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In situ gastrointestinal protection against anthrax edema toxin by single-chain antibody fragment producing lactobacilli.

Andersen KK, Marcotte H, Álvarez B, Boyaka PN, Hammarström L - BMC Biotechnol. (2011)

Bottom Line: Cell wall display on lactobacilli and PA binding of the anchored constructs was confirmed by flow cytometry analysis.Utilising engineered lactobacilli therapeutically for neutralising toxins in the gastrointestinal tract can potential be expanded to provide protection against a range of additional gastrointestinal pathogens.The ability of lactobacilli to colonise the gastrointestinal tract may allow the system to be used both prophylactically and therapeutically.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden.

ABSTRACT

Background: Anthrax is caused by the bacterium Bacillus anthracis and is regarded as one of the most prominent bioterrorism threats. Anthrax toxicity is induced by the tripartite toxin complex, composed of the receptor-binding anthrax protective antigen and the two enzymatic subunits, lethal factor and edema factor. Recombinant lactobacilli have previously been used to deliver antibody fragments directed against surface epitopes of a variety of pathogens, including Streptococcus mutans, Porphyromonas gingivalis, and rotavirus. Here, we addressed whether or not anthrax toxins could be targeted and neutralised in the gastrointestinal tract by lactobacilli producing recombinant antibody fragments as a model system for toxin neutralisation in the gastrointestinal lumen.

Results: The neutralising anti-PA scFv, 1H, was expressed in L. paracasei as a secreted protein, a cell wall-anchored protein or both secreted and wall-anchored protein. Cell wall display on lactobacilli and PA binding of the anchored constructs was confirmed by flow cytometry analysis. Binding of secreted or attached scFv produced by lactobacilli to PA were verified by ELISA. Both construct were able to protect macrophages in an in vitro cytotoxicity assay. Finally, lactobacilli producing the cell wall attached scFv were able to neutralise the activity of anthrax edema toxin in the GI tract of mice, in vivo.

Conclusion: We have developed lactobacilli expressing a neutralising scFv fragment against the PA antigen of the anthrax toxin, which can provide protection against anthrax toxins both in vitro and in vivo. Utilising engineered lactobacilli therapeutically for neutralising toxins in the gastrointestinal tract can potential be expanded to provide protection against a range of additional gastrointestinal pathogens. The ability of lactobacilli to colonise the gastrointestinal tract may allow the system to be used both prophylactically and therapeutically.

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(A) Detection of the scFv expressed by recombinant L. paracasei by immunoblotting. Cell extract (c) of cell wall anchored strain (KKA307), secreted strain (KKA308) and attached strain (KKA317). Culture supernatant (s) from cell wall anchored strain (KKA307), secreted strain (KKA308) and attached strain (KKA317). The expected size of L. paracasei produced scFvs was 57.1, 29.2 and 42.2 KDa for the anchored, secreted and attached constructs respectively. (B) Binding and quantification of anti-PA scFv secreted into the growth media of the recombinant lactobacilli as measured by ELISA, with 1H scFv purified from E. coli as a reference (average of 4 experiments).
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Figure 2: (A) Detection of the scFv expressed by recombinant L. paracasei by immunoblotting. Cell extract (c) of cell wall anchored strain (KKA307), secreted strain (KKA308) and attached strain (KKA317). Culture supernatant (s) from cell wall anchored strain (KKA307), secreted strain (KKA308) and attached strain (KKA317). The expected size of L. paracasei produced scFvs was 57.1, 29.2 and 42.2 KDa for the anchored, secreted and attached constructs respectively. (B) Binding and quantification of anti-PA scFv secreted into the growth media of the recombinant lactobacilli as measured by ELISA, with 1H scFv purified from E. coli as a reference (average of 4 experiments).

Mentions: Expression and correct localisation of the three constructs upon transformation into Lactobacillus paracasei was verified by Western blot analysis of the supernatant and cell fractions of cultures grown in MRS (Figure 2A). The scFv expressed by both the secreted (KKA308) and anchored constructs (KKA307) were found primarily in the expected fraction, the supernatant for KKA308 and the cell fraction for KKA307. Some scFv were found in the supernatant fraction of the lactobacilli expressing the anchored construct, which is likely to be due either to saturation of anchoring sites or inefficient anchoring of the scFv. For lactobacilli expressing the attached construct, KKA317, the scFv was found to be bound to the cell wall but also secreted into the media in significant amounts. This is probably due to the weaker nature of the non-covalent binding to the cell wall of the APF binding domain. The total scFv production for the lactobacilli expressing the attached construct was 2-3 fold higher, relative to the two other constructs, despite being expressed from the same promoter. We have previously observed this effect for scFv fusions to the APF anchoring domain [25] suggesting that the fusion could be beneficial for the secretion or stability of the scFv fragments in the supernatant.


In situ gastrointestinal protection against anthrax edema toxin by single-chain antibody fragment producing lactobacilli.

Andersen KK, Marcotte H, Álvarez B, Boyaka PN, Hammarström L - BMC Biotechnol. (2011)

(A) Detection of the scFv expressed by recombinant L. paracasei by immunoblotting. Cell extract (c) of cell wall anchored strain (KKA307), secreted strain (KKA308) and attached strain (KKA317). Culture supernatant (s) from cell wall anchored strain (KKA307), secreted strain (KKA308) and attached strain (KKA317). The expected size of L. paracasei produced scFvs was 57.1, 29.2 and 42.2 KDa for the anchored, secreted and attached constructs respectively. (B) Binding and quantification of anti-PA scFv secreted into the growth media of the recombinant lactobacilli as measured by ELISA, with 1H scFv purified from E. coli as a reference (average of 4 experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3295704&req=5

Figure 2: (A) Detection of the scFv expressed by recombinant L. paracasei by immunoblotting. Cell extract (c) of cell wall anchored strain (KKA307), secreted strain (KKA308) and attached strain (KKA317). Culture supernatant (s) from cell wall anchored strain (KKA307), secreted strain (KKA308) and attached strain (KKA317). The expected size of L. paracasei produced scFvs was 57.1, 29.2 and 42.2 KDa for the anchored, secreted and attached constructs respectively. (B) Binding and quantification of anti-PA scFv secreted into the growth media of the recombinant lactobacilli as measured by ELISA, with 1H scFv purified from E. coli as a reference (average of 4 experiments).
Mentions: Expression and correct localisation of the three constructs upon transformation into Lactobacillus paracasei was verified by Western blot analysis of the supernatant and cell fractions of cultures grown in MRS (Figure 2A). The scFv expressed by both the secreted (KKA308) and anchored constructs (KKA307) were found primarily in the expected fraction, the supernatant for KKA308 and the cell fraction for KKA307. Some scFv were found in the supernatant fraction of the lactobacilli expressing the anchored construct, which is likely to be due either to saturation of anchoring sites or inefficient anchoring of the scFv. For lactobacilli expressing the attached construct, KKA317, the scFv was found to be bound to the cell wall but also secreted into the media in significant amounts. This is probably due to the weaker nature of the non-covalent binding to the cell wall of the APF binding domain. The total scFv production for the lactobacilli expressing the attached construct was 2-3 fold higher, relative to the two other constructs, despite being expressed from the same promoter. We have previously observed this effect for scFv fusions to the APF anchoring domain [25] suggesting that the fusion could be beneficial for the secretion or stability of the scFv fragments in the supernatant.

Bottom Line: Cell wall display on lactobacilli and PA binding of the anchored constructs was confirmed by flow cytometry analysis.Utilising engineered lactobacilli therapeutically for neutralising toxins in the gastrointestinal tract can potential be expanded to provide protection against a range of additional gastrointestinal pathogens.The ability of lactobacilli to colonise the gastrointestinal tract may allow the system to be used both prophylactically and therapeutically.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden.

ABSTRACT

Background: Anthrax is caused by the bacterium Bacillus anthracis and is regarded as one of the most prominent bioterrorism threats. Anthrax toxicity is induced by the tripartite toxin complex, composed of the receptor-binding anthrax protective antigen and the two enzymatic subunits, lethal factor and edema factor. Recombinant lactobacilli have previously been used to deliver antibody fragments directed against surface epitopes of a variety of pathogens, including Streptococcus mutans, Porphyromonas gingivalis, and rotavirus. Here, we addressed whether or not anthrax toxins could be targeted and neutralised in the gastrointestinal tract by lactobacilli producing recombinant antibody fragments as a model system for toxin neutralisation in the gastrointestinal lumen.

Results: The neutralising anti-PA scFv, 1H, was expressed in L. paracasei as a secreted protein, a cell wall-anchored protein or both secreted and wall-anchored protein. Cell wall display on lactobacilli and PA binding of the anchored constructs was confirmed by flow cytometry analysis. Binding of secreted or attached scFv produced by lactobacilli to PA were verified by ELISA. Both construct were able to protect macrophages in an in vitro cytotoxicity assay. Finally, lactobacilli producing the cell wall attached scFv were able to neutralise the activity of anthrax edema toxin in the GI tract of mice, in vivo.

Conclusion: We have developed lactobacilli expressing a neutralising scFv fragment against the PA antigen of the anthrax toxin, which can provide protection against anthrax toxins both in vitro and in vivo. Utilising engineered lactobacilli therapeutically for neutralising toxins in the gastrointestinal tract can potential be expanded to provide protection against a range of additional gastrointestinal pathogens. The ability of lactobacilli to colonise the gastrointestinal tract may allow the system to be used both prophylactically and therapeutically.

Show MeSH
Related in: MedlinePlus