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Lactococcus lactis Expressing either Staphylococcus aureus 1
Fibronectin-Binding Protein A or Listeria monocytogenes Internalin A can 2
efficiently internalize and deliver DNA in human epithelial cells 3
4
Silvia Innocentin a
, Valeria Guimarães a, Anderson Miyoshi
b, Vasco Azevedo
b, Philippe 5
Langella c Jean-Marc Chatel
c,*and François Lefèvre
a 6
7
a Unité de Virologie et Immunologie Moléculaire, INRA, Domaine de Vilvert, 78352 Jouy en 8
Josas cedex, France 9
b Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG-ICB), Belo 10
Horizonte – MG, Brasil 11
c Unité d’Ecologie et Physiologie du Système Digestif, INRA, Domaine de Vilvert, 78352 Jouy 12
en Josas cedex, France 13
14
* Corresponding author: E-mail: [email protected] 15
Phone: 33 01 34 65 24 68 16
Fax: 33 01 34 65 24 62 17
18
Running title: DNA delivery by Lactococcus lactis FnBPA+ strains 19
20
Key words: Lactococcus lactis, internalin, Staphylococcus aureus, fibronectin binding protein 21
A, Lactobacillus acidophilus, internalization 22
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00825-09 AEM Accepts, published online ahead of print on 29 May 2009
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Abstract 1
Lactococci are non invasive bacteria frequently used as protein delivery vectors and, more 2
recently, as in vitro and in vivo DNA delivery vehicles. We previously showed that a 3
functional eukaryotic eGFP expression plasmid vector was delivered in epithelial cells by 4
Lactococcus lactis producing Listeria monocytogenes Internalin A (LL-InlA+) but this 5
strategy is limited in vivo to transgenic mice and guinea pigs. In this study, we compare the 6
internalization ability of LL-InlA+ and L. lactis producing either the fibronectin-binding 7
protein A of Staphylococcus aureus (LL-FnBPA+) or its fibronectin binding domains C and 8
D (LL-CD+). LL-FnBPA+, and LL-InlA+ showed comparable internalization rates in Caco-2 9
cells while internalization rate observed with LL-CD+ was lower. As visualized by 10
conventional and confocal fluorescence microscopy, big clusters of LL-FnBPA+, LL-CD+ 11
and LL-InlA+ were present in the cytoplasm of Caco-2 cells after internalization. Moreover, 12
the internalization rates of Lactobacillus acidophilus NCFM and of a NCFM mutant strain 13
inactivated for the gene coding for the fibronectin-binding protein (fbpA) were also evaluated 14
in Caco-2 cells. Similar, low internalization rates were observed for both Lb acidophilus 15
NCFM wt and the fbpA mutant, suggesting that commensal fibronectin binding proteins have 16
a role in adhesion but not in invasion. LL-FnBPA+, LL-CD+ and LL-InlA+ were then used 17
to deliver a eukaryotic eGFP expression plasmid in Caco-2 cells: flow cytometry analysis 18
showed that the highest percentage of green fluorescent Caco-2 cells was observed after co-19
culture with either LL-FnBPA+ or with LL-InlA+. Analysis of in vivo efficiency of these 20
invasive recombinant strains is currently in progress to validate their potential as DNA 21
vaccine delivery vehicles. 22
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Introduction 1
The mucosal administration of bacterial carriers to deliver antigens and plasmid DNA 2
constitutes a promising vaccination strategy. Pathogenic bacteria that have the capacity to 3
invade cells, such as Listeria, Salmonella, and Shigella have been used to deliver DNA 4
constructs into mammalian cells (23). Nevertheless, the risk associated with possible 5
reversion to a virulent phenotype of these pathogens is a major concern (5). Lactococcus 6
lactis, the food-grade Gram-positive non-invasive model bacterium, has been intensively used 7
to deliver antigens and cytokines at the mucosal level (30). We previously showed i) that 8
native L. lactis can deliver a eukaryotic expression cassette coding for the bovine β-9
lactoglobulin (BLG), one of the major cow’s milk allergen, into mammalian epithelial Caco-2 10
cells and ii) that these cells were able to express and secrete BLG protein in its native 11
conformation (10). Recently, we demonstrated the ability of native non-invasive L. lactis to 12
deliver in vivo a fully functional plasmid to murine intestinal cells (2). 13
The internalization of the bacterial carrier is a fundamental step to achieve efficient DNA 14
delivery in eukaryotic cells (7). In order to increase DNA delivery by lactic acid bacteria 15
(LAB), invasin genes were expressed in L. lactis. Due to LAB safety profile, recombinant 16
lactococci expressing invasin genes from intracellular bacteria are attractive potential DNA 17
delivery vectors compared to attenuated pathogens presently used. 18
In this field, we previously demonstrated that L. lactis expressing the main Listeria 19
monocytogenes invasin, Internalin A (InlA), were able to invade eukaryotic cells and 20
efficiently deliver a functional GFP expression plasmid into epithelial/endothelial cells (9). 21
Even though attractive, the experimental use of lactococci InlA+ in a mouse model has a 22
major bottleneck: InlA, which binds to human E-cadherin (15), does not interact with murine 23
E-cadherin. Consequently, the in vivo experimental studies using lactococci InlA+ as DNA 24
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delivery vehicles are limited to transgenic mice expressing human E-cadherin or to guinea 1
pigs (13). 2
Fibronectin binding protein A (FnBPA) of Staphylococcus aureus is another bacterial 3
invasin involved in S. aureus intracellular spreading in the host (27). It is a multi-functional 4
adhesion protein gathering both fibrinogen and fibronectin binding capacities (24). Its N-5
terminal part, also called A domain, is responsible for fibrinogen (29) and elastin (20) binding, 6
whereas its C-terminal part including domains B, C, and D binds to fibronectin (25). FnBPA 7
is known to mediate adhesion to host tissue and bacterial uptake into non-phagocytic host 8
cells (27). Its expression by L. lactis was previously shown to be sufficient to confer 9
invasivity into non phagocytic cells in vitro and in vivo, while the expression of the domains 10
C and D confers invasivity only in vitro (19). 11
In this study, we show that L. lactis expressing full-length FnBPA (LL-FnBPA+) or a 12
truncated form encompassing only its C and D domains (LL-CD+) are internalized as 13
efficiently as L. lactis expressing InlA (LL-InlA+) in the human intestinal cell line Caco-2. 14
We also provide for the first time direct microscopic evidences of the intracellular location of 15
the internalized lactococci showing that the bacteria are heterogeneously distributed among 16
the cell monolayer and that their number per cell can reach a surprisingly high number. 17
However, we demonstrate that Fbpa, a fibronectin binding protein from the commensal 18
Lactobacillus acidophilus NCFM, does not mediate bacterial internalization: no difference in 19
invasivity was observed between the wild type strain and the mutant fbpa-. This result 20
indicates that, although widely distributed among bacteria, fibronectin binding proteins are 21
not universal mediators of bacterial internalization, even at low level. Finally, we also 22
demonstrate that, similarly to LL-InlA+, the LL-FnBPA+ and LL-CD+ can efficiently deliver 23
a eukaryotic GFP expression plasmid in Caco-2 cells and trigger GFP expression in these 24
cells. Consequently, LL-FnBPA+ can be used for further DNA delivery experiments in vivo. 25
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Materials and Methods 1
Bacterial strains, plasmids, media and growth conditions. Bacterial strains and plasmids 2
used in this study are listed in Table 1. L. lactis subsp. cremoris strains were grown in M17 3
medium containing 0.5% glucose (GM17) at 30°C. E. coli strains were grown in Luria–4
Bertani medium and incubated at 37°C with vigorous shaking. Lb. acidophilus NCFM was 5
grown in MRS broth at 37°C. Antibiotics were added at the indicated concentrations as 6
necessary: erythromycin, 500 µg/ml for E. coli, and 5 µg/ml for L. lactis and Lb. acidophilus 7
NCFM; chloramphenicol, 10 µg/ml for E. coli and L. lactis. 8
DNA manipulations. DNA manipulations were performed as described previously (21) with 9
the following modifications: for plasmid DNA extraction from L. lactis, TES (25% sucrose, 1 10
mM EDTA, 50 mM Tris–HCl pH 8) containing lysozyme (10 mg/ml) was added for 30 min 11
at 37 °C to prepare protoplasts. Enzymes were used as recommended by suppliers. 12
Electroporation of L. lactis was performed as described (11). Transformants were plated on 13
GM17 agar plates containing the required antibiotic and were counted after 2-day incubation 14
at 30 °C. 15
Invasiveness assays of bacteria into human epithelial cells. Bacterial entry into human 16
epithelial cells was assayed using the human colon carcinoma cell line Caco-2 (ATCC 17
number HTB37), as described (4). Eukaryotic cells were cultured in RPMI supplemented with 18
2 mM L-glutamine (BioWhittaker, Cambrex Bio Science, Verviers, Belgium) and 20% fetal 19
calf serum. Caco-2 cell lines maintained in these conditions without antibiotics were used 20
between passages 9 and 12. The gentamicin survival assay was used to estimate bacteria 21
survival: L. lactis strains were grown to an OD600 of 0.9–1.0, washed, and diluted such that 22
the multiplicity of infection (MOI) was about 103 bacteria per cell. The bacterial suspension 23
was added to mammalian cells grown in P-24 plates (Corning Glass Works). 2 × 105 cells per 24
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well were used. After 1 h of co-incubation, 20 µg/ml of gentamicin was added to kill non 1
internalized bacteria. After 2 h .of gentamicin treatment, cells were washed and lyzed in 0.2% 2
Triton X100 and serial dilutions of the lysate were plated for bacterial counting. Results 3
presented correspond to the average of three independent gentamicin assays done in triplicate. 4
Preparation and analysis of protein extracts by Western blots. L. lactis protein extracts 5
were prepared as previously described (3) from 2 ml of stationary phase cultures (OD600 0.9–6
1.0). Equivalent amounts of protein extracts were resolved in denaturing conditions (SDS–7
PAGE 8%) and transferred onto a nitrocellulose membrane as described previously (16). The 8
membrane was incubated overnight at 4°C in PBS containing 10 % (v/v) non fat dry milk and 9
0.1% Tween 20, then 1 hour at room temperature with a 1/2000 dilution of a rabbit antiserum 10
directed against the D region of FnBPA .The membrane was rinsed and incubated in PBS 11
containing non fat dry milk (5% v/v) in PBS, 0.1% Tween 20, and horseradish peroxidase 12
(HRP) conjugated secondary antibody (P.A.R.I.S) for 1 hour at room temperature. Signals 13
were detected using an enhanced chemiluminiscence kit (Amersham Bioscience). 14
Analysis of bacterial internalization by confocal microscopy. 2 ml of stationary phase 15
cultures of L. lactis, LL-FnBPA+, LL-CD+, LL-InlA+ were washed twice in PBS and stained 16
with the green fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) at 50 µM for 20 17
min at 37°C under constant shaking in the dark (14). Bacteria were washed in PBS and used 18
to perform invasiveness assay in Caco-2 cells grown as described above on 10 µg/ml poly-D-19
lysine (PDL, Sigma-Aldrich) pre-treated glass coverslips. After 1 h of infection and 2 h of 20
incubation in the presence of gentamicin (20 µg/ml), cells were fixed 20 min with 21
paraformaldehyde (2.5% in PBS), permeabilized with Triton-X100 (0.1%) for 5 min at RT 22
and actin cytoskeleton was stained with Alexa Fluor®
594-labelled phalloidin (Invitrogen) in 23
PBS containing BSA (1%), for 20 min at RT. Samples were mounted with Fluoromount G 24
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medium (Interchim) and the images were captured by using a Zeiss LSM 510 META inverted 1
confocal laser scanning microscope equipped with an argon and a helium–neon lasers (for 2
double fluorescence at 488 and 543 nm) and with a Zeiss 63 X Plan-Apachromat lens. A 3
composite picture of 39 to 44 sections (0.41 µm or 0.8 µm apart in the z axis) of each sample 4
was collected and analyzed using the Zeiss LSM Image Browser software (version 4.2). 5
Analysis of bacterial internalization by conventional immunofluorescence microscopy. 6
An antiserum directed against L. lactis was prepared by immunizing a rabbit with bacterial 7
wall extracts. Specific response against bacteria was checked using an agglutination test and 8
indirect immunofluorescence as described below. Caco-2 cells were grown on glass 9
coverslips pre-treated with poly-D-lysine. Invasiveness assay with CFSE stained L. lactis was 10
performed as described above. After 1 h of infection and 2 h of incubation in the presence of 11
gentamicin (20 µg/ml), cells were fixed with 2.5% paraformaldehyde in PBS. Non 12
internalized L. lactis were visualized using the rabbit anti-L. lactis serum (diluted 1/100) as 13
primary antibody followed by an Alexa Fluor®
594-labelled goat anti rabbit immunoglobulin 14
(Invitrogen) as secondary antibody. The nuclei were stained with 4,6-diamidinophenylindole 15
(DAPI). Samples were mounted as described above and analyzed with a Zeiss AXIOVERT 16
200M inverted fluorescence microscope equipped with a 20 X Zeiss LD A-Plan phase 17
contrast lens and a CoolSNAP HQ camera (Roper Scientific). For each field observed, a 18
phase contrast image and fluorescence images in the blue, red and green channels were 19
acquired and processed using MetaVue (version 6.3, Molecular Devices) and ImageJ (version 20
1.38, http://rsb.info.nih.gov/ij/) softwares. 21
Analysis of GFP expression by Caco-2 cells using flow cytometry. The gfp ORF under 22
control of the eukaryotic immediate early promoter of the human Cytomegalovirus (IE 23
HCMV promoter) was used to demonstrate the potential of L. lactis to deliver a functional 24
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gene into the Caco-2 mammalian cell line. L. lactis InlA+GFP, L. lactis FnBPA+GFP, L. 1
lactis CD+GFP and L. lactis GFP were grown to an OD600 of 0.9–1.0 and bacteria were 2
added to cells at a MOI of 103. Internalization assays with these strains were performed as 3
described above. After 1h or 3 h of infection, cells were incubated in RPMI medium with 4
gentamicin (20 µg/ml) for 2 hours. Cells were collected at 24, 48 and 72 h, rinsed with PBS 5
and fixed (Cell fix, BD Biosciences). Quantification of fluorescent cells was performed using 6
a FACSCalibur flow cytometer and the CellQuest Pro software (Becton Dickinson). For each 7
interaction experiment, 500,000 cells were analyzed. Assays were performed in triplicates. 8
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Results 1
Production of FnBPA and its CD domains by recombinant L. lactis strains. We first 2
checked the production of full-length FnBPA and its CD domains by LL-FnBPA+ and LL-3
CD+ strains, respectively. Western blot analysis was performed using a polyclonal antiserum 4
specific for the D domain of FnBPA. Neither FnBPA, nor CD form were detected in L. lactis 5
wild type (LL-wt; negative control strain) (Fig 1; lane 1). However, proteins with the 6
expected size were clearly detected for LL-FnBPA+ (Fig. 1, lane 2) and LL-CD+ (Fig. 1, lane 7
3). 8
9
L. lactis strain producing FnBPA internalizes in Caco2 cells as efficiently as the same 10
strain expressing InlA. The ability of LL-FnBPA+, LL-CD+ and LL InlA+ strains to invade 11
Caco-2 cells was evaluated using the classical gentamicin survival assay that allows global 12
quantification of viable intracellular bacteria. Figure 2 clearly shows that LL-FnBPA+ and 13
LL-InlA+ displayed similar internalization rates in Caco-2 cells whereas LL-CD+ internalized 14
less efficiently than LL-FnBPA+. 15
16
Microscopic visualization of internalized L. lactis producing InlA, FnBPA and CD 17
reveals cytoplasmic bacterial clusters unevenly distributed in Caco-2 cells. To confirm 18
our previous result and to determine more precisely the fate and cellular location of the 19
different Lactococcus strains after internalization in Caco-2 cells, we tried to localize bacteria 20
inside the cells using fluorescence microscopy techniques. Bacteria were stained with the 21
green vital fluorescent dye CFSE. After staining, the 4 different strains (LL-FnBPA+, LL-22
CD+, LL-InlA+ and control LL-wt) were incubated 1 hour with Caco-2 cells seeded at low 23
density to obtain small isolated islets of cells. Non-internalized bacteria were then killed by 24
treatment with gentamicin. 25
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We first used conventional fluorescence microscopy to visualize in situ internalized bacteria 1
(Fig. 3). Cell monolayers were thus fixed without permeabilization and non-internalized, 2
extracellular bacteria were specifically stained by indirect immunofluorescence using an anti-3
L. lactis serum and a red fluorophore. Consequently, on 3 colors (green, red and blue) overlay 4
photographs, doubly stained non-internalized bacteria appeared orange-yellow and single 5
stained intracellular bacteria appeared green. After incubation with LL-wt, almost no 6
intracellular bacteria could be detected. In contrast, intracellular bacteria were detected in the 7
cytoplasm of Caco-2 cells after co-culture with the three invasive lactococci strains. They 8
were not evenly distributed in the cell islets but preferentially detected in cells located at the 9
periphery of the islets where they form intracytoplasmic clusters that seem to contain up to 10
several tens of bacteria. This heterogeneous location suggests that InlA and FnBPA receptors 11
are more accessible at the periphery of the islets. Moreover, intracellular bacteria appeared 12
more homogeneously distributed for the LL-InlA+ strain. Non-internalized bacteria could be 13
observed almost exclusively outside the cellular islets or attached at the surface of the cells. 14
To eliminate the possibility of an artifact of the detection method, we confirmed these results 15
by confocal microscopy. To clearly visualize intracellular CFSE-stained bacteria, Caco-2 cells 16
were stained for cytoplasmic filamentous actin with phalloidin labeled with a red fluorophore. 17
Virtual z-sections generated by confocal analysis (Fig. 4) clearly showed that co-culture of 18
Caco-2 cells with the 3 internalizing lactococci strains results in the formation of big clusters 19
of internalized bacteria in Caco-2 cells. In contrast, neither intracellular nor cell associated 20
bacteria could be detected after co-culture with LL-wt. 21
22
Fibronectin binding protein of Lactobacillus acidophilus NCFM does not mediate 23
internalization in Caco-2 cells. Fibronectin binding proteins are widely distributed among 24
bacteria, including LAB such as lactococci and lactobacilli, in which they have a clear role in 25
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adhesion to epithelial cells (1). In order to test whether fibronectin binding could represent a 1
more general mean of bacterial entry into cells, we tested whether such protein from the 2
commensal Lactobacillus acidophilus NCFM could be involved, even at a very low level, in 3
internalization. We thus compared the invasiveness rates in Caco-2 cells of the wild type 4
strain and one mutant strain deleted in the gene encoding fibronectin-binding protein FbpA 5
(fbpA-). The LL-wt (which also carries one FbpA encoding gene) and our recombinant 6
invasive strains (LL-FnBPA+ and LL-InlA+) were also included in this comparison assay. As 7
shown in Figure 5, LL-wt, Lb. acidophilus NCFM and Lb. acidophilus fbpA- showed very 8
similar internalization rates, which were significantly lower than the rates obtained with LL-9
FnBPA+ or LL-InlA+. This showed that Lb. acidophilus FbpA protein, despite its well 10
documented role in adhesion, does not confer invasive properties to this bacterium. Even if 11
there is “natural” background internalization of Lb. acidophilus in Caco2 cells, it is not 12
mediated by the fbpa gene product. 13
14
Efficient in vitro delivery of a eukaryotic expression vector by L. lactis strains expressing 15
FnBPA. To demonstrate that FnBPA and its CD domains can mediate in vitro gene delivery 16
when produced by L. lactis and to compare DNA delivery ability between the different 17
invasive strains, we transformed LL-FnBPA+, LL-CD+, LL-InlA+ and non invasive strains 18
LL-wt, with pValac:GFP, a plasmid carrying a GFP eukaryotic expression cassette (8). The 19
resulting strains were called LL-FnBPA+GFP, LL-CD+GFP, LL-InlA+GFP and LL-GFP, 20
respectively (Table 1). Caco-2 cells were co-cultivated with these strains for 1 or 3 hours and 21
the percentage of Caco-2 cells expressing GFP was monitored by flow cytometry after 24, 48 22
and 72 hrs of gentamicin treatment. Almost no GFP expression could be detected with non-23
invasive strain (Fig. 6A). The one hour incubation condition elicited a transitory GFP 24
expression with a maximum at 48 hrs after gentamicin treatment (Fig. 6B). After 3 hours of 25
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co-cultivation, the percentage of Caco-2 cells expressing GFP reached a maximum 24 hrs 1
after gentamicin treatment and then remained constant when strains LL-FnBPA+GFP and LL-2
InlA+GFP were used. When strain LL-CD+GFP was used, GFP expression reached its 3
maximum only 48 hrs after gentamicin treatment. The percentage of Caco-2 cells expressing 4
GFP was the same 3 days after gentamicin treatment with the three invasive strains used. 5
These results clearly demonstrate that i) L. lactis strain expressing either full-length FnBPA or 6
its CD domains can efficiently mediate in vitro intracellular delivery of a functional 7
eukaryotic expression plasmid, and ii) the highest percentage of green fluorescent Caco-2 8
cells was obtained after co-culture with LL-FnBPA+GFP or LL-InlA+GFP strains. 9
10
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12
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Discussion 1
LAB represents an attractive alternative to attenuated pathogenic bacteria as DNA delivery 2
vehicles. We demonstrated previously for the first time that a fully functional plasmid can be 3
transferred with low efficiency from a LAB transiting in the gut to intestinal cells: the oral 4
administration of L. lactis strains carrying a plasmid containing a eukaryotic BLG expression 5
cassette elicited transitory BLG production by the epithelial cells of the intestinal wall. Mice 6
treated with this non invasive strain developed a TH1 BLG specific immune response and 7
were protected from further sensitization (2). 8
To improve plasmid DNA delivery by lactococci, we developed lactococcal invasive 9
strains like LL-InlA+ (9) whose internalization is mediated through the binding of InlA to E-10
cadherin expressed on epithelial/endothelial cells (15). This interaction is species specific 11
since InlA interacts with the human E-cadherin, but not with the homolog of some rodents, 12
including mouse (12). This observation excludes mouse as an experimental model to study 13
InlA mediated DNA delivery improvement in vivo, although it was recently reported that two 14
single substitutions in InlA increased binding affinity to formerly incompatible murine E-15
cadherin by four orders of magnitude extending its binding specificity (31). This recent 16
finding opens the possibility to express this mutated InlA in lactococci and to use them in 17
conventional mice. As this limitation does not exist in guinea pig, we used this animal model 18
in our previous study to demonstrate the ability of LL-InlA+ to invade intestinal cells in vivo 19
(7). 20
In order to explore the potential use of other bacterial internalins allowing further animal 21
experiments for mucosal DNA delivery, we analyze here the binding and internalizing 22
properties of S. aureus FnbpA and its ability to confer DNA delivery properties to L. lactis. 23
This invasin would be more suitable for further in vivo studies because it has many binding 24
regions and it could be used in a wider range of animal models. Here, we show that cell wall 25
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anchored full length FnBPA promotes the entry of L. lactis into Caco-2 cells as efficiently as 1
InlA and confers to the bacteria very similar plasmid DNA delivery properties, as judged by 2
its ability to transfer a pValac:GFP plasmid in Caco-2 cells to elicit GFP expression. 3
Moreover, we provide microscopic evidences of FnbpA- and InlA-mediated L. lactis 4
internalization that raise interesting questions about the process of internalization of these 5
genetically modified bacteria. 6
Staphylococcus aureus fibronectin-binding protein A (FnBPA) was the first surface protein of 7
bacterial pathogens shown to be sufficient to confer invasivity to L. lactis into human 293 8
embryonic kidney cell line (27). In our study, we confirm in the intestinal human Caco2 cell 9
line that full-length FnBPA and its CD domain can mediate L. lactis internalization. 10
Moreover, we compare the internalization and DNA delivery ability of L. lactis expressing S. 11
aureus FnBPA or CD and L. lactis expressing L. monocytogenes InlA. The rates of 12
invasiveness of LL-InlA+ and LL-FnBPA+ strains determined after the gentamicin treatment 13
were similar and about 1,000-fold higher than the one obtained with L. lactis wt strain. In 14
contrast, LL-CD+ exhibited a rate of invasiveness significantly lower than LL-FnBPA+ one. 15
In order to explore the possibility that bacterial fibronectin binding protein, especially those 16
from non pathogenic bacteria, could be more widely involved in bacterial internalization, we 17
analyzed whether the Fbpa protein from the non pathogenic commensal organism Lb. 18
acidophilus NCFM could mediate a “natural” low level internalization, as we previously 19
showed for L. lactis (9, 10). It would have been subsequently possible to improve this 20
internalization by constructing recombinant strains overexpressing this protein. However, our 21
results with fbpa- mutant indicate that if this background internalization exists, it is not 22
mediated by Fbpa but by other proteins. Thus, fibronectin binding proteins are not universal 23
promoters of bacterial internalization and those from pathogenic bacteria probably trigger 24
specific signals in host cell necessary for efficient entry. This suggests that two different 25
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classes of fibronectin binding proteins can be distinguished: those from pathogenic bacteria 1
conferring both cell adhesion and internalization and those from either food-grade or 2
commensal bacteria conferring only cell adhesion. 3
One of the two main original interests of our paper is i) to confirm internalization data by 4
direct visualization of recombinant lactococci expressing InlA and FnBPA inside the cells 5
using conventional and confocal microscopy and ii) to analyze precisely their cellular and 6
subcellular location after internalization in vitro, which was never done before. In previous 7
papers (9, 10, 27), the internalization process was evaluated using only an antibiotic-8
resistance assay similar to the one used in the present study. However, this assay suffers some 9
bias: when a high MOI is used, bacteria can form a biofilm at the surface of the cells and 10
remain protected from the antibiotic treatment although being not internalized. The 11
visualization experiments performed in our work clearly discard this artifact. Moreover, they 12
provide interesting information about the localization and number of bacteria inside the Caco2 13
cell islets. Our images clearly show that both invasins drive lactococci to a similar 14
cytoplasmic location in Caco2 cells. The preferential distribution of internalized bacteria at 15
the periphery of the Caco-2 cells islets can be explained by the fact that E-cadherin and α5β1 16
integrins (15, 28), the respective InlA and FnbpA receptors, are accessible at the periphery but 17
not at the center of the Caco-2 cells islets. However, the surprisingly high number of bacteria 18
detected in the cells in which internalization occurred is a point that remains difficult to 19
explain. Two non exclusive mechanisms could be involved: 1) massive internalization of 20
several tens of bacteria in a single cell through their binding to fibronectin or E-cadherin 21
leading to the formation of a complex subsequently internalized by the eukaryotic cells as a 22
single unit and 2) intracellular multiplication of few internalized bacteria. Although the latter 23
possibility appears less probable, as the time left after the beginning of the gentamicin 24
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treatment is probably too short to allow lactococci multiplication under the unfavorable 1
intracytoplasmic environment, further work is required to clarify the mechanisms involved. 2
Another important point of our work is to demonstrate for the first time that L. lactis 3
expressing FnBPA can efficiently deliver a eukaryotic expression plasmid with subsequent 4
protein expression thus indicating that this bacterial invasin can be used for plasmid delivery. 5
It is known that FnBPA increases both L. lactis adherence to immobilized fibronectin and 6
infectivity in experimental endocarditis to levels similar to those observed for S. aureus, thus 7
identifying FnBPA as a critical virulence factor in endovascular infection (18). This raises the 8
question of the safety of Lactococcus strains expressing this protein. A previous study 9
showed that L. lactis expressing FnBPA or its CD domains can cause valvular lesions in rats. 10
However, this occurs only when lactococci are injected by the parenteral route to animals that 11
have preexisting valvular lesions. The conditions used in mucosal immunization, i.e. local 12
administration at a mucosal surface of a healthy animal cannot result in a massive release of 13
recombinant bacteria in the bloodstream and can consequently be considered as much safer. 14
Flow cytometry analysis indicates that around 1% of Caco-2 cells expressed GFP after co-15
incubation with the FnBPA expressing invasive strains bearing the pValac:GFP plasmid 16
while no fluorescence was detectable with the non-invasive strain. These results prove the 17
ability of these invasive strains to transfer functional plasmids in Caco-2 cells as efficiently as 18
those expressing InlA. Consequently, these results indicate that L. lactis expressing FnBPA 19
represents a promising tool for mucosal DNA delivery that will be tested in vivo in future 20
experiments using the mouse model. 21
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Acknowledgements. 1
We are very grateful to Dr. Philippe Moreillon (Department of Fundamental 2
Microbiology, University of Lausanne, Switzerland) for providing us with the L. lactis strains 3
expressing S. aureus FnBPA and its derivatives. We also thank Dr. Todd Klaenhammer 4
(Department of Food, Bioprocessing, and Nutrition Sciences, North Carolina State University, 5
USA) for the Lactobacillus acidophilus NCFM strain and its fbpA mutant, and Professor 6
Martin McGavin (Department of Laboratory Medicine and Pathobiology, University of 7
Toronto, Toronto, Canada) for the anti-FnBPA antiserum. We are very grateful to Dr. René 8
L’Haridon (INRA Jouy-en-Josas) for the preparation and testing of the anti-L. lactis serum 9
and to Mrs. Céline Urien (INRA Jouy-en-Josas) for confocal microscopy analyses that were 10
performed at the MIMA2 microscopy platform of the INRA Jouy-en-Josas research centre. 11
Valeria Guimaraes received grants from the Region of Ile-de-France and Animal Health 12
Division of INRA (convention # E1511). Silvia Innocentin is a recipient of a European PhD 13
Marie Curie grant from the LABHEALTH program (MEST-CT-2004-514428). 14
15
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Figure legends 1
2
Figure 1. Detection of S. aureus Fibronectin binding protein A (FnBPA) or its CD 3
domains in L. lactis by Western blot analysis. Cell extracts from cultures of recombinant L. 4
lactis producing either S. aureus fibronectin binding protein A (FnBPA) or CD domains were 5
analyzed by Western blot using a rabbit antiserum against the D domain of S. aureus FnBPA. 6
Lane 1, LL-wt; lane 2, LL-FnBPA+; lane 3, LL-CD+. 7
8
Figure 2. Invasiveness assays on Caco-2 cells of LL-FnBPA+, LL-CD+ and LL-InlA+. 2 9
× 105 Caco-2 cells were used for each assay. LL-wt: L. lactis used as negative control; LL-10
FnBPA+: L. lactis strain producing the native S. aureus FnBPA; LL-CD+: L. lactis strain 11
producing the CD domains of S. aureus FnBPA; LL-InlA+:L lactis strain producing the native 12
L. monocytogenes InlA. Results are mean ± standard deviation of triplicates. Bars represent 13
standard deviations, asterisk represents survival rates significantly different from the one 14
obtained for LL-wt one and # represents statistically comparable survival rates (t-student, 15
p<0.05). The results presented are from one experiment representative of three performed 16
independently. 17
18
Figure 3. L. lactis internalization in Caco-2 cells analyzed by conventional fluorescence 19
microscopy. The CFSE stained (green) bacteria (LL-wt, LL-FnBPA+, LL-CD+ and LL-20
InlA+) were co-incubated with Caco-2 cells for 1 hour to perform internalization and fixed 21
without permeabilization. Extracellular, non-internalized bacteria were stained with Alexa 22
Fluor®
594 (red) by indirect immunofluorescence using an anti-L. lactis serum. Nuclei were 23
stained with DAPI (blue). The samples were analyzed by conventional fluorescence 24
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microscopy. Each panel represents the overlay of the inverted phase contrast image of the 1
field and of the three fluorescence images acquired in the blue, red and green channels. 2
Intracellular bacteria appear in green and extracellular bacteria in orange to yellow. Big 3
clumps of intracellular bacteria are clearly visible with internalizing strains. Bar, 50 µm. 4
5
Figure 4. L. lactis internalization in Caco-2 cells analyzed by confocal microscopy. The 6
CFSE stained (green) bacteria (LL-wt, LL-FnBPA+, LL-CD+ and LL-InlA+) were co-7
incubated with Caco-2 cells for 1 hour to perform internalization and the actin cytoskeleton 8
was stained by phalloidin (red) after fixation and permeabilization. The fluorescent samples 9
were analyzed by confocal microscopy. Each panel represents a section from the stack on the 10
z axis appropriately chosen to visualize both extracellular and intracellular bacteria. For each 11
field, two three-dimensional reconstruction sections perpendicular to the plane of the 12
monolayer and parallel to the x and y axis are shown on the lower part (x–z section, green line) 13
and right part (y–z section, red line) of each panel, respectively. Clumps of several tens of 14
bacteria per Caco-2 cell (white arrows) are clearly visible with internalizing strains. Bar, 20 15
µm. 16
17
Figure 5. Invasiveness assays on Caco-2 cells of Lb. acidophilus NCFM, Lb. acidophilus 18
NCFM fbpA-, LL-FnBPA+ and LL-InlA+. 2 × 105 Caco-2 cells were used in each assay. 19
LL-wt: L. lactis used as negative control; LL-FnBPA+: L. lactis strain expressing the full-20
length S. aureus FnBPA gene; Lb. acidophilus NCFM: Lb. acidophilus NCFM wt; Lb. 21
acidophilus NCFM fbpA-: Lb. acidophilus NCFM deleted in gene encoding fibronectin-22
binding protein; LL-InlA+: L. lactis strain expressing the native L. monocytogenes inlA gene. 23
Results are mean ± standard deviation of triplicates.. Bars represent standard deviations and # 24
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represents statistically comparable survival rates (t-student, p<0.05). The results presented are 1
from one experiment representative of three performed independently. 2
3
Figure 6. In vitro gene transfer assessment following invasion of Caco-2 cells with L. 4
lactis strain carrying pValac:GFP plasmid. The percentage of GFP expressing Caco-2 cells 5
was determined by flow cytometry after co-incubation 1 hour or 3 hours with LL-wt, LL-6
FnBPA+, LL-CD+, and LL-InlA+, previously transformed with pValac:GFP. The percentages 7
of GFP expressing Caco-2 cells were quantified by flow cytometry after 24, 48 and 72 hours 8
following co-incubation using the positivity threshold indicated on the FL1/FSC dot-plots 9
(Fig. 5A). The values obtained were reported on the two graphs of Fig. 5B for a better 10
comparison. The flow cytometry assays shown here are from one experiment representative of 11
three performed independently. 12
13
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Table 1 1
Strains or Plasmids Properties Reference
L. lactis InlA+ L. lactis strain expressing the inlA gene Guimarães et al.,
2005(9)
L. lactis FnBPA+ L. lactis strain expressing the fnbpA gene Que et al., 2001(17)
L. lactis CD+ L. lactis strain expressing the C and D
domains of fnbpA gene
Que et al., 2005(19)
L. lactis wt L. lactis strain carrying pIL253 plasmid Simon & Chopin,
1988(26)
L. lactis GFP L. lactis strain carrying pIL253 and
pValac:GFP plasmid
This study
L. lactis InlA+ GFP L. lactis strain expressing inlA gene and
carrying pValac:GFP
This study
L. lactis FnBPA+ GFP L. lactis strain expressing fnbpA gene and
carrying pValac:GFP
This study
L.lactis CD+ GFP L. lactis strain expressing the C and D
domains of fnbpA gene and carrying
pValac:GFP
This study
pIL253 High-copy number lactococcal vector, Eryr Simon & Chopin,
1988(26)
pGM10 pUC19/pAT19 derivatives carrying inlA
promoter and inlA gene of L.
monocytogenes, Eryr
Guimaraes et al.,
2005(9)
pOri23-FnBPA L. lactis-E. coli shuttle vector carrying
fnBPA gene from S. aureus, Eryr
Que et al., 2001(17)
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pOri23-CD L. lactis-E. coli shuttle vector carrying
sequence coding for the C and D domains
of FnBPA from S. aureus, Eryr
Que et al., 2005(19)
pValac:GFP L. lactis-E. coli shuttle vector carrying the
gfp gene under the control of the
eukaryotic promoter CMV, Cmr
Guimarães et al.,
2009(8)
pTRK833 E. coli/ Lb acidophilus NCFM shuttle vector
734-bp internal region of fbpA (LBA1148)
cloned into BglII-XbaI sites of pORI28 Eryr
Buck et al., 2005(1)
Lb. acidophilus NCFM
fbpA
NCFM mutant inactivated in fbpA gene Buck et al., 2005(1)
Lb. acidophilus NCFM Human intestinal isolate Sanders &
Klaenhammer,
2001(22)
L. lactis MG1363 Wild type strain Gasson, 1983(6)
Eryr, Erythromycin; Cm
r, Chloramphenicol 1
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39
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1 2 3
FnBPA f. l. 173
117
76
51
38
26
CD
kD
Figure 1
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1
2
3
4
5
LL wt LL- FnBpA+ LL-CD+ LL-InLA+
Lo
g C
FU *
**
* #
#
Fig 2
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L. lactis-CD+
L. lactis wt
L. lactis-InlA+
L. lactis-FnBPA+
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L. lactis wt L. lactis-FnBPA+
L. lactis-CD+ L. lactis-InlA+
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Figure 5
1
2
3
4
5
L. lactis Lb. acido.NCFM
Lb. acido.NCFMFbpA-
LL-FnBPA LL-InlA
Lo
g C
FU
#
# #
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0.01%
0.62%
0.40%
0.27%
0.02%
1.17%
0.77%
0.99% 0.28%
0.57%
0.88%
0.02% 0.03%
1.10%
0.89%
0.94%0.98%
0.80%
0.94%
0.02%0.02%
0.84%
0.49%
0.97%
1 hour of co-incubation 3 hours of co-incubation
LL-GFP
LL-FnBPA+ GFP
LL-CD+ GFP
LL-InlA+ GFP
24h 48h 72h 24h 48h 72h
Hours of expression Figure 6A
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Figure 6B
0.0
0.5
1.0
1.5
24 48 72 24 48 72 24 48 72 24 48 72
% o
f G
FP
+ c
aco
-2 c
ells A
B
0.0
0.5
1.0
1.5
24 48 72 24 48 72 24 48 72 24 48 72LL-GFP LL-FnBPA+
GFP
LL-CD+
GFP
LL-InlA+
GFP
% o
fG
FP
+ c
ac
o-2
ce
lls
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