p.i. (c) EGFP-expressing DC, 24 h p.i. (calculated as percentage of all CD83-positivecells measured by flow cytometry). (d) Rate of viable cells after infection incomparison with uninfected cells (set to 1), measured with MTT.

Figure 6 Bacterial load in liver and spleen of orogastrically infectedBALB/c mice (4 days p.i.) with 109 bacteria of (a) L. monocytogenes wild-type, (b) DtrpS strain (WL-140) with plasmid pSP0, (c) DtrpS straincarrying plasmid pSP118 and (d) D(trpS aroA) strain (WL-141) carryingplasmid pSP0.

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and inefficient bactofection rates of the macrophages inthe latter case.

Virulence of the L. monocytogenes plasmid-deliverystrains in the mouse modelPrevious bactofection studies with L. monocytogenes ascarrier of eucaryotic expression plasmids have usedwild-type strains in order to obtain significant transfec-tion rates.9 Such bacterial carriers can hardly be appliedfor gene therapy or vaccination due to their highvirulence.

To test the virulence of our new L. monocytogenesplasmid delivery strains, we performed orogastricinfections in BALB/c mice using 2� 109 bacteria ofL. monocytogenes EGD-e wild-type and the DtrpS mutantstrain each carrying pSP0 or pSP118 and the D(trpS aroA)strain with plasmid pSP0. At 4 days after infection, thebacterial load was determined in spleen and liver. Asshown in Figure 6, roughly equal viable bacterial countswere observed in both organs after infection with thewild-type strain and the DtrpS strain carrying pSP0. Noviable bacteria were detected in both organs afterinfection with the DtrpS strain carrying pSP118, indicat-ing strong virulence attenuation of the DtrpS carrierstrain with pSP118. Similar high attenuation wasobtained with the D(trpS aroA) mutant carrying pSP0;this attenuation is expected to be further enhanced in thepSP118-carrying strain. Thus the DaroA mutation of theL. monocytogenes carrier WL-141 (D(trpS aroA) harboringpSP118 as expression plasmid may represent not onlya very efficient vector system for bactofection, at leastin vitro, but may warrant also the highest biosafety forin vivo studies.

Mechanism of bacterial lysis and plasmid releasein the cytosol of mammalian cellsThe above-described data indicate that release ofplasmid DNA can occur in the absence of phage lysin.The extent of this apparently spontaneous bacterial lysiswith subsequent plasmid release seems to differ in theused mammalian cells (Figure 2); similar observationswere also made with other bacterial carriers.9 One maytherefore argue that disruption of the listerial carrierdoes not solely occur in the cytosol of the bactofected cellbut may be initiated already by partial damage of thebacteria in the phagosomal compartment which theyhave to pass before entering the cytosol.

To avoid passage of the bacterial carrier throughphagosomal compartments, we microinjected a spread-ing-deficient DactA mutant of the L. monocytogenes DtrpSstrain (WL-142) harboring either pSP0-EGFP or pSP118-EGFP into Caco-2 cells as recently described.23 Bycoinjection of Texas red-labelled dextran together withthe bacterial carrier strains (Figure 7, right panel) weobserved that EGFP expression occurred in the micro-injected cell 24 h after microinjection (indicated by theyellow fluorescence caused by the overlap of the greenEGFP and the red fluorescence of Texas red). Assummarized in Figure 7, about 5% of the cells micro-injected with the L. monocytogenes D(trpS actA) strainharboring pSP0 and more than 13% of those harboringpSP118 expressed EGFP. There was no further increasebut even a decrease in the number of EGFP-expressingcells after extended incubation (48 and 72 h) of the

microinjected cells, which seems to be due to enhancedcell death with time. These data support the assumptionthat DNA release is initiated in the cytosol by sponta-neous lysis of at least some carrier bacteria.

To further analyze the mechanism of lysis ofL. monocytogenes in bactofected cells, we replaced inpSP118 the cDNA of EGFP by cDNA of the redfluorescent protein (DsRed) or by the gene encodingthe Leishmania antigen KMP-11 (the expression of whichwas monitored by a red-fluorescent LRSC-labelledmonoclonal antibody). The intracellular bacteria weretagged either with a plasmid containing the previouslyreported PactA-gfp cassette which results in GFP expres-sion when L. monocytogenes replicates in the mammaliancell cytosol13 or with an FITC-labelled anti-ActA anti-serum. After infection into HeLa cells, we could nowsimultaneously determine the fate of the carrier bacteriaand the expression of the two delivered genes. As shownin Figure 8, most infected HeLa cells that did not expressDsRed or KMP-11 carried many bacteria (green fluor-escent) after 24 h, whereas those cells that expressedeither of the two (red fluorescent) proteins containedonly few or no detectable bacteria. Qualitatively similarresults were obtained regardless of whether the bacteriaharbored pSP0 or pSP118 suggesting that spontaneouslysis of a single carrier bacterium may release not onlyplasmid DNA but also the phage lysin (in the case ofpSP118) or other bacteriolytic enzymes (in the case ofpSP0 and pSP118), which subsequently lyse all othercarrier bacteria in the cytosol of the mammalian cell. Themore efficient lysis of the pSP118-harboring bacteria thenleads to higher bactofection rates.

Bactofection rate is strongly enhanced by spreading-competent bacteriaThe rather low bactofection of Caco-2 cells observed bymicroinjection of the spreading-incompetent L. monocy-togenes D(trpS actA) mutant strain (Figure 7) prompted usto further analyze the effect of spreading on bactofection.For this, we microinjected the actAþ listerial carrier

Figure 7 EGFP expression of Caco-2 cells after microinjection with D(trpSactA) mutant Listeriae (WL-142) carrying pSP0-EGFP or pSP118-EGFP.EGFP-expressing Caco-2 cells were counted 24, 48 and 72 h afterbactofection. The micrograph shows Caco-2 cells 24 h after microinjectionwith the D(trpS actA) strain carrying pSP118-EGFP. Primarily micro-injected Caco-2 cells appear red due to coinjection of Texas red-labelleddextran together with the bacteria, and yellow cells indicate EGFPexpression in the primarily microinjected cells due to the overlay of red(Texas red) and green (EGFP) fluorescence.

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strain (WL-140) harboring pSP0-EGFP or pSP118-EGFPinto Caco-2 cells as described in Figure 7 for the D(trpSactA) mutant. As shown in Figure 9, low numbers ofEGFP-expressing cells were obtained 24 h after micro-injection, but in contrast to the D(trpS actA) mutant strainthese numbers increased very significantly at later timepoints (48 and 72 h). Interestingly, at these time points,EGFP expression was rarely found in the primarymicroinjected Caco-2 cells (red fluorescent due tocoinjected Texas red) but rather in cells in the vicinityof the microinjected ones (green fluorescent Caco-2 cellsdue to EGFP expression). These cells can be accessedonly by spreading carrier bacteria. Such events werenever observed with the isogenic D(trpS actA) mutantstrain, ruling out the possibility that reinfection of Caco-2cells by bacteria released from lysed primary micro-injected cells is responsible for these events. In addition,reinfection is highly unlikely due to the presence ofgentamicin in the culture media during the entireexperiment.

To better quantify this ‘spreading effect’, we comparedthe efficiency of bactofection of Caco-2 cells by the L.monocytogenes D(trpS actA) mutant strain harboring pSP0-EGFP or pSP118-EGFP with that of the isogenic actAþ

strain. Both carrier strains (applied at an MOI of 5) wereinitially taken up by about 20% of the Caco-2 cells atsimilar frequencies (Figure 10a). While the D(trpS actA)mutant strain remained in the primarily infected cells(Figure 10c), the actAþ carrier strain infected virtually allcells in the cell layer by spreading during the course ofinfection (Figure 10d). The number of EGFP-expressingcells obtained after 48 h postinfection with the D(trpS

Figure 8 Colocalization of HeLa cells expressing either red fluorescentprotein (DsRed) or the leishmanial antigen KMP-11 and the intracellularListeriae (green) 24 h after bactofection. (a) The listerial DtrpS carrier wasequipped with plasmid pSP0-DsRed for eucaryotic DsRed expression andplasmid PactA-gfp for bacterial expression of GFP. The phase contrastmicroscopic picture shows cell infected with many green fluorescentbacteria and one cell expressing DsRed with no or few green fluorescentbacteria. (b) The DtrpS carrier strain harbored plasmid pSP118-KMP foreucaryotic expression of the leishmanial antigen KMP-11, which wasdetermined by staining with an LRSC-labelled anti-KMP-11 mAb (red)and the bacteria were visualized with FITC-labelled anti-ActA antiserum(green). The picture shows one cell expressing KMP-11 visualized with redfluorescent antibodies but no bacteria and neighboring cells containingmany bacteria visualized by green fluorescent antibodies, but notexpressing KMP-11.

Figure 9 EGFP expression of Caco-2 cells after microinjection with thespreading-competent DtrpS strain (WL-140) carrying pSP0-EGFP orpSP118-EGFP. The experiment was performed as described in the legendto Figure 2. Primarily microinjected Caco-2 cells appear red due tocoinjection of Texas red-labelled dextran together with the bacteria, andgreen fluorescent cells indicate EGFP-expressing Caco-2 cells in thevicinity of the primarily microinjected cell.

Figure 10 Efficiencies of bactofection of Caco-2 cells by the L.monocytogenes DtrpS (WL-140) and D(trpS actA) (WL-142) mutantstrains both carrying plasmids pSP0-EGFP or pSP118-EGFP afterinfection of Caco-2 cells with 5 or 100 bacteria per cell. (a) Averagenumber of intracellular bacteria per well containing 5� 105 Caco-2 cells2 h after bactofection (performed in triplicate). (b) EGFP-expressing cells(% of all cells measured by flow cytometric analysis) 48 h afterbactofection. (c) L. monocytogenes D(trpS actA) (WL-142) mutantbacteria, growing in microcolonies in initially infected Caco-2 cells(applied at an MOI of 5). (d) DtrpS (WL-140) bacteria, also applied at anMOI of 5, infected nearly all Caco-2 cells by cell-to-cell spread. (e) D(trpSactA) (WL-142) mutant bacteria infect nearly all cells when applied at anMOI of 100. All pictures were taken 6 h p.i. after Giemsa staining.Bacteria were highlighted in red.

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actA) mutant strain was more than 20-fold lower (Figure10b) than with the actAþ strain.

However, even infection with 100 D(trpS actA) mutantbacteria per Caco-2 cell, which resulted in at least asmany infected Caco-2 cells (Figure 10e) and even moreintracellular bacteria than obtained with the actAþ strainby spreading (Figure 10a), yielded still at least 10-foldless EGFP-expressing Caco-2 cells than the actAþ strain(Figure 10b). This result further supports the notion thatnot only the number of intracellular carrier bacteria orthe number of initially infected cells but also theirspreading capacity are important factors determining theefficiency of bacterial lysis and release of plasmid DNA.

Discussion

This report describes a greatly improved L. monocyto-genes-mediated plasmid DNA delivery system for mam-malian cells (bactofection15). Our new bactofectionsystem consists of the L. monocytogenes EGD-e strain21

with deletion in trpS and for additional attenuation inaroA, carrying a plasmid pSP118 with four characteristicfeatures: (1) a reasonably small size (9.2 kb); (2) an originof replication highly stable in L. monocytogenes; (3) thetrpS gene with its own promoter allowing in vitro and invivo growth of the thus stabilized DtrpS carrier strain at asimilar rate as the wild-type strain; (4) the previouslyreported lysis cassette13 which expresses the phage lysin11819 once the carrier bacteria enter the cytosol of theinfected mammalian cells. Although this phage lysinremains in the cytoplasm of the producing carrierbacteria and does not lyse all intracellular bacteria, itproved to be sufficient for lysis of the intracellularbacteria, particularly in combination with the DaroAmutation.

Bactofection of several nonphagocytic and phagocyticmammalian cells with the optimized L. monocytogenessystem (determined mainly by the expression of theplasmid-encoded EGFP cDNA under the control ofPCMV) was more than 10-fold enhanced compared toour previously described system13 and at least compar-able to other reported bactofection systems.9,14 Bactofec-tion efficacy was highly dependent on the mammaliancell lines used as also observed in other studies9,14 andwas between 5 and 20% for the nonphagocytic cellstested.

Bactofection of phagocytic cell lines, primary murinemacrophages and human DC with this new system wasalso much higher than with our previously reportedsystem,13 but still less efficient than that of thenonphagocytic cell lines. The fact that lipofection ordirect microinjection of the same plasmid into these cellsalso yielded lower transfection rates than into nonpha-gocytic cells (data not shown) suggests that enhanceddegradation or inefficient expression of the introducedDNA may be responsible for the reduced bactofection ofthe phagocytic cells. Nevertheless, efficient antigen(EGFP) presentation together with MHC class I mole-cules was observed in bactofected macrophages asdetermined by the activation of an EGFP-specific CD8T cell line.

Bactofection requires the disruption of the carrierbacteria. Our data obtained by microinjection andinfection of Caco-2 cells with the new vector system

indicate that bactofected cells expressing DsRed or aleishmanial antigen contained only few or no detectableintracellular bacteria, whereas the nonexpressing cellscarried large numbers of bacteria. This suggests thatbacterial cell disruption and subsequent plasmid releaseis an autocatalyzing process probably initiated by thespontaneous lysis of a single bacterial cell which releasesphage lysin (in the case of pSP118) and other peptido-glycan-hydrolyzing enzymes subsequently disruptingmost other carrier bacteria present in the target cell thusresulting in the release of sufficient plasmid DNA towarrant expression of the plasmid-encoded gene by thetarget cell. The observation that microinjection of S.typhimurium carrying the same plasmid (pSP118) yieldsconsiderably fewer EGFP-expressing cells than even theL. monocytogenes DtrpS strain with pSP0 supports thisassumption; S. typhimurium is virtually unable toreplicate in Caco-2 cell cytosol after microinjection23

and hence the amount of plasmid DNA released byspontaneous lysis of these carrier bacteria will be ratherlow and hence EGFP expression less efficient.

A highly interesting observation is the stronglyincreased bactofection rate (defined by the number ofEGFP-expressing mammalian cells) by spreading-com-petent L. monocytogenes carrier bacteria compared toisogenic spreading-deficient ones. This is not solely dueto the larger number of infected target cells obtained byspreading carrier bacteria since the same number of cellsinfected with spreading-deficient bacteria (obtained by ahigher MOI of the latter carrier bacteria) yield still aconsiderably lower number of bactofected cells. Inaddition, microinjection of Caco-2 cells with spreading-competent L. monocytogenes carrying pSP118 showed thatmost EGFP-positive cells were not the microinjected cellsbut cells located in the vicinity of primary microinjectedcells. Such EGFP-positive satellite cells were not ob-served with spreading-incompetent actA mutant carrierbacteria ruling out the possibility that reinfection ofCaco-2 cells by bacteria released from lysed primarymicroinjected cells is responsible for the generation ofthese EGFP-expressing satellite cells. These resultssuggest that spreading L. monocytogenes bacteria (espe-cially when harboring pSP118) are (for yet unknownreasons) more lysis sensitive than bacteria remaining inthe infected target cell.

It seems difficult to imagine a mechanism by which alisterial strain yields the optimal DNA release when ithas (a) to be lysed in the cytosol, (b) to growintracellularly and (c) to spread from cell to cell. Apossible explanation may be that more the bacteria are inthe cytosol, the higher the probability of destructing abacterial cell of this intracellular bacterial populationwhich will release the produced phage lysin. This lysinwill destruct more bacteria thus amplifying the release ofadditional phage lysin and of plasmid DNA. Spreadingbacteria that have to pass again phagosomal compart-ments may become more sensitive to lysis than bacteriathat remain only in the host cells cytosol. Thus wepropose that intracellular listeriae are gradually lysedduring the course of infection of a cell layer. During an invivo infection, all these processes are occurring and theproduction of the phage lysin causes a very efficientdestruction of the carrier bacteria.

Taken together, our results indicate that bactofectionwith the optimized L. monocytogenes vector system is

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highly efficient in transfering eucaryotic expressionplasmids into mammalian cells. In addition to the highDNA delivery capacity, the L. monocytogenes DtrpS carrierstrain harboring pSP118 and the D(trpS aroA) carrierstrain harboring pSP0 are well attenuated in the mousemodel. If the attenuation of this carrier should prove tobe too rigorous under in vivo conditions, we can lower itsattenuation by modulating the expression of the phagelysin within the host cell cytosol by using differentvariants of the actA promoter. This live vector system,which seems to be not only suitable for the release ofDNA but also of RNA (C Schoen et al, unpublishedresults) and of biologically active proteins (J Stritzkeret al, unpublished results), may thus be applicable with-out risk in genetic therapy approaches17,18 and vaccinedevelopment against infectious agents and tumors.31

Experimental protocol

Bacterial strains and plasmidsThe bacterial strains and plasmids used in this work arelisted in Table 1. E. coli DH10b was used as host for allDNA manipulations.

For bactofection, Listeria strains were grown to a latelogarithmic growth phase (180 Klett units resp. 150 Klettunits using the D(trpS aroA) strain) at 371C in BHI,washed with phosphate-buffered saline pH 7.4 (PBS),resuspended in 20% (v/v) glycerol/PBS and stored at�801C.

Plasmid pUNK1 was constructed by amplifyingfragment OriE1 (681 bp) with primers ori1 (50-AAAAAAGAATTCGCCAGCAAAAGGCCAGGA-30)and ori2 (50-AAAAAAGAATTC-ACTGAGCGTCAGACCCCG-30) and pUC18 as template. The fragmentOriE1 and plasmid pIL253 (Gene Bank accession no.AF041239) were cut with EcoRI and ligated, resultingin pUNK1 (5586 bp). To construct the complementa-tion plasmid pTRPS, the trpSþ expression cassettewas amplified using primers trpS1 (50-TGTTATGTCGACTAGTATTTTATG-30) and trpS2 (50-GGTAACGTCGACGTGGAAATT-AAA-30) and genomic DNA ofL. monocytogenes EGD-e as template. After digestionwith SalI, this cassette was cloned into shuttle plasmidpFLO1 (with a tetracycline resistance marker), resultingin pTRPS.

Construction of Listeria mutant strainsThe DtrpS and the D(trpS actA) deletion mutant(called WL-140 and WL-142) were constructed usingL. monocytogenes EGD-e as parental strain. Deletionmutagenesis were performed by a homologous recombi-nation technique using constructs derived from themutagenesis vector pLSV1.24 Primers trpS-A1 (50-AAGAAA-TGTGGATCCGAATTACTATTT-30) and trpS-A2(50-AGTTTACAACCCGGGTTGTCA-ATCACA-30) wereused to amplify a 342 bp fragment which was localizedupstream of the deletion locus and a second down-stream fragment (382 bp) was amplified with the primerstrpS-B1 (50-CGATGTTACCCCGGGTTGCTTTAGAAT-30)and trpS-B2 (50-AATTAGGAG-GAATTCAAAATGAAAAAA-30). Both fragments were digested with PspAI,ligated and further amplified with primers trpS-A1 andtrpS-B2 mentioned above. The double fragment was thencloned into pLSV1 using EcoRI and BamHI restriction

and the resulting knockout plasmid was transformedinto L. monocytogenes EGD-e. After cultivation at 421Cand with 5 mg/ml erythromycin, clones with a chromo-somally integrated knockout plasmid could be selectedwhich were additionally transformed with a trpSþ

expression plasmid (pTRPS) for trans-complementationof the gene. These resulting clones were furthercultivated at 301C without erythromycin to obtain theDtrpS deletion mutant, which has a chromosomaldeletion of the trpS gene but harbors a plasmid-codedtrpSþ expression cassette.

WL-140 (DtrpS) was used as parental strain for theconstruction of the double-mutant strain WL-142which harbors an additional DactA mutation. First,a specific knockout plasmid was made with a simi-lar strategy illustrated above: primers actA-A1 (50-AAAAAAGGATCC-AATCGCTTCCACTCACAGAGG-30)and actA-A2 (50-AAAAAACCCGGGCACTTATACTCCCTCCTCGTG-30) were used to amplify an upstreamfragment (467 bp), and a second 346 bp fragment whichis localized downstream of the actA gene locus wasamplified with primers actA-B1 (50-AAAAAACCCGGGAATAATTAAAAACAC-AGAACG-30) and actA-B2 (50-AAAAAAGAATTCCCTTGAGCTATTTGTTTATCG-30).Both fragments were cut with PspAI, ligated and a largefragment was then obtained by PCR amplification withprimers actA-A1 and actA-B2 using the ligation mixtureas template. The large fragment was introduced intopLSV1 with the same restriction enzymes mentionedabove, resulting in a specific DactA knockout plasmid.After electroporation of this plasmid into WL-140,plasmid integration and excision were executed asdescribed above, resulting in the D(trpS actA) double-mutant strain WL-142.

Cell culture and infection experimentsCaco-2 (human colon adenocarcinoma), COS-1 (monkeyAfrican green kidney), HeLa (human cervix epitheloidcarcinoma) and P388.D1 (murine lymphoid macrophage)were cultured in RPMI 1640 medium supplemented with2 mM L-glutamine (Gibco) and 10% fetal calf serum (FCS,Biochrom, Berlin, Germany); HepG2 (human hepatocytecarcinoma) were grown in Eagle’s minimal essentialmedium with Earl’s salts supplemented with 2 mM L-glutamine, 1% nonessential amino acids, 1 mM sodiumpyruvate, 0.15% sodium bicarbonate (all from Gibco) and10% FCS. All cell lines were maintained at 371C in a 5%CO2 atmosphere. For bactofection, cells were seeded into24-well plates 1 day prior to infection. After a wash stepwith PBS containing 1 mM CaCl2 and 0.5 mM MgCl2

(PBS(Ca2þ/Mg2þ )), 2� 105 cells were infected with anMOI of 5 (if not otherwise indicated) bacteria per cellfor 1 h. The cells were washed three times withPBS(Ca2þ/Mg2þ ) and cultivated with gentamicin-con-taining medium (100 mg/ml) which was replaced withmedium containing 10 mg/ml gentamicin after 1 h.Viable bacterial counts of intracellular bacteria weredetermined by plating serial dilutions of mechanicallylysed cell suspensions on BHI-agar.

Isolation of human DCs from peripheral bloodand infection assayHuman MoDC were prepared from peripheral bloodmononuclear cells (PBMC) as described in detail in

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Kolb-Maurer et al.25 On day 7, cells were transfered to 24-well plates in RPMI supplemented with 3% human auto-logous plasma at a density of 5� 105 cells/ml and bacteriawere added. After incubation for 1 h, medium wasreplaced by RPMI 1640 medium containing 3% humanautologous plasma and 100 mg/ml gentamicin. After 1 h,medium was replaced by RPMI 1640 medium containing3% human autologous plasma and 15 mg/ml gentamicin.

Cell viability assayThe amount of viable cells after infection with bacteriawas measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) (Sigma), followingthe manufacturer’s instructions. At 1, 2 or 3 days afterinfection of cells, medium was replaced by 0.2 ml MTTsolution at a concentration of 2.5 mg/ml MTT dissolvedin RPMI 1640 without phenol red and incubated for 4 hat 371C in a 5% CO2 atmosphere. After removal of theMTT solution, the color reaction was stopped by adding1 N HCl diluted in isopropanol. The probes were measuredat a wavelength of 570 nm. Uninfected cells were used asa reference and were considered as 100% viable.

Flow cytometry analysisAt 1, 2 or 3 days after infection, cells were washed withPBS, trypsinized and resuspended in PBS. Cell viabilitywas determined by staining cells with propidium iodide(PI) (1 mg/ml). Since nonviable cells tend to fluoresce at asimilar wave length as EGFP, PI-positive cells were gatedout from the measurement. A minimum of 5� 104 cells(or 105 phagocytic cells) were then measured using anEpics XL flow cytometer (Beckman Coulter). DC wereadditionally stained with PE anti-human CD83 (BDBiosciences Pharmingen).

Immunofluorescence analysisAccording to a previously described procedure,26 cellswere grown on glass cover slides and infected asdescribed above. After 12 h, the cell layer was washedand fixed as described in Kuhn et al.26 Cells were stainedwith an antiserum against ActA27 or monoclonalantibodies (mAb) directed against kinetoplastid mem-brane protein-11 (KMP-11) (Cedarlane Laboratories) for1 h, followed by a wash step with PBS and afterwardsstained with an FITC- or Lissamine Rhodamine (LRSC)-labelled goat anti-rabbit IgG or (Dianova) for anotherhour. Cells were then pictured with a fluorescence-equipped microscope (Leica DMR HC) and an electroniccamera (Diagnostic Instruments Inc.). Digital imageswere processed using META-MORPH software (Univer-sal Imaging, Media, PA, USA).

Microinjection protocolMicroinjection procedure was exactly performed asrecently described.23 Additionally, bacteria were micro-injected after resuspension with PBS buffer containing0.5 mg/ml Texas red-labelled dextran (70 000 MW, Mole-cular Probes).

T cell lines and antigen presentation assayThe CD8 T cell line specific for the gfp-derived H-2Kd-restricted epitope HYLSTQSAL22 was established fromspleens of DBA/2 mice 3 weeks after immunizationwith 5� 106 P815 cells transfected with the pEGFP-N1vector (according to the instructions supplied by the

manufacturer; Clontech Laboratories, Palo Alto, CA,USA). Spleen cells (15� 106 cells/well) were cultured in24-well plates in 2 ml alpha-modified Eagle’s medium(Gibco) supplemented with 1 mM Hepes, 1 mM gluta-mine, 2� 10�5 M 2-mercaptoethanol, penicillin, strepto-mycin, 10% FCS and 10�9 M synthetic HYLSTQSALpeptide (Jerini Biotools, Berlin, Germany). After 5 daysof culture, 1 ml of medium was exchanged with IL-2medium supplemented with 10 ng/ml murine recombi-nant IL-2 (R&D, Wiesbaden, Germany). CD8 T cell lineswere further restimulated every 3–4 weeks.28 In 2 ml IL-2medium, 0.8� 106 T cells were cultured with 0.4� 106

mitomycin D-inactivated P815/B7 cells (P815 cellstransfected with the human B7.1 gene29) in the presenceof 10�9 M HYLSTQSAL peptide. T cell recognition ofListeria-infected P388.D1 as APC was measured by thedetection of IFN-g in culture supernatants as described.28

Briefly, APC were infected in 96-well flat bottommicrowell plates by 10 min� 200 g centrifugation. After2 h at 371C, infected APC were washed twice and culturemedium supplemented with 10 mg/ml gentamicin wasadded. After 18 h at 371C, cells were fixed for 10 min with1% paraformaldehyde in PBS and after thorough wash-ing, 5� 104 T cells were added to each well. Supernatantswere harvested 12–18 h after addition of T cells and theIFN-g concentration was measured by means of an IFN-g-specific ELISA that binds and detects IFN-g with a pairof specific mAb. Results were corrected for dilution of thesample to yield the sample concentration in ng/ml. Thesensitivity and specificity of T cell lines was monitoredwith APC loaded with graded amounts of the targetpeptide. The detection limit of T cells was between 10�10

and 10�11 M HYLSTQSAL (data not shown).

Infection of animalsAll animal experiments were approved by the govern-ment of Unterfranken and conducted according to theGerman animal protection guidelines. Female BALB/cmice (6–8 weeks old) were purchased from Charles RiverLaboratories, Germany. Mice in groups of five animalswere fed with 109 bacteria via a flexible orogastricfeeding tube. The number of viable bacteria in theinoculum and in liver and spleen homogenates wasdetermined by plating serial dilutions on BHI agarplates. Plates were incubated at 371C and numbers ofcolony forming units (CFU) were counted after 24 h.

Acknowledgements

We thank M Kuhn and B Joseph for the critical readingof the manuscript and Ch Berberich for the kind gift ofKMP-11 cDNA. This work was supported by a grantfrom the Deutsche Forschungsgemeinschaft (Go168/27-1) and the Fonds der Chemischen Industrie. SP thanksthe DFG Graduate College (GK520), and CS The BMBF(IZKF Wurzburg, 01KS9603) for financial support.

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