Abstract
BackgroundAnthrax 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.ResultsThe 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.ConclusionWe 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.
Highlights
Anthrax is caused by the bacterium Bacillus anthracis and is regarded as one of the most prominent bioterrorism threats
To evaluate the therapeutic potential of Lactobacillus expressing a scFv against anthrax toxin, a series of expression cassettes was constructed with the 1H scFv encoding gene placed under the control of the apf promoter and fused to the apf signal peptide at the N-terminal and with a C-terminal Etag for detection (Figure 1)
In pAF100-1HscFv, a stop codon just terminal of the E-tag leaves the scFv secreted into the media, pAF900-1HscFv has the C-terminal E-tag fused to the prtP anchoring domain leading the scFv to be covalently bound and displayed on the cell wall upon secretion from the cell, and lastly, pAF400-1HscFv, where the E-tag is fused to the anchoring domain of the apf gene which attaches the scFv non-covalently to the cell wall upon secretion
Summary
Anthrax is caused by the bacterium Bacillus anthracis and is regarded as one of the most prominent bioterrorism threats. Spores of Bacillus anthracis have for long been regarded as one of the most powerful bioterrorism threats due to their stability and high lethality [1]. Previous deliberate spread of anthrax spores as agent of biowarfare has been as aerosol. They could be Anthrax infections fall into three different categories, reflecting the route of entry; inhalational, gastrointestinal or cutaneous in order of severity of the infection. Natural occurrence of human gastrointestinal anthrax in the western world is rare due to the high standard of the food supply chain but is more common than inhalational anthrax in the developing world [6]
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