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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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Re-attachment of 1H scFv to the cell wall of L. paracasei detected by immunoblotting. Pellet fraction of attached strain (KKA317) and (KKA101) grown in conditioned media containing 1H scFv (lane 1 and 3 respectively). Pellet fraction of strain KKA101 grown in conditioned media containing no 1H scFv, lane 2.
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Figure 5: Re-attachment of 1H scFv to the cell wall of L. paracasei detected by immunoblotting. Pellet fraction of attached strain (KKA317) and (KKA101) grown in conditioned media containing 1H scFv (lane 1 and 3 respectively). Pellet fraction of strain KKA101 grown in conditioned media containing no 1H scFv, lane 2.

Mentions: To examine if the attached 1H scFv could re-attach to the cell wall of lactobacilli we therefore grew a non-expressing strain of L. paracasei (KKA101) in media with and without the attached 1H scFv. Western blot analysis of the cell pellet of the cultures showed a clear re-attachment of the 1H scFv on the cell wall of the non-expressing strain (KKA101) when grown in media containing the scFv (Figure 5). Homologous binding domains are also found in other Gram-positive bacteria [26,27] but further studies would be needed to determine if re-attachment also occur on other bacteria and if this is a parameter for neutralisation with the attached construct.


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)

Re-attachment of 1H scFv to the cell wall of L. paracasei detected by immunoblotting. Pellet fraction of attached strain (KKA317) and (KKA101) grown in conditioned media containing 1H scFv (lane 1 and 3 respectively). Pellet fraction of strain KKA101 grown in conditioned media containing no 1H scFv, lane 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Re-attachment of 1H scFv to the cell wall of L. paracasei detected by immunoblotting. Pellet fraction of attached strain (KKA317) and (KKA101) grown in conditioned media containing 1H scFv (lane 1 and 3 respectively). Pellet fraction of strain KKA101 grown in conditioned media containing no 1H scFv, lane 2.
Mentions: To examine if the attached 1H scFv could re-attach to the cell wall of lactobacilli we therefore grew a non-expressing strain of L. paracasei (KKA101) in media with and without the attached 1H scFv. Western blot analysis of the cell pellet of the cultures showed a clear re-attachment of the 1H scFv on the cell wall of the non-expressing strain (KKA101) when grown in media containing the scFv (Figure 5). Homologous binding domains are also found in other Gram-positive bacteria [26,27] but further studies would be needed to determine if re-attachment also occur on other bacteria and if this is a parameter for neutralisation with the attached construct.

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