Expression and Secretion of Cholera Toxin B Subunit in Lactobacilli

Abstract

Lactic acid bacteria (LAB) are used in various fields, including in food and medical supplies. There has been a great deal of research into vaccine development using LAB as carriers due to their "generally recognized as safe" status. Cholera is an infectious disease that causes diarrhea due to cholera toxin (CT) produced by Vibrio cholerae. The pentameric cholera toxin B (CTB) subunit has no toxicity, and is used as an antigen in cholera vaccines and as a delivery molecule in vaccines to various diseases. In this study, we generated recombinant LAB expressing and secreting CTB. Here, we first report that CTB expressed and secreted from LAB bound to GM1 ganglioside. The secreted CTB was purified, and its immunogenicity was determined by intranasal administration into mice. The results of the present study suggested that it may be useful as the basis of a new oral cholera vaccine combining LAB and CTB.

Recombinant fusion protein of cholera toxin B subunit with YVAD secreted by Lactobacillus caseiinhibits lipopolysaccharide-induced caspase-1 activation and subsequent IL-1 beta secretion in Caco-2 cells

“…In contrast, Dertzbaugh and Cox reported that CTB can bind to nickel ions without a His-tag because of a specific histidine residue within its sequence [17]. However, we recently showed that the purification of rCTB with a His-tag from the culture supernatant of L. casei was 20 times more efficient than the purification of rCTB without a His-tag [5]. The GM1-ganglioside-binding activity of rCTB with a His-tag, secreted by L. casei , was similar to those of rCTB without a His-tag and native CT from V. cholerae [5].…”

“…However, we recently showed that the purification of rCTB with a His-tag from the culture supernatant of L. casei was 20 times more efficient than the purification of rCTB without a His-tag [5]. The GM1-ganglioside-binding activity of rCTB with a His-tag, secreted by L. casei , was similar to those of rCTB without a His-tag and native CT from V. cholerae [5]. Therefore, to avoid any steric effects on the structure and GM1-ganglioside-binding activity of rCTB secreted by L. casei , we fused YVAD and the His-tag to the C-terminus of CTB.…”

“…Therefore, to avoid any steric effects on the structure and GM1-ganglioside-binding activity of rCTB secreted by L. casei , we fused YVAD and the His-tag to the C-terminus of CTB. The rCTB–YVAD secretion vector pSCTB–YVAD includes the promoter region of the lactate dehydrogenase (LDH) gene, the secretory signal sequence (SS) and terminator region (Term) of the β- N -acetylglucosaminidase gene from L. casei , the CTB-coding sequence from V. cholerae , the YVAD-coding sequence (tatgttgctgat; this nucleotide sequence was determined by reference to the codon usage of L. casei ATCC 334 and ATCC 393), and the His-tag-coding sequence in the Lactobacillus – E. coli shuttle vector pHIL253 [5] (Figure 1). Protein bands of about 12–14 kDa were detected in the supernatant of L. casei transformed with pSCTB–YVAD or pSCTB, when immunoblotted with an antibody directed against CT (Figure 2A).…”

“…L. casei was grown at 37°C in MRS broth to produce the recombinant strain or at 30°C in MRS/K (MRS with 0.2 M potassium phosphate buffer) to produce the cell culture supernatant [5]. Erythromycin (5 μg/ml) was added to MRS or MRS/K to select the recombinant strain.…”

“…Many groups have reported that recombinant CTB (rCTB) expressed in various bacteria, yeasts, and plants also binds to GM1 ganglioside. Previously, we constructed a recombinant Lactobacillus casei that secretes CTB, and showed that the rCTB secreted by L. casei has GM1-ganglioside-binding activity similar to that of CT from V. cholerae [5]. Recombinant fusion proteins of CTB with functional proteins and peptides, such as vaccine antigens [6] and the insulin B chain peptide [7], have been used as carriers to deliver these proteins and peptides to IECs, because they also bind to GM1 ganglioside.…”