Lactobacillli expressing llama VHH fragments neutralise Lactococcusphages
© Hultberg et al; licensee BioMed Central Ltd. 2007
Received: 10 May 2007
Accepted: 17 September 2007
Published: 17 September 2007
Bacteriophages infecting lactic acid bacteria (LAB) are widely acknowledged as the main cause of milk fermentation failures. In this study, we describe the surface-expression as well as the secretion of two functional llama heavy-chain antibody fragments, one binding to the major capsid protein (MCP) and the other to the receptor-binding proteins (RBP) of the lactococcal bacteriophage p2, by lactobacilli in order to neutralise lactococcal phages.
The antibody fragment VHH5 that is directed against the RBP, was fused to a c-myc tag and expressed in a secreted form by a Lactobacillus strain. The fragment VHH2 that is binding to the MCP, was fused to an E-tag and anchored on the surface of the lactobacilli. Surface expression of VHH2 was confirmed by flow cytometry using an anti-E-tag antibody. Efficient binding of both the VHH2 and the secreted VHH5 fragment to the phage antigens was shown in ELISA. Scanning electron microscopy showed that lactobacilli expressing VHH2 anchored at their surface were able to bind lactococcal phages. A neutralisation assay also confirmed that the secreted VHH5 and the anchored VHH2 fragments prevented the adsorption of lactococcal phages to their host cells.
Lactobacilli were able to express functional VHH fragments in both a secreted and a cell surface form and reduced phage infection of lactococcal cells. Lactobacilli expressing llama heavy-chain antibody fragments represent a novel way to limit phage infection.
Llamas, a member of the Camelidae family, produce heavy chain antibodies, a type of antibodies that lack the CH1 domain and light chains . The antigen binding portion of these antibodies, called VHH, can be expressed at high levels in Saccharomyces cerevisiae . VHH antibody fragments have already shown a considerable potential in several biotechnological applications such as decreasing the amount of smooth surface caries in a rat model , shortening disease duration, severity and viral load in a mouse model of rotavirus-induced diarrhea , and preventing phage infection of Lactococcus cells during milk fermentation [5, 6].
Virulent bacteriophages infecting lactic acid bacteria (LAB) are widely acknowledged as the main cause of milk fermentation failures and they are also responsible for the downgrade of fermented dairy products such as cheeses [7, 8]. Their ubiquity in dairy environments, biodiversity, and genomic plasticity are largely responsible for the difficulty in controlling phage infection [9, 10]. Consequently, several tactics have been proposed to curtail their proliferation in industrial settings . The generation of phage neutralising VHH antibodies is one of the latest antiviral strategies that have been proposed to inhibit lactococcal phages [5, 6]. As a proof of concept, a panel of non-neutralising and neutralising VHH antibody fragments targeting the lactococcal isometric-headed 936-type phage p2, was recently obtained . The direct addition of one of them (VHH5) to milk prevented the infection of the strain Lactococus lactis subsp. cremoris C2 by the virulent phage p2 during the manufacture of a Gouda-type cheese . The VHH5 fragment effectively inhibited lactococcal phage infection by directly binding to the receptor-binding protein (RBP/ORF18) located at the distal part of the phage tail . Recently, it was shown that other phages belonging to the predominant lactococcal 936 species, could also be neutralised by this antibody . Moreover, some of the non-neutralising fragments, such as VHH2, were shown to bind to the major structural capsid protein (ORF11) of phage p2 .
Lactobacilli are also Gram-positive lactic acid bacteria that normally colonize the oro-gastrointestinal tract [12, 13]. Some Lactobacillus strains are believed to have health promoting properties and are used as supplements in dairy products, either alone or in combination with other microorganisms [14, 15]. Similarly to Lactococcus lactis strains, other carefully selected Lactobacillus strains are an integral part of industrial starter cultures that are added to milk for the manufacture of an array of fermented dairy products. Thus, their large-scale used in the food industry is well established and their long history of safe use has led to their status as a Generally Regarded As Safe (GRAS) microorganism. This GRAS status has led to reports in which lactobacilli were suggested as carriers for passive immunization through surface expression or secretion of various antibodies . Recently, functional antibody fragments targeting pathogenic bacteria (Streptococcus mutans and Porphyromonas gingivalis) and a human virus (rotavirus) have been produced in lactobacilli [4, 16–18] and shown to have an antimicrobial potential.
In this study, we have explored the possibility of producing functional VHH antibody fragments by lactobacilli in order to neutralise lactococcal phages. The in situ VHH production, in a secreted form or anchored to the cell surface, could potentially alleviate the need to add the VHH fragments directly to the fermentation medium, thereby reducing the costs of the technology.
Results and discussion
Construction of Lactobacillus paracasei strains expressing VHH fragments that bind to structural proteins of Lactococcus lactisphage p2
The presence of pLP402-VHH5-secreted in L. paracasei was found to mediate the secretion of VHH5 into the medium (Fig 1A). The introduction of pLP401-VHH2-anchored into L. paracasei led to the cell surface expression of VHH2 by fusion to the C-terminal cell wall anchored domain of proteinase P (Fig. 1B). In these vectors, the VHH expression is under the control of the promoter of the amy gene (Fig. 1). This regulatable promoter is repressed by sugars transported by the phosphoenolpyruvate-dependent phosphotransferase systems (PTS). De-repression of the promoter and protein expression is obtained by growing the cells in the presence of non-PTS sugars such as mannitol. Both VHH fragments were tagged for detection with anti-E-tag or anti-myc-tag antibodies. The theoretical molecular mass of the anchored VHH2 and secreted VHH5 is 43 kDa and 23 kDa respectively after cleavage of the signal peptide (4 kDa) but containing the N-terminus (26 amino acids, 3.9 kDa) of the amylase protein fused to the VHH (Fig. 1).
Expression of the VHH fragments
Using purified VHH5 and VHH2 fragments as positive controls, we also estimated that the L. paracasei strain containing the vector pLP401-VHH2-anchored expressed about 103–104 molecules per cell (calculated from the VHH standard) while the L. paracasei strain carrying pLP402-VHH5-secreted, grown to an OD600 of 0.8, expressed about 500 ng of VHH fragments per ml of supernatant, corresponding to a production rate of roughly 5 × 104 VHH fragments/bacterium/hour.
Binding of the expressed VHH fragments to phage p2
Binding of the expressed VHH fragments to phage p2 antigens by ELISA
Neutralisation of phage p2 by VHH2 and VHH5 expressed in lactobacilli
Percentage of phage p2 inhibition by bacterially produced VHH
L. paracasei parental
1.8 ± 5.9
2.1 ± 1.0
L. paracasei + pLP402
4.6 ± 4.0
-4.4 ± 6.4
L. paracasei + pLP402-VHH5-secreted
86.0 ± 5.1
L. paracasei + pLP402-VHH2-anchored
3.4 ± 6.6
31.4 ± 2.8
It was previously shown that VHH5 fragments bind to some but not all 936-like phages , which is the most prevalent lactococcal phage group in the dairy industry [5, 9]. Moreover, it was recently shown that phage mutants no longer neutralised by VHH5 could be readily isolated in the laboratory . Further strategies are thus needed to improve the broadness of the VHH protection and to prevent the emergence of new virulent phages. One possible approach could be the identification of more potent neutralising VHH fragments. Alternatively, the expression of multiple VHH fragments could enhance the protection and the applicability of the system. It should be noted that the VHH fragment should preferably be constitutively produced using an expression system devoid of antibiotic selection marker. Nonetheless, the proof of concept reported here univocally showed that the expression of anti-phage VHH by a LAB represents a novel tool to prevent phage infection. This method would be much more cost effective as the VHH fragments would be produced in situ in the fermentation medium, eliminating the need for additional purification of the VHH fragments.
Bacterial strains, phage, culturing conditions, and VHH expression
Escherichia coli DH5α was used as the cloning host strain and cells were grown in Luria-Bertani (LB) medium (10 g tryptone/litre, 5 g NaCl/litre, 5 g yeast extract/litre). E. coli transformants were selected on LB plates containing 100 μg/ml ampicillin. Lactobacillus paracasei (previously known as L. casei or L. zeae ATCC 393 pLZ15-) , transformed with the plasmids pLP402 , pLP402-VHH5-secreted or pLP401-VHH2-anchored, were selected on MRS (Difco) plates with 3 μg/ml erythromycin after cultivation anaerobically at 37°C for 48 h. The pLP402-VHH5-secreted and pLP401-VHH2-anchored vectors, respectively mediated the secretion of VHH5 fragments and the surface expression of VHH2 fragments under the transcriptional control of the regulatable α-amylase promoter. The α-amylase promoter is regulated by a negative feedback. It is repressed by PTS sugars such as glucose and lactose in L. paracasei. Growth in presence of non-PTS sugars, such as mannitol, derepresses the promoter and activate gene expression. Pre-cultures of L. paracasei were made by growing the cultures in LCM medium  supplemented with 1% glucose and 3 μg/ml of erythromycin when needed and incubating them at 37°C overnight. These cultures were used to inoculate (2%) LCM-Man medium supplemented with 0.5% mannitol and 3 μg/ml of erythromycin, which were then incubated at 37°C. Cells were harvested in the exponential growth phase at an optical density at 600 nm (OD600) of 0.8 (108 cfu/ml) [16, 21]. Lactococcus lactis MG1363 was grown at 30°C in M17 broth  supplemented with 0.5% glucose (GM17) (Difco). Lysate of the lactococcal phage p2 was prepared as described previously .
Construction of the Lactobacillus paracasei expression vectors pLP401-VHH2-anchor and pLP402-VHH5-secreted
The VHH2 encoding gene was cut out from the phagemid vector pUR3824  at the restriction sites SfiI and NotI and ligated into pCANTAB 5E (Amersham Pharmacia Biotech) in order to fuse it with the E-tag. PCR amplification of the VHH2-E-tag was performed to add restriction sites for ClaI and XhoI to the VHH2-E-tag using primers ClaI-VHH: 5'-GCCATTGGAACTTACTCTGAAAA-3' and XhoI-VHH: 5'-CCGCTCGAGTGCGGCACGCGGTTCC-3'. Similarly, the VHH5-c-myc fragment was amplified by PCR from the pUR3825  vector using the primers ClaI-VHH and c-myc-stop (5'-CCGCTCGAGTTATGCGGCACGCGGTTCC-3') and adding in the process the restriction sites ClaI and XhoI with a stop codon (TAA) after the c-myc gene. The VHH2-E-tag and VHH5-c-myc fragments were, after restriction cutting and purification, ligated into the Lactobacillus expression vector pLP401 (previously named pLP402) [16, 21] at the ClaI and XhoI sites to generate the vectors pLP401-VHH2-anchored and pLP402-VHH5-secreted. Transformation of L. paracasei was performed as previously described [16, 21]. Selection of positive clones was performed using MRS plates containing 3 μg/ml erythromycin. Lactobacilli containing pLP402-VHH2-secreted was also constructed, similarly to the pLP401-VHH2-anchored construct but with a stop (TAA) before the E- tag and was used as control in the Western blot assays.
Preparation of samples for the enzyme-linked immunosorbent assay and Western blot analysis
After growth in presence of 0.5% mannitol, cells were washed, treated with lysosyme and disrupted by sonication as previously described . For ELISA, cell debris were removed by centrifugation for 10 min at 10,000 × g and the supernatant containing the protein extracts were stored at -20°C before use. For the Western blot analyses, Laemmli loading buffer was added to the sonicated cell extracts and the samples were boiled for 5 min, centrifuged (15 min, 10,000 × g) after which the supernatants were stored at -20°C. Supernatants from cultures of L. paracasei secreting VHH5 were filtered (0.45 μm) and concentrated 50 times using an ultrafiltration unit (Amicon). Protein concentrations were determined by the BioRad protein assay (BioRad Laboratories). For Western blot, the concentrated supernatant was boiled for 5 min in Laemmli buffer.
After growth in presence of mannitol, 100 μl of each lactobacilli culture (107 bacteria) containing the vectors pLP402 or pLP401-VHH2-anchored were washed three times in PBS by centrifugation (10,000 × g for 15 min) before resuspension in 100 μl of PBS. An equal amount of mouse anti-E-tag antibody (Amersham Bioscience) diluted 1/200 was added and the samples were incubated on ice for 1 h. The washing procedure in PBS was repeated and the samples were resuspended in 100 μl of PBS and mixed with 100 μl cy2-labeled donkey anti-mouse antibodies (Jackson Immunoresearch Laboratories) (final dilution 1/200) and incubated on ice for 30 min. After washing, the samples were resuspended in one ml of PBS and analysed in a FACSCalibur machine (Becton Dickinson).
Samples were run on a 12% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham-Biosciences). The membranes were blocked with 5% (w/v) milk powder diluted in PBS-Tween 20 (0.05% v/v) (PBS-TM) and incubated for 1 h at room temperature with monoclonal mouse anti-myc 9E10 (Abcam Inc.) for VHH5-secreted or anti-E-tag antibodies for VHH2-anchored, both diluted 1/1,000 in PBS-TM, followed by HRP (horse radish peroxidase) labelled goat anti-mouse antibodies (1/1,000) (DAKO) for 1 h. The washed membrane was then developed with ECL Plus Western Blotting Kit (Amersham-Bioscience) according to the manufacturer's instructions.
Enzyme-linked immunosorbent Assay
First, 96-well (Maxisorp) plates were coated with 10 μg/ml of recombinant major structural capsid protein (MCP, the VHH2 antigen) or recombinant receptor-binding protein (RBP, the VHH5 antigen)  and left overnight at 4°C. After washing with PBS containing 0.05% Tween 20 (PBS-T), dilutions of the concentrated supernatants from L. paracasei cultures secreting VHH5 as well as of the extract from cells anchoring VHH2, were added and incubated at room temperature for 1 h. Concentrated supernatants and cell extracts from cultures of L. paracasei containing only the vector pLP402 were used as negative controls. Purified VHH2 and VHH5 from E. coli, respectively with an E-tag and a c-myc tag , were used in standard curves to evaluate the amount of VHH produced by various L. paracasei transformants.
Plates were washed twice and a mouse anti-E-tag antibody (1/1,000) was added to the wells previously incubated with the extracts from L. paracasei VHH2-anchored. A mouse anti-myc antibody 9E10 (1/1,000) (Abcam Inc.) was added to the wells previously incubated with the purified VHH2 and VHH5 as well as supernatant containing VHH5 secreted by L. paracasei cells. After 1 h incubation at room temperature, plates were washed twice and an alkaline phosphatase conjugated rabbit anti-mouse antibody (1/1,000) (DAKO) was added to the plates. Following incubation for 1 h at room temperature, diethanolamine buffer (1 M, pH 10.0) containing 1 mg/ml of pNPP substrate (Sigma-Aldrich) was added to the wells. The absorbance was read at 405 nm in a Vmax Kinetic Microplate reader (Molecular Devices).
Scanning electron microscopy (SEM)
A culture of L. paracasei anchoring VHH2 was washed in PBS and 100 μl (106 bacteria/ml) was mixed with 500 μl (5 × 1010 pfu/ml) of the lactococcal phage p2 and left at room temperature for 1 h. The mixture was fixed in 2% glutaraldehyde diluted in 0.1 M sodium cacodylate and 0.1 M sucrose, and finally added onto a 0.1 mg/ml poly-L-lysine coated RC58 filter. After dehydration (70% ethanol 10 min, 95% ethanol 10 min, 99% ethanol 10 min), the samples were sputtered and analysed by SEM (JEOL JSM-820) at 15 kV.
Phage inhibition assay
The protocol for this assay was adapted from the lactococcal 936-phage adsorption experiments of Geller et al. . L. paracasei strains were grown to an optical density of 0.8 at 600 nm (OD600) in LCM-Man medium containing the appropriate antibiotic. Cells were harvested and the supernatant was filtered (0.45 μm) for immediate use. Cell pellets were suspended in 0.25 volume of LCM. Approximately 1,000 plaque-forming unit (pfu) of lactococcal phage p2 (30 μl) were mixed on ice with 10 μl of L. paracasei supernatant (about 5 ng VHH5), cells (4 × 106 bacteria) or LCM. After incubation of 4 h on ice, the mixture was centrifuged 5 min at 16,100 × g at 4°C. Phage titer was determined as followed in triplicate using 10 μl of the supernatant. Ten μl were added to 3 ml of GM17 supplemented with 0.75% agar and containing 100 μl of an overnight culture of the host strain Lactococcus lactis MG1363. The mixture was then poured onto a GM17 plate (1% agar), incubated overnight at 30°C, and the number of plaques were counted. The percentage of inhibition was calculated by dividing the titer of the phage with L. paracasei bacteria or supernatant by the phage titer in LCM. The quotient was subtracted from 1 and multiplied by 100. The experiment was repeated at least three times and the means were calculated for the pool of experiments. Because the amy promoter is repressed by lactose, co-culture of both strains in milk to test inhibition of phage infection was not tested with the present system.
We thank Pim Hermans (Unilever research and Development, Vlaardingen, The Netherlands) for help with culturing the phages and Neha Pant (Division of Clinical Immunology, Department of Laboratory Medicine, Karolinska Institutet, Sweden) for help with flow cytometry. We are grateful to Claudia Bergeron for her technical work on the phage neutralization assays. This study was supported by the Swedish Research Council (VR). S. M. would like to acknowledge the Natural Sciences and Engineering Research Council (NSERC) of Canada for funding part of this study.
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